US20240019449A1 - Screening Kit and Diagnosis System for Primary Aldosteronism - Google Patents
Screening Kit and Diagnosis System for Primary Aldosteronism Download PDFInfo
- Publication number
- US20240019449A1 US20240019449A1 US18/304,915 US202318304915A US2024019449A1 US 20240019449 A1 US20240019449 A1 US 20240019449A1 US 202318304915 A US202318304915 A US 202318304915A US 2024019449 A1 US2024019449 A1 US 2024019449A1
- Authority
- US
- United States
- Prior art keywords
- markers
- angiotensin
- aldosterone
- sample
- magnetic bead
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000016998 Conn syndrome Diseases 0.000 title claims abstract description 73
- 208000013846 primary aldosteronism Diseases 0.000 title claims abstract description 73
- 238000003745 diagnosis Methods 0.000 title claims abstract description 54
- 238000012216 screening Methods 0.000 title claims abstract description 49
- 239000011324 bead Substances 0.000 claims abstract description 104
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 claims abstract description 70
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 claims abstract description 70
- 229960002478 aldosterone Drugs 0.000 claims abstract description 70
- 238000012360 testing method Methods 0.000 claims abstract description 62
- 229920000642 polymer Polymers 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 42
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 13
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 65
- 230000000694 effects Effects 0.000 claims description 63
- 101800000734 Angiotensin-1 Proteins 0.000 claims description 55
- 102400000344 Angiotensin-1 Human genes 0.000 claims description 55
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 claims description 55
- 229960000890 hydrocortisone Drugs 0.000 claims description 34
- 210000004369 blood Anatomy 0.000 claims description 33
- 239000008280 blood Substances 0.000 claims description 33
- 239000003480 eluent Substances 0.000 claims description 32
- 101800000733 Angiotensin-2 Proteins 0.000 claims description 26
- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 claims description 26
- 229950006323 angiotensin ii Drugs 0.000 claims description 25
- 108090000783 Renin Proteins 0.000 claims description 24
- 102100028255 Renin Human genes 0.000 claims description 24
- 239000003550 marker Substances 0.000 claims description 19
- 206010020772 Hypertension Diseases 0.000 claims description 11
- 238000004458 analytical method Methods 0.000 claims description 9
- 238000007405 data analysis Methods 0.000 claims description 9
- 230000001225 therapeutic effect Effects 0.000 claims description 8
- 102000005862 Angiotensin II Human genes 0.000 claims 3
- 108090000746 Chymosin Proteins 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 63
- 230000008569 process Effects 0.000 abstract description 12
- 238000011285 therapeutic regimen Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 118
- 239000000243 solution Substances 0.000 description 107
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 69
- 238000005406 washing Methods 0.000 description 58
- 238000000605 extraction Methods 0.000 description 54
- 239000007788 liquid Substances 0.000 description 52
- 239000003795 chemical substances by application Substances 0.000 description 35
- 239000000047 product Substances 0.000 description 33
- 230000003213 activating effect Effects 0.000 description 30
- 239000012071 phase Substances 0.000 description 29
- 238000011534 incubation Methods 0.000 description 28
- 230000035945 sensitivity Effects 0.000 description 28
- 102400000345 Angiotensin-2 Human genes 0.000 description 23
- 239000007864 aqueous solution Substances 0.000 description 22
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 21
- 238000002360 preparation method Methods 0.000 description 20
- 239000000654 additive Substances 0.000 description 18
- 239000000872 buffer Substances 0.000 description 18
- 102000015427 Angiotensins Human genes 0.000 description 16
- 108010064733 Angiotensins Proteins 0.000 description 16
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 16
- 239000011159 matrix material Substances 0.000 description 15
- 239000012472 biological sample Substances 0.000 description 14
- 229940079593 drug Drugs 0.000 description 14
- 239000003814 drug Substances 0.000 description 14
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 13
- 201000010099 disease Diseases 0.000 description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 13
- 238000001179 sorption measurement Methods 0.000 description 11
- 239000012224 working solution Substances 0.000 description 11
- 239000011148 porous material Substances 0.000 description 10
- 239000007790 solid phase Substances 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 9
- 230000002596 correlated effect Effects 0.000 description 9
- 238000010828 elution Methods 0.000 description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 8
- 230000000996 additive effect Effects 0.000 description 8
- 238000005349 anion exchange Methods 0.000 description 8
- 235000019253 formic acid Nutrition 0.000 description 8
- 238000004949 mass spectrometry Methods 0.000 description 8
- 238000003908 quality control method Methods 0.000 description 8
- DDFHBQSCUXNBSA-UHFFFAOYSA-N 5-(5-carboxythiophen-2-yl)thiophene-2-carboxylic acid Chemical compound S1C(C(=O)O)=CC=C1C1=CC=C(C(O)=O)S1 DDFHBQSCUXNBSA-UHFFFAOYSA-N 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 210000002381 plasma Anatomy 0.000 description 7
- 238000005070 sampling Methods 0.000 description 7
- 201000004239 Secondary hypertension Diseases 0.000 description 6
- 239000000090 biomarker Substances 0.000 description 6
- 239000006185 dispersion Substances 0.000 description 6
- 239000000945 filler Substances 0.000 description 6
- 238000011533 pre-incubation Methods 0.000 description 6
- 238000004885 tandem mass spectrometry Methods 0.000 description 6
- 239000005541 ACE inhibitor Substances 0.000 description 5
- 230000001919 adrenal effect Effects 0.000 description 5
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 5
- 125000000129 anionic group Chemical group 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 4
- 230000003044 adaptive effect Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 3
- 241001251200 Agelas Species 0.000 description 3
- 101710129690 Angiotensin-converting enzyme inhibitor Proteins 0.000 description 3
- 101710086378 Bradykinin-potentiating and C-type natriuretic peptides Proteins 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 3
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000000274 adsorptive effect Effects 0.000 description 3
- 229920006318 anionic polymer Polymers 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 230000002146 bilateral effect Effects 0.000 description 3
- 238000010241 blood sampling Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 238000005341 cation exchange Methods 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000011258 core-shell material Substances 0.000 description 3
- 238000003748 differential diagnosis Methods 0.000 description 3
- 239000002934 diuretic Substances 0.000 description 3
- 229940030606 diuretics Drugs 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 102000008873 Angiotensin II receptor Human genes 0.000 description 2
- 108050000824 Angiotensin II receptor Proteins 0.000 description 2
- 102000004881 Angiotensinogen Human genes 0.000 description 2
- 108090001067 Angiotensinogen Proteins 0.000 description 2
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 208000007530 Essential hypertension Diseases 0.000 description 2
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 2
- 244000303040 Glycyrrhiza glabra Species 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 206010020571 Hyperaldosteronism Diseases 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 208000007271 Substance Withdrawal Syndrome Diseases 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000004100 adrenal gland Anatomy 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 229960000830 captopril Drugs 0.000 description 2
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 229920006317 cationic polymer Polymers 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 229960001484 edetic acid Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 235000011477 liquorice Nutrition 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000007886 magnetic bead extraction Methods 0.000 description 2
- 239000002395 mineralocorticoid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940037129 plain mineralocorticoids for systemic use Drugs 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 201000009395 primary hyperaldosteronism Diseases 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013062 quality control Sample Substances 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000013102 re-test Methods 0.000 description 2
- 239000003087 receptor blocking agent Substances 0.000 description 2
- 230000036454 renin-angiotensin system Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 239000000275 Adrenocorticotropic Hormone Substances 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 239000002083 C09CA01 - Losartan Substances 0.000 description 1
- 239000002053 C09CA06 - Candesartan Substances 0.000 description 1
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
- 101800000414 Corticotropin Proteins 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- 208000014311 Cushing syndrome Diseases 0.000 description 1
- 206010057615 Endocrine hypertension Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 208000019025 Hypokalemia Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 208000026709 Liddle syndrome Diseases 0.000 description 1
- 102000003979 Mineralocorticoid Receptors Human genes 0.000 description 1
- 108090000375 Mineralocorticoid Receptors Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 206010037113 Pseudoaldosteronism Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010041277 Sodium retention Diseases 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- PQSUYGKTWSAVDQ-IVGHLNRDSA-N [2H]C1=C2C([2H])([2H])C[C@H]3[C@@H]4CC[C@H](C(=O)C([2H])([2H])O)[C@]4(C[C@H](O)[C@@H]3[C@@]2(C)CC([2H])([2H])C1=O)C=O Chemical compound [2H]C1=C2C([2H])([2H])C[C@H]3[C@@H]4CC[C@H](C(=O)C([2H])([2H])O)[C@]4(C[C@H](O)[C@@H]3[C@@]2(C)CC([2H])([2H])C1=O)C=O PQSUYGKTWSAVDQ-IVGHLNRDSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 206010001323 adrenal adenoma Diseases 0.000 description 1
- 208000014594 aldosterone-producing adenoma with seizures and neurological abnormalities Diseases 0.000 description 1
- 150000001325 aldosterones Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000002333 angiotensin II receptor antagonist Substances 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 208000037849 arterial hypertension Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 229960000932 candesartan Drugs 0.000 description 1
- SGZAIDDFHDDFJU-UHFFFAOYSA-N candesartan Chemical compound CCOC1=NC2=CC=CC(C(O)=O)=C2N1CC(C=C1)=CC=C1C1=CC=CC=C1C1=NN=N[N]1 SGZAIDDFHDDFJU-UHFFFAOYSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 1
- 229960000258 corticotropin Drugs 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 208000028208 end stage renal disease Diseases 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- AAXVEMMRQDVLJB-BULBTXNYSA-N fludrocortisone Chemical compound O=C1CC[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 AAXVEMMRQDVLJB-BULBTXNYSA-N 0.000 description 1
- 229960002011 fludrocortisone Drugs 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- LNEPOXFFQSENCJ-UHFFFAOYSA-N haloperidol Chemical compound C1CC(O)(C=2C=CC(Cl)=CC=2)CCN1CCCC(=O)C1=CC=C(F)C=C1 LNEPOXFFQSENCJ-UHFFFAOYSA-N 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229960004773 losartan Drugs 0.000 description 1
- KJJZZJSZUJXYEA-UHFFFAOYSA-N losartan Chemical compound CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C=2[N]N=NN=2)C=C1 KJJZZJSZUJXYEA-UHFFFAOYSA-N 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- MBHOQEWPITZVIX-UHFFFAOYSA-N methanol Chemical compound OC.OC.OC.OC.OC MBHOQEWPITZVIX-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229960002582 perindopril Drugs 0.000 description 1
- IPVQLZZIHOAWMC-QXKUPLGCSA-N perindopril Chemical compound C1CCC[C@H]2C[C@@H](C(O)=O)N(C(=O)[C@H](C)N[C@@H](CCC)C(=O)OCC)[C@H]21 IPVQLZZIHOAWMC-QXKUPLGCSA-N 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 208000024896 potassium deficiency disease Diseases 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000002627 tracheal intubation Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
- G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
- G16H50/70—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for mining of medical data, e.g. analysing previous cases of other patients
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
- G01N2410/02—Angiotensins; Related peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/321—Arterial hypertension
Definitions
- the present invention relates to the field of medical diagnosis, and in particular to a screening kit, and a confirmed and typing diagnosis system for primary aldosteronism.
- PA Primary Aldosteronism
- PA aldosterone-producing adenoma
- IHA bilateral idiopathic hyperaldosteronism
- APA can be cured by adrenal gland surgery to improve arterial hypertension; and for the patients who refuse or are not suitable for surgery, drug intervention is an effective choice.
- PA diagnosis includes screening, confirmed and typing diagnosis.
- Aldosterone/renin activity is the preferred screening indicator for PA.
- Positive patients screened can be confirmed by oral administration of diet high-sodium, fludrocortisone test (FST), saline solution test (SIT) or captopril test (CCT), and unilateral dominant or bilateral equilibrium PA is distinguished by adrenal CT, adrenal venous sampling (AVS) and other ways.
- FST fludrocortisone test
- SIT saline solution test
- CCT captopril test
- PA unilateral dominant or bilateral equilibrium PA is distinguished by adrenal CT, adrenal venous sampling (AVS) and other ways.
- Test for aldosterone/renin activity is the common PA biochemical test index at present, but different test methods will affect the accuracy of the ARR test value, especially, the quantitative result of aldosterone; the test is throughout the whole process of screening, diagnosis and typing diagnosis.
- the measurement of aldosterone mainly includes radioimmunoassay and chemiluminescent immunoassay. Even though the method is rapid and easy to be implemented, the method has the disadvantages of cross-reactivity and poor interlaboratory test comparability.
- the present invention provides a screening kit and a confirmed and typing diagnosis system for primary aldosteronism.
- An object to be detected in a sample is extracted by a magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof, and the content of the five markers of aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone in the sample is detected by liquid chromatography-tandem mass spectrometry for one time; an ARR value is calculated; an judgment is made in combination with a cut-off value of 20.4 of the ARR and a concentration of a hypertension therapeutic affecting the ARR value; when a positive result is judged, PA typing is performed according to the test values of the markers, thereby achieving the simultaneous detection of the content of the five markers such as, aldosterone on the same platform, and establishing the clinical cut-off value for the screening and confirmed diagnosis of PA, thus achieving the accurate
- the present invention provides a detection kit for detecting primary aldosteronism;
- the kit includes an activating agent, a balanced solution, a washing liquid and an eluent solution;
- the activating agent is a solution including a magnetic bead with a surface, and a balanced hydrophilic-lipophilic polymer is bonded on the surface of the magnetic bead.
- the markers in a blood sample can be captured by the magnetic bead at one time; the marker is any one or more of aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
- the content of a series of specific markers in a blood sample is detected accurately to provide good basis for the subsequent screening and typing.
- markers in the blood sample need to be accurately extracted firstly, and then combined with the following accurate detection, which may reflect the real content of each marker in the blood sample indeed.
- the markers in the present invention include 5 types of aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
- the existing PA detection kits may be used to diagnose primary aldosteronism by detecting 3 markers at most only.
- the present invention innovates a one-step detection of 5 markers for the first time to achieve more accurate screening, confirmed and typing diagnosis of primary aldosteronism.
- this research group performed the detection originally in a way of pretreatment or single sample introduction; in the synchronous detection of three biomarkers of aldosterone, angiotensin I and angiotensin 1l, 96-well SPE plates (Agela PEP 96 Well Microplates) are used for extraction on a solid phase extraction column.
- the biological sample solution to be detected is loaded on the SPE plate, and the target component is selectively extracted and adsorbed by a solid phase, and partial interference elements are washed off by drip washing, and then the target component is eluted from the extraction column with an eluting agent with a stronger binding capacity to the solid phase, thus achieving the purposes of separation and purification.
- the disadvantages are as follows: the operating steps, including activation, sampling, drip washing, and elution, are more tedious and time-consuming; a single batch of sample treatment takes two hours around.
- the inter-well extraction efficiency of the sample greatly varies from the difference of the biological sample matrix; all the indexes need to be calibrated via isotope internal standards.
- dispersion solid phase extraction is used to treat samples.
- a solid absorbent material with specific adsorptive selectivity is dispersed into a biological sample solution, oscillated and mixed well such that the target component in the sample solution achieves a balance between the liquid phase and the selective adsorption solid phase.
- the dispersion solid phase extraction achieves the purpose of selective adsorption and extraction of the target markers according to the selectivity of the solid absorbent material.
- the major disadvantage is comparatively complex process of separating the solid phase from the solution.
- the magnetic bead with specific extraction and adsorption property is ultimately applied in this present invention to improve the separation process of the dispersion solid phase extraction.
- the magnetic bead is extracted and transferred with a magnetic bead extractor.
- the present invention rapidly and efficiently achieves the steps such as, activation, drip washing, sampling, elution and waste discharge, overcomes the disadvantages of the above-mentioned extraction by an extraction column and the dispersion solid phase extraction, and greatly improves the automation efficiency of the pretreatment, effectively reduces the blocking of SPE column possibly caused by the specificity of the biological sample matrix and other problems.
- the present invention reduces the matrix effects during the mass spectrometric detection of biological sample to shorten the pretreatment time of the same from 2 h to 10 min, and can further enhance the extraction effects of low-content indexes (aldosterone and 18-hydrocorticosterone) to improve the detection sensitivity.
- the solid phase extraction column bonded with a balanced hydrophilic-lipophilic polymer (Agela PEP 96 Well Microplates) filler is compared with the solid phase extraction column bonded a mixed anion exchange polymer (AgelaCleanert PAX 96 Wellplates) filler firstly.
- the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof has obvious advantages to improve the adsorption effects on the 5 markers.
- the balanced hydrophilic-lipophilic polymer may not only achieve the adsorption on high polar compounds, but also may effectively extract and adsorb the low polar compounds; but anionic and cationic polymers have stronger adsorptive selectivity and only selectively achieve the adsorption on high polar anions or cations, and hardly achieve the adsorption on low polar compounds simultaneously, leading to a low extraction efficiency of small molecules such as, aldosterone and 18-hydrocorticosterone, incapable of achieving the sensitivity requirements of clinical test.
- a method for magnetic bead is applied.
- the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is used to adsorb the 5 markers from a blood sample, and then a magnetic bead extractor may extract the magnetic bead which absorbs the markers from the biological sample matrix, thus effectively removing the interference elements from the biological sample. Therefore, after extraction and enrichment via the magnetic bead, compared with the solid phase extraction column, the eluent solution extracted by the magnetic bead has more purified components; the markers have smaller disturbing influences in the detection process; the mass spectrometry ionization efficiency is higher, and better detection sensitivity may be achieved.
- the pretreatment process may achieve high degree of automation and a single batch of sample pretreatment only takes 10 min around.
- a novel method for magnetic bead extraction is applied in this present invention, which may more adequately and efficiently extract the markers in a blood sample for one time.
- the magnetic bead is screened and pretreated.
- the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is applied, which may selectively adsorb 5 markers with great differences in physical and chemical properties from the sample, thus achieving efficient extraction and accurate detection.
- the eluent solution is an aqueous solution containing 50% methanol.
- the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof needs to be washed firstly, and then the target markers adsorbed are eluted, thus obtaining the content of each marker during the extraction of the blood sample.
- the washing liquid includes a washing liquid 1 and washing liquid 2;
- the washing liquid 1 is an aqueous solution with 10% methanol and the washing liquid 2 is isooctane.
- the activating agent is a solution of 50% ethanol including the magnetic bead
- the magnetic bead is a magnetic granule which is a core-shell solid phase and has a granularity of 30-50 ⁇ m, a specific surface area of 600 m 2 /g and a pore diameter of 80A.
- the present invention further contains a balanced solution and a liquid chromatogram mobile phase;
- the balanced solution is an aqueous solution with 1% formic acid.
- the liquid chromatogram mobile phase includes a mobile phase A and a mobile phase B; the mobile phase A is an aqueous solution containing an additive, and the mobile phase B is a methanol solution containing an additive (with 5% isopropanol).
- the additive is 1 mM ammonium fluoride.
- the balanced solution can simply and rapidly wrap the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof, thus completing the improvement of the activity of the magnetic bead. Moreover, markers in the sample can be further wrapped by the balanced solution, thus enhancing the bonding of the magnetic bead to the markers more, and greatly improving the enrichment effect of the magnetic bead.
- An additive 1 mM ammonium fluoride, is added to the mobile phase, which is capable of significantly improving the detection sensitivity of the markers in the sample, especially for the ionization efficiency of a low content index, aldosterone.
- the present invention provides an application of a kit in the preparation of a screening, confirmed and typing diagnosis kit for primary aldosteronism;
- the kit includes an activating agent, a washing liquid and an eluent solution;
- the activating agent is a solution containing a magnetic bead, and a balanced hydrophilic-lipophilic polymer is bonded on the surface of the magnetic bead;
- the markers in a blood sample can be captured by the magnetic bead at one time, and the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
- the present invention provides a method for detecting markers in a blood sample; the method includes the following steps:
- HPLC-tandem mass spectrometry is applied in this present invention to detect the 5 markers.
- the HPLC-tandem mass spectrometry has the advantages of high sensitivity and high specificity, and can achieve the combined detection of multiple clinical indexes in the same standard, thus reducing the result deviation caused by the difference between different detection systems and greatly improving the sensitivity and specificity in the diagnosis of secondary hypertension and other internal secretion-associated diseases.
- the blood sample in the step (1) firstly needs to be incubated in a buffer formation solution with an angiotensin converting enzyme inhibitor (PMSF) under acidic conditions, and at the end of the incubation, a stop buffer is added to stop the incubation, and then the incubated blood sample is treated (mixed or contacted) by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof.
- PMSF angiotensin converting enzyme inhibitor
- the buffer formation solution has a pH value of 5-6.
- angiotensinogen in the plasma sample will be converted into angiotensin I under the catalysis of renin activity, and due to the addition of the angiotensin converting enzyme inhibitor PMSF, the conversion of the angiotensin I into the angiotensin II is blocked. Renin activity is calculated by the change in the content of the angiotensin I.
- the pH value of the sample must be controlled within a suitable scope, or, the catalytic activity of renin will be affected seriously, leading to a larger deviation in the angiotensin I generated after the incubation.
- the measured renin activity is low under nonideal pH conditions, and there is a false-positive result when ARR value is calculated by aldosterone/renin activity.
- the buffer formation solution has a pH value of 5.4-5.6.
- the pH value of the incubated blood sample is associated with the pH values of the buffer solution, the buffer formation solution, the stop buffer, diluting agent for the preparation of the sample, and the like.
- the buffer formation solution has a pH value of 5.5.
- the step (1) is as follows: adsorbing the blood sample with the magnetic bead that is treated by an activating agent and a balanced solution in order, and then washed with a washing liquid before using an eluent solution to elute the markers from the magnetic bead.
- the activating agent is a solution of 50% ethanol including the magnetic bead.
- the magnetic bead is a magnetic granule which is a core-shell solid phase and has a granularity of 30-50 ⁇ m, a specific surface area of 600 m 2 /g and a pore diameter of 80A.
- the balanced solution is an aqueous solution with 1% formic acid.
- the washing liquid includes a washing liquid 1 and a washing liquid 2;
- the washing liquid 1 is an aqueous solution with 10% methanol and the washing liquid 2 is isooctane.
- the eluent solution in the step (2) is an aqueous solution containing 50% methanol.
- the LC mobile phase in the step (2) includes a mobile phase A and a mobile phase B;
- the mobile phase A is an aqueous solution containing an additive
- the mobile phase B is a methanol solution containing an additive (with 5% isopropanol);
- the additive is 1 mM ammonium fluoride.
- the present invention provides a screening, confirmed and typing diagnosis system for primary aldosteronism;
- the system includes a marker test module, a data input/output interface and a data analysis module;
- the marker test module is used for testing a test value of each marker obtained by the above method;
- the data input/output interface is used for inputting a test value of each marker;
- the data analysis module is used for analyzing the test value of each marker;
- the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone; after the analysis of the data analysis module, the data input/output interface is used for outputting a screening, confirmed and typing diagnosis result of primary aldosteronism.
- the quantitative determination of aldosterone is throughout the screening, confirmed and typing diagnosis.
- Aldosterone and renin activity are preferred biochemical indexes to screen PA; the confirmed test mainly replies on the quantitative results of aldosterone, and typing diagnosis relies on the reliable determination of aldosterone and cortisol more.
- the existing methods are chemiluminescent immunoassay or radioimmunoassay; there is no detection system capable of achieving simultaneous detection under the same standard.
- the system provided by the present invention may be used to simultaneously detect 5 markers by HPLC-tandem mass spectrometry, and then complete the screening, confirmed and typing diagnosis of PA. The whole process is more efficient. Moreover, the system of the present invention has the advantages of high sensitivity, good specificity, quantitative and qualitative determination of interference drugs, and the like during the diagnosis and identification of primary aldosteronism, thus achieving more accurate and reliable screening and typing results.
- the method for analyzing the test value of the marker by the data analysis module is as follows: calculating ARR value based on aldosterone and renin activity and making a judgment in combination with a cut-off value of the ARR and a concentration of a hypertension therapeutic affecting the ARR value; when a positive result is judged, confirmed and typing diagnosis is performed according to the test values of the aldosterone, the angiotensin I, the angiotensin II, the cortisol and the 18-hydrocorticosterone; the renin activity is a yield of the angiotensin I per unit time.
- the ARR has a cut-off value of 20.4.
- the cut-off value of ARR is commonly believed as 30 in the prior art.
- the cut-off value of ARR needs to be adjusted 20.4 when the screening, confirmed and typing diagnosis system for primary aldosteronism provided in the present invention is applied.
- the ARR value is directly correlated to the detection sensitivity of aldosterone and renin activity.
- Aldosterone is universally detected by chemiluminescent immunoassay previously, but the test value is higher such that the calculated result of the ARR value is greater than the actual value. Therefore, it needs to set a cut-off value of 30 to judge whether PA is positive or negative accurately more.
- the test value may reflect the content of the aldosterone reagent more accurately.
- the previous cut-off value of 30 of ARR has not conformed to the PA screening and typing system provided by the present invention; the cut-off value of ARR needs to be adjusted 20.4 such that the screening and typing result has a higher sensitivity, and the diagnostic result is more accurate.
- the concentration of the pre-incubation angiotensin I is very low and thus, may be basically ignored.
- the binding affects the concentration of the ARR hypertension therapeutic for judgment, which is aimed at eliminating interference from the therapeutic.
- Drugs which will interfere the screening and diagnosis of PA are angiotensin converting enzyme (ACE) inhibitors (e.g., Perindopril and Captopril), angiotensin II receptor blocking agents (e.g., Losartan and Candesartan), calcium ion antagonists, 3-receptor blocking agents, diuretics and the like.
- ACE angiotensin converting enzyme
- angiotensin II receptor blocking agents e.g., Losartan and Candesartan
- calcium ion antagonists e.g., Losartan and Candesartan
- 3-receptor blocking agents e.g., 3-receptor blocking agents, diuretics and the like.
- the present invention provides an application of the magnetic bead in extracting 5 markers in a blood sample simultaneously; the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone; a balanced hydrophilic-lipophilic polymer is bonded on the surface of the magnetic bead (HLB magnetic bead from Biosepur).
- the magnetic bead is a magnetic granule which is a core-shell solid phase and has a granularity of 30-50 ⁇ m, a specific surface area of 600 m 2 /g and a pore diameter of 80A.
- the screening kit, and the confirmed and typing diagnosis system for primary aldosteronism constructed in this present invention have the following beneficial effects:
- Diagnosis or detection refers to test or assay on a biomarker in a sample, or on a content of a target biomarker, for example, an absolute content or a relative content, and then the presence or the amount of the target marker serves to indicate whether an individual providing the sample has or suffers from a certain disease, or has the possibility of a certain disease.
- the meaning of the diagnosis or detection here may be exchanged.
- the result of such detection or diagnosis may not be directly used as a direct result of a disease, but an intermediate result. If it is desired to obtain the direct result, pathological or anatomic or other auxiliary means is required to confirm whether patients suffer from a certain disease.
- the present invention provides a plurality of novel biomarkers correlated to primary aldosteronism; the change of the content of these markers is directly correlated to the presence of primary aldosteronism.
- Markers or biomarkers have the same meanings in the present invention.
- the correlation here refers that the presence or content change of a certain biomarker in a sample is directly correlated to a specific disease, for example, the relative increase or decrease of the content indicates that the possibility of suffering from the disease is higher than that for healthy people.
- the plurality of different markers are present simultaneously or the content changes relatively in a sample, it indicates that the possibility of suffering from the disease is also higher than that for healthy people. That is, for the different types of markers, some markers are strongly correlated to the disease, while some markers are poorly correlated to the disease, or some are not correlated to a specific disease.
- One or more markers with strong correlation may be used as the markers for the diagnosis of a disease; the markers with weak correlation may be used to diagnose a certain disease in combination with the markers with strong correlation, thus improving the accuracy of the test results.
- the series of markers in blood plasma are closely correlated to PA, and the series of markers can be detected for one time by high performance liquid chromatography (HPLC)-tandem mass spectrometry, which is jointly useful for the screening, confirmed and typing diagnosis of PA.
- HPLC high performance liquid chromatography
- FIG. 1 A - FIG. 1 E are test chromatogram of 5 markers in a blood sample provided in Example 1 and wherein FIG. 1 A is the chromatogram of angiotensin I; FIG. 1 B is the chromatogram of angiotensin II; FIG. 1 C is the chromatogram of 18OH-Corticosterone; FIG. 1 D is the chromatogram of aldosterone; FIG. 1 E is the chromatogram of Cortisol.
- FIG. 2 is a ROC curve graph showing that the ARR cut-off value is 20.4 in Example 10.
- PMSF phenylmethylsulfonyl fluoride
- Buffer formation solution 12.11 g TRIS and 7.4 g ethylene diamine tetraacetic acid (EDTA) were added to a 100 mL volumetric flask, and added with deionized water to 90 mL, and then subjected to ultrasonic treatment for 30 min to be evenly dissolved. Deionized water was added to the scale line and mixed well. The solution was transferred to a reservoir vessel made of polypropylene. The PH value of the solution was adjusted within 5.45-5.60, and then the solution was stored at ⁇ 20° C.
- EDTA ethylene diamine tetraacetic acid
- Buffer formation solution A the solution was prepared at the same day of the detection analysis; 100 ⁇ L of 100 mM PMSF (angiotensin converting enzyme inhibitor) solution was added to 10 mL of the buffer formation solution, thus preparing the buffer formation solution A (pH value ranges from 5.4 to 5.6).
- PMSF angiotensin converting enzyme inhibitor
- Stop buffer containing internal standard 1 mL of the internal standard working solution was mixed with 9 mL water and 250 ⁇ L formic acid into the stop buffer containing internal standard.
- the magnetic bead is the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 ⁇ m; specific surface area: 600 m 2 /g and pore diameter: 80 A).
- the activating agent is a solution of 50% ethanol including the magnetic bead; the balanced solution is an aqueous solution containing 1% formic acid; the washing liquid 1 is an aqueous solution containing 10% methanol; the washing liquid 2 is isooctane, and the eluent solution is an aqueous solution containing 50% methanol.
- chromatographic column was a Phenomenex C8 chromatographic column; flow rate was 0.4 mL/min; column temperature was 40° C.; where the mobile phase A was an aqueous solution containing 1 mM ammonium fluoride, and the mobile phase B was a methanol solution containing 1 mM ammonium fluoride (with 5% isopropanol); the volume ratio of the mobile phase A to the mobile phase B was 90-5%:10-95%.
- the gradient elution program is shown in Table 7.
- Mass spectrometry conditions Value Curtain gas CUR 25 psi Atomized gas GS1 55 psi Auxiliary heating gas GS2 55 psi Ion source heating temperature 500° C. Collision gas CAD 10 psi Spray voltage 5500 V/ ⁇ 4500 V
- the standard curve was formulated by the PBS buffer solution matrix containing 4% BSA and subjected to synchronous treatment with the sample to be tested for detection.
- the test chromatogram is shown in FIG. 1 , representing angiotensin I (Ang I), angiotensin II (Ang II), 18-hydrocorticosterone (18-OH CORT), aldosterone (Aldo) and Cortisol from top to bottom in order.
- Ang I angiotensin I
- Ang II angiotensin II
- 18-OH CORT 18-hydrocorticosterone
- Aldo aldosterone
- Cortisol Cortisol from top to bottom in order.
- the method provided by the example may accurately detect the 5 markers simultaneously.
- the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1.
- the extraction was performed respectively by the different three methods: 1, extraction by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.
- the solid phase extraction is featured by easy blocking, complex operation and too stronger matrix effect, and the like. Therefore, the method of magnetic bead is applied.
- the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is used to adsorb the 5 markers, and then the magnetic bead extractor may extract the magnetic bead which absorbs the markers from the biological sample matrix, thus effectively removing the interference elements in the biological sample. Therefore, after extraction and enrichment of the magnetic bead, compared with the solid phase extraction column, the eluent solution extracted by the magnetic bead has more purified components; the markers have smaller disturbing influences in the detection process; the mass spectrometry ionization efficiency is higher, and better detection sensitivity may be achieved.
- the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1.
- the mixed anion exchange polymer SPE may be used in the sample pretreatment of the angiotensin detection. Therefore, comparisons were further made on the solid phase extraction column bonded a mixed anion exchange polymer (AgelaCleanert PAX 96 Wellplates) filler, the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.
- the 5 markers were adsorbed and extracted in the 4 ways, and after elution, the sample was subjected to liquid chromatography tandem-mass spectrometry; the test result of the treated sample was surveyed to measure the peak areas of the 5 markers in the sample S1, as shown in Table 13.
- the extraction effect of the MAX magnetic bead is obviously superior to that of the AgelaCleanert PAX 96 Wellplates solid phase extraction column; meanwhile, different materials are bonded on the surface of the magnetic bead, which has a large impact on the extraction effects of the 5 markers in the blood sample.
- the magnetic bead bonded the hydrophilic-lipophilic polymer may significantly improve the extraction effect on a portion of low-content indexes (aldosterone and 18-hydrocorticosterone) and may achieve better balance on the extraction and enrichment effects of the 5 markers with greater differences in physical and chemical properties.
- the balanced hydrophilic-lipophilic polymer may not only achieve the adsorption on high polar compounds, but also may effectively adsorb the low polar compounds; but anionic and cationic polymers have stronger adsorptive selectivity and thus, only selectively achieve the adsorption on high polar anions or cations, and hardly achieve the adsorption on low polar compounds simultaneously, leading to a low extraction efficiency of small molecules such as, aldosterone and 18-hydrocorticosterone, incapable of achieving the sensitivity requirements of clinical test.
- the 96-well SPE plate (Agela PEP 96 Well Microplates) was firstly extracted by the solid phase extraction column for activation and balance, and then the biological sample solution to be tested was loaded on the SPE plates; the target compound was extracted and adsorbed by a PEP filler selectively; a portion of inorganic salt and other impurities were washed from the SPE column via drip washing, and then the target component was eluted from the extraction column by an eluting agent with stronger binding capacity to the solid phase, afterwards, the eluent solution was blown-dried with nitrogen gas, redissolved and loaded for sample detection.
- the operation process has more manual steps, is complex and time-consuming; it takes about 2 h to treat a batch of samples.
- the inter-well extraction efficiency of the sample greatly varies from the difference of the biological sample matrix; all the indexes need to be calibrated via isotope internal standards.
- partial samples in especial hemolysis, lipemia and other samples may cause the blocking of the SPE column, leading to reduced pretreatment efficiency of the total batch of the samples and retest of the blocked sample.
- the magnetic bead is the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 ⁇ m; specific surface area: 600 m 2 /g and pore diameter: 80 A).
- the activating agent is a solution of 50% ethanol including the magnetic bead; the balanced solution is an aqueous solution containing 1% formic acid; the washing liquid 1 is aqueous solution containing 10% methanol; the washing liquid 2 is isooctane, and the eluent solution is an aqueous solution containing 50% methanol.
- the sample pretreatment step of the magnetic bead extractor is shown in Table 15 (the same as Table 6); the pretreatment time of each batch of samples is about 10 min.
- the solution to be tested in the columns 6 and 12 of the 96-well plate may be transferred to the 96-well loading plate for detection on the machine.
- the existing magnetic bead extractor may accommodate two 96-well plates for parallel operation for one time. Therefore, the pretreatment flux is 32 samples/batch; a 96-channel magnetic bead extractor may be also available; and the extraction efficiency is kept same.
- the pretreatment of the sample 96 may be achieved in 10 min. The pretreatment efficiency is greatly improved to achieve the high degree of automation of clinical sample pretreatment.
- angiotensinogen in the plasma sample will be converted into angiotensin I under the catalysis of renin activity, and due to the addition of the angiotensin converting enzyme, the content of the angiotensin I increases. Therefore, the incubation process will directly affect the content of the angiotensin I in the sample.
- the preparation, extraction and detection of the low, moderate and high-quality control samples were performed by the method provided in Example 1.
- the pH values of the buffer formation solution added to the pre-incubation samples were respectively 4.0, 5.0, 5.5, 6.0 and 7.4; the post-incubation samples were absorbed and extracted by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.
- the pH value of the buffer formation solution must be controlled within a suitable scope during the incubation, or, the catalytic activity of renin will be affected seriously, leading to a larger deviation in the angiotensin I generated after the incubation.
- the measured renin activity is low under nonideal pH conditions, and when ARR is calculated by aldosterone/renin activity, it is easy to cause a higher value of ARR, leading to a false-positive result.
- the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1.
- Different additives were added to the eluting agent to prepare different eluting agents, thus eluting the magnetic bead sample; after being eluted, the sample was subjected to liquid chromatography tandem-mass spectrometry to survey the test results of the sample after being eluted by different eluting agents, thus measuring the peak areas of the 5 markers in the sample S1, as shown in Table 17.
- each index has poor shape of detection peak and there are serious solvent effects; the situation is improved to some extent when the aqueous solution with 75% methanol is used for elution; and the aqueous solution with 50% methanol may effectively improve the peak shape; the elution efficiency is inadequate when 25% methanol is used; after an acid is added to the eluting agent, the ionization efficiency of aldosterone is obviously inhibited and the peak area decreases, thus affecting the detection sensitivity.
- Example 1 the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1.
- Mobile phase A was an aqueous solution and mobile phase B was a methanol solution (with 5% isopropanol) as basic mobile phases.
- Different additives were added to the mobile phase A and mobile phase B to prepare different mobile phases for liquid chromatography tandem-mass spectrometry; the test results of the sample detected under different mobile phases were surveyed, thus measuring the peak areas of the 5 markers in the sample S1, as shown in Table 18.
- the addition of 1 mM ammonium fluoride both in the mobile phase A and the mobile phase B may effectively improve the detection sensitivity; but the test result of the sample greatly varies from the type of the additives added; compared with the addition of 0.03% formic acid, the addition of 1 mM ammonium fluoride will cause decreased Ang I response to some extent, but may further improve the detection sensitivity of the other 4 indexes, especially for the low-content aldosterone. Therefore, the detection sensitivity of the 5 markers may satisfy the clinical demand more.
- the cationic mode should be chosen to detect angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone, while the anionic mode needs to be chosen to detect aldosterone; this is because when the cationic mode is chosen, there exists a peak diagram of cortisone, an isomer of aldosterone, nearby the detection peak of aldosterone to cause larger interference, and CV % is greater than 15%; but when the anionic mode is applied for detection, the test result is more stable and accurate, and CV % is less than 8.33%.
- Example 9 Screening, Confirmed and Typing Diagnosis System for Primary Aldosteronism
- a screening, confirmed and typing diagnosis system for primary aldosteronism is used for the screening, confirmed and typing diagnosis of primary aldosteronism; the specific method is as follows:
- the patients whose standing position PAC was greater than 15 ng/dL, standing position ARR was greater than 30 and aldosterone inhibition ratio after CCT was less than 30% were brought into the group PA.
- the patients were requested to withdraw diuretics or aldosterone receptor antagonists at least for 4 weeks, and other antihypertensive drugs such as, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor inhibitors (ARB), calcium ion antagonists (CCB) and beta receptor blockers at least for 2 weeks.
- ACEI angiotensin converting enzyme inhibitors
- ARB angiotensin receptor inhibitors
- CCB calcium ion antagonists
- beta receptor blockers at least for 2 weeks.
- serum potassium should be corrected to the normal range as much as possible.
- the patients who were grouped into the PA group kept in a standing position at 05:00 a.m. in the following day, and 2 h later, the blood sample was collected in the standing position at 07:00 a.m
- Bilateral synchronous blood sampling stimulated by non-adrenocorticotrophic hormone was applied.
- the content of the 5 markers in the blood sample was detected by the method provided by Example 1.
- a ratio of cortisol in adrenal veins to arterio-venous cortisol is defined as a selectivity index (SI); SI>2 indicates successful blood sampling.
- SI selectivity index
- a ratio of the aldosterone-cortisol ratio at the dominant side (a standardized aldosterone value) to the contralateral standardized aldosterone value is defined as a lateralized index (LI); when LI is greater than 2, it is believed that there is unilateral dominant secretion, and when LI is less than 2, it is believed that there is no obvious unilateral dominant secretion.
- sample Collection for all the sample collection, the subject was requested to receive the treatment of overnight fasting for 8 h above; sample transfer, centrifugation and separation should be ensured to be completed within 1 h, thus avoiding possible pre-analysis factors. All the samples were kept at ⁇ 80° C. for test before being analyzed.
- the screening, confirmed and typing diagnosis system for primary aldosteronism provided in Example 8 was used for the screening, confirmed and typing diagnosis of primary aldosteronism.
- the data was processed by R language software. Based on the analysis of the correlation between the ARR value and the presence of PA patients or not, the analysis result is shown in Table 19.
- the detection sensitivity, degree of sensitivity and 95% CI are in higher levels and have obvious advantages relative to other cut-off values.
- the effect of sensitivity is the most crucial index to judge whether ARR is negative or positive by the screening, confirmed and typing diagnosis system for primary aldosteronism provided by the present invention. Missing detection may be prevented effectively only by high sensitivity. Even though it is false-positive, the false-positive result may be further confirmed by PA typing according to the test values of the subsequent aldosterone, angiotensin II, cortisol and 18-hydrocorticosterone.
- the cut-off value of ARR is commonly believed as 30 in the prior art.
- the cut-off value of ARR needs to be adjusted 20.4 when the screening, confirmed and typing diagnosis system for primary aldosteronism provided in the present invention is applied. This is mainly because the ARR value is directly correlated to the detection sensitivity of aldosterone and renin activity. Aldosterone is universally detected by chemiluminescent immunoassay previously, but the test value is higher such that the calculated result of the ARR value is greater than the actual value. Therefore, it needs to set a cut-off value of 30 to judge whether PA is positive or negative accurately more.
- the test value may reflect the content of the aldosterone reagent more accurately. Therefore, the previous cut-off value of 30 of ARR has not conformed to the PA screening and typing system provided by the present invention; the cut-off value of ARR needs to be adjusted 20.4 such that the screening and typing result has a higher sensitivity, and the diagnostic result is more accurate.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Medical Informatics (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Data Mining & Analysis (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biophysics (AREA)
- Primary Health Care (AREA)
- Epidemiology (AREA)
- Databases & Information Systems (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
In a screening kit and a confirmed and typing diagnosis system for primary aldosteronism, a sample is pretreated by a magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof, and process conditions are optimized and the content of each the five markers such as, aldosterone in the sample is accurately detected by liquid chromatography-tandem mass spectrometry for one time, thus finding the optimal screening cut-off value of 20.4; when a positive result is judged, PA is confirmed and subjected to typing diagnosis according to the test values of the markers, thereby achieving the simultaneous detection of the content of each the five markers such as, aldosterone on the same platform. Therefore, the screening kit and confirmed and typing diagnosis system for primary aldosteronism are integrated with screening, confirmed and typing diagnosis functions, thus providing a reliable laboratory examination basis for clinicians to formulate an effective therapeutic regimen.
Description
- The present application claims priority to a Chinese prior application No. CN202210819638.2 and filed on Jul. 13, 2022; the entire contents of the above application, including the description, claims, abstract and abstract drawings of which are incorporated herein as a portion of the present invention.
- The present invention relates to the field of medical diagnosis, and in particular to a screening kit, and a confirmed and typing diagnosis system for primary aldosteronism.
- Primary Aldosteronism (PA) refers that excessive aldosterone is spontaneously secreted by adrenal gland to cause sodium retention and potassium discharge in vivo as well as increased blood volume, and the activity of the renin-angiotensin system is inhibited. PA is clinically manifested as hypertension accompanied with or without hypokalemia. The latest domestic study has shown that the incidence of PA exceeds 4% in the newly diagnosed hypertension and 17-23% in the refractory hypertension group. Compared with the essential hypertension patients, PA patients have a higher risk of suffering from atherosclerosis, myocardial infarction, peripheral vascular diseases, stroke and chronic renal damage, and it is easy to cause missed diagnosis and misdiagnosis. Therefore, early diagnosis and early treatment are of great importance. The most common two forms of PA are aldosterone-producing adenoma (APA) and bilateral idiopathic hyperaldosteronism (IHA); APA can be cured by adrenal gland surgery to improve arterial hypertension; and for the patients who refuse or are not suitable for surgery, drug intervention is an effective choice.
- PA diagnosis includes screening, confirmed and typing diagnosis. Aldosterone/renin activity (ARR) is the preferred screening indicator for PA. Positive patients screened can be confirmed by oral administration of diet high-sodium, fludrocortisone test (FST), saline solution test (SIT) or captopril test (CCT), and unilateral dominant or bilateral equilibrium PA is distinguished by adrenal CT, adrenal venous sampling (AVS) and other ways. There are the following aspects of reasons to interfere the confirmed diagnosis of PA:
- 1) Test Method:
- Test for aldosterone/renin activity (ARR) is the common PA biochemical test index at present, but different test methods will affect the accuracy of the ARR test value, especially, the quantitative result of aldosterone; the test is throughout the whole process of screening, diagnosis and typing diagnosis. Currently, the measurement of aldosterone mainly includes radioimmunoassay and chemiluminescent immunoassay. Even though the method is rapid and easy to be implemented, the method has the disadvantages of cross-reactivity and poor interlaboratory test comparability. Besides no comparability and lack of standardization in the test method, in case of renal function damage, the use of immunoassay will cause significantly overestimated test result of aldosterone; IA indicates that the median positive bias is 127%; it is presumably caused by the cross reaction between antibodies and non-eliminated aldosterone metabolites. Such an action is not only limited to end-stage renal failure, but also occurs in patients with mild or moderate renal insufficiency in a grading way. Therefore, there is frequently a phenomenon of inconsistency between the test result and clinical manifestation, which affects the screening and differential diagnosis for the pathogenesis of secondary hypertension by a clinician, and even leads to misdiagnosis or missed diagnosis of PA patients.
- 2) Interference from Therapeutic Drugs
- Recommended by domestic and overseas guidelines, test for aldosterone/renin activity is the preferred biochemical index to screen primary aldosteronism. However, the renin-angiotensin-aldosterone system of a human body is highly susceptible to diet and drugs; therefore, patients need to withdraw the conventional hypotensive drugs (such as, calcium ion antagonists, angiotoninase inhibitors, angiotensin II receptor antagonists and diuretics) for 4-6 weeks during screening, which causes a false-positive or false-negative screening result and causes certain difficulties to the clinical implementation of screening items. This is solved by the applicant in the patent invention 2021106374412 of the prior application, namely, how to eliminate the false-positive or false-negative drug interference.
- 3) Differential Diagnosis
- In the differential diagnosis of endocrine hypertension, there is always hypertension caused by other excessive mineralocorticoids. There exist clinical symptoms similar to primary aldosteronism, which causes certain disturbances and difficulties for clinical diagnosis to some extent; for example, low renin hypertension caused by the increase of other mineralocorticoids due to the inhibition of hereditary or acquired (from liquorice ingestion) 11-3HSD enzyme, pseudo-aldosteronism caused by the excessive consumption of liquorice, Cushing's syndrome, DOC secretion-producing tumors, and the like. These clinical symptoms are similar to PA, but are not PA, which causes lots of interference on the confirmed diagnosis of PA.
- Therefore, it is urgent to find a more accurate kit for diagnosis, screening and typing as well as an integrated system of PA.
- In view of the problems existing in the prior art, the present invention provides a screening kit and a confirmed and typing diagnosis system for primary aldosteronism. An object to be detected in a sample is extracted by a magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof, and the content of the five markers of aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone in the sample is detected by liquid chromatography-tandem mass spectrometry for one time; an ARR value is calculated; an judgment is made in combination with a cut-off value of 20.4 of the ARR and a concentration of a hypertension therapeutic affecting the ARR value; when a positive result is judged, PA typing is performed according to the test values of the markers, thereby achieving the simultaneous detection of the content of the five markers such as, aldosterone on the same platform, and establishing the clinical cut-off value for the screening and confirmed diagnosis of PA, thus achieving the accurate diagnosis and typing of PA such that patients with hypertension secondary to PA can be screened and confirmed in early stage. Therefore, the present invention provides a reliable laboratory examination basis for clinicians to formulate an effective therapeutic intervention.
- In one aspect, the present invention provides a detection kit for detecting primary aldosteronism; the kit includes an activating agent, a balanced solution, a washing liquid and an eluent solution; the activating agent is a solution including a magnetic bead with a surface, and a balanced hydrophilic-lipophilic polymer is bonded on the surface of the magnetic bead. The markers in a blood sample can be captured by the magnetic bead at one time; the marker is any one or more of aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
- In this present invention, the content of a series of specific markers in a blood sample is detected accurately to provide good basis for the subsequent screening and typing. To achieve accurate detection, markers in the blood sample need to be accurately extracted firstly, and then combined with the following accurate detection, which may reflect the real content of each marker in the blood sample indeed.
- The markers in the present invention include 5 types of aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone. The existing PA detection kits may be used to diagnose primary aldosteronism by detecting 3 markers at most only. The present invention innovates a one-step detection of 5 markers for the first time to achieve more accurate screening, confirmed and typing diagnosis of primary aldosteronism.
- Directed to the detection of aldosterone and angiotensin I, this research group performed the detection originally in a way of pretreatment or single sample introduction; in the synchronous detection of three biomarkers of aldosterone, angiotensin I and angiotensin 1l, 96-well SPE plates (Agela PEP 96 Well Microplates) are used for extraction on a solid phase extraction column. After the SPE plate is activated and balanced, the biological sample solution to be detected is loaded on the SPE plate, and the target component is selectively extracted and adsorbed by a solid phase, and partial interference elements are washed off by drip washing, and then the target component is eluted from the extraction column with an eluting agent with a stronger binding capacity to the solid phase, thus achieving the purposes of separation and purification. The disadvantages are as follows: the operating steps, including activation, sampling, drip washing, and elution, are more tedious and time-consuming; a single batch of sample treatment takes two hours around. Moreover, the inter-well extraction efficiency of the sample greatly varies from the difference of the biological sample matrix; all the indexes need to be calibrated via isotope internal standards. Further, due to the specificity of the biological sample matrix, partial samples (in especial hemolysis, lipemia and other samples) may cause the blocking of the SPE column, leading to reduced pretreatment efficiency of the total batch of the sample and retest of the blocked sample. To further achieve the typing diagnosis of PA patients, new test indexes of 18-hydrocorticosterone and cortisol are added. Since both content and mass spectrum response of 18-hydrocorticosterone are lower, there is a higher demand for the enrichment efficiency and purification effect in the pretreatment process.
- Therefore, dispersion solid phase extraction is used to treat samples. In the dispersion solid phase extraction, a solid absorbent material with specific adsorptive selectivity is dispersed into a biological sample solution, oscillated and mixed well such that the target component in the sample solution achieves a balance between the liquid phase and the selective adsorption solid phase. The dispersion solid phase extraction achieves the purpose of selective adsorption and extraction of the target markers according to the selectivity of the solid absorbent material. However, the major disadvantage is comparatively complex process of separating the solid phase from the solution.
- To further solve the problem of the dispersion solid phase extraction, namely, too complex process of separating the solid phase from the solution, the magnetic bead with specific extraction and adsorption property is ultimately applied in this present invention to improve the separation process of the dispersion solid phase extraction. After the object to be detected is adsorbed by the magnetic bead specifically, the magnetic bead is extracted and transferred with a magnetic bead extractor. The present invention rapidly and efficiently achieves the steps such as, activation, drip washing, sampling, elution and waste discharge, overcomes the disadvantages of the above-mentioned extraction by an extraction column and the dispersion solid phase extraction, and greatly improves the automation efficiency of the pretreatment, effectively reduces the blocking of SPE column possibly caused by the specificity of the biological sample matrix and other problems. Moreover, the present invention reduces the matrix effects during the mass spectrometric detection of biological sample to shorten the pretreatment time of the same from 2 h to 10 min, and can further enhance the extraction effects of low-content indexes (aldosterone and 18-hydrocorticosterone) to improve the detection sensitivity.
- Moreover, in terms of the solid phase extraction, different properties of polymer fillers are bonded on the surface of the solid phase, which will cause large impact on the adsorption and extraction effects of the object to be detected. Based on the search result of the literature on the single and separate detection of 5 different markers (the mixed anion exchange polymer SPE may be used in the sample pretreatment of the angiotensin detection), in this present invention, the solid phase extraction column bonded with a balanced hydrophilic-lipophilic polymer (Agela PEP 96 Well Microplates) filler is compared with the solid phase extraction column bonded a mixed anion exchange polymer (AgelaCleanert PAX 96 Wellplates) filler firstly. Through the comparison, it is found that the extraction effects of the solid phase extraction column bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof on the 5 markers are not lower than those of the solid phase extraction column of the mixed anion exchange polymer, especially, it has better enrichment effect on the lower content of aldosterone and 18-hydrocorticosterone. Meanwhile, comparisons further are made to the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof, the magnetic bead bonded with a mixed anion exchange polymer and the magnetic bead bonded with a mixed cation exchange polymer. It is found that the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof has obvious advantages to improve the adsorption effects on the 5 markers. The reason is probably as follows: the balanced hydrophilic-lipophilic polymer may not only achieve the adsorption on high polar compounds, but also may effectively extract and adsorb the low polar compounds; but anionic and cationic polymers have stronger adsorptive selectivity and only selectively achieve the adsorption on high polar anions or cations, and hardly achieve the adsorption on low polar compounds simultaneously, leading to a low extraction efficiency of small molecules such as, aldosterone and 18-hydrocorticosterone, incapable of achieving the sensitivity requirements of clinical test.
- A method for magnetic bead is applied. The magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is used to adsorb the 5 markers from a blood sample, and then a magnetic bead extractor may extract the magnetic bead which absorbs the markers from the biological sample matrix, thus effectively removing the interference elements from the biological sample. Therefore, after extraction and enrichment via the magnetic bead, compared with the solid phase extraction column, the eluent solution extracted by the magnetic bead has more purified components; the markers have smaller disturbing influences in the detection process; the mass spectrometry ionization efficiency is higher, and better detection sensitivity may be achieved. Moreover, the pretreatment process may achieve high degree of automation and a single batch of sample pretreatment only takes 10 min around.
- To sum up, directed to the extraction of the 5 markers in the blood sample, a novel method for magnetic bead extraction is applied in this present invention, which may more adequately and efficiently extract the markers in a blood sample for one time. The magnetic bead is screened and pretreated. The magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is applied, which may selectively adsorb 5 markers with great differences in physical and chemical properties from the sample, thus achieving efficient extraction and accurate detection.
- Further, the eluent solution is an aqueous solution containing 50% methanol.
- After fully absorbing these markers for one time, the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof needs to be washed firstly, and then the target markers adsorbed are eluted, thus obtaining the content of each marker during the extraction of the blood sample.
- In this present invention, research shows that when the aqueous solution containing 50% methanol is used as the eluent solution, the efficient elution of the 5 markers may be achieved, thus improving the sensitivity of the test results and effectively avoiding the solvent effect caused by too high concentration of organic phase content simultaneously.
- Further, the washing liquid includes a
washing liquid 1 andwashing liquid 2; thewashing liquid 1 is an aqueous solution with 10% methanol and thewashing liquid 2 is isooctane. - Further, the activating agent is a solution of 50% ethanol including the magnetic bead, and the magnetic bead is a magnetic granule which is a core-shell solid phase and has a granularity of 30-50 μm, a specific surface area of 600 m2/g and a pore diameter of 80A.
- Further, the present invention further contains a balanced solution and a liquid chromatogram mobile phase; the balanced solution is an aqueous solution with 1% formic acid.
- Further, the liquid chromatogram mobile phase includes a mobile phase A and a mobile phase B; the mobile phase A is an aqueous solution containing an additive, and the mobile phase B is a methanol solution containing an additive (with 5% isopropanol).
- Further, the additive is 1 mM ammonium fluoride.
- The balanced solution can simply and rapidly wrap the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof, thus completing the improvement of the activity of the magnetic bead. Moreover, markers in the sample can be further wrapped by the balanced solution, thus enhancing the bonding of the magnetic bead to the markers more, and greatly improving the enrichment effect of the magnetic bead.
- An additive, 1 mM ammonium fluoride, is added to the mobile phase, which is capable of significantly improving the detection sensitivity of the markers in the sample, especially for the ionization efficiency of a low content index, aldosterone.
- In the other aspect, the present invention provides an application of a kit in the preparation of a screening, confirmed and typing diagnosis kit for primary aldosteronism; the kit includes an activating agent, a washing liquid and an eluent solution; the activating agent is a solution containing a magnetic bead, and a balanced hydrophilic-lipophilic polymer is bonded on the surface of the magnetic bead; the markers in a blood sample can be captured by the magnetic bead at one time, and the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
- In a further aspect, the present invention provides a method for detecting markers in a blood sample; the method includes the following steps:
-
- (1) treating the blood sample with a magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof to absorb the markers in the blood sample;
- (2) using an eluent solution to elute the markers from the magnetic bead and using a liquid chromatography tandem-mass spectrometry to test the numbers of the markers in the blood sample; the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
- High performance liquid chromatography (HPLC)-tandem mass spectrometry is applied in this present invention to detect the 5 markers. The HPLC-tandem mass spectrometry has the advantages of high sensitivity and high specificity, and can achieve the combined detection of multiple clinical indexes in the same standard, thus reducing the result deviation caused by the difference between different detection systems and greatly improving the sensitivity and specificity in the diagnosis of secondary hypertension and other internal secretion-associated diseases.
- Further, the blood sample in the step (1) firstly needs to be incubated in a buffer formation solution with an angiotensin converting enzyme inhibitor (PMSF) under acidic conditions, and at the end of the incubation, a stop buffer is added to stop the incubation, and then the incubated blood sample is treated (mixed or contacted) by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof.
- Further, the buffer formation solution has a pH value of 5-6.
- During the incubation, angiotensinogen in the plasma sample will be converted into angiotensin I under the catalysis of renin activity, and due to the addition of the angiotensin converting enzyme inhibitor PMSF, the conversion of the angiotensin I into the angiotensin II is blocked. Renin activity is calculated by the change in the content of the angiotensin I.
- During the incubation, the pH value of the sample must be controlled within a suitable scope, or, the catalytic activity of renin will be affected seriously, leading to a larger deviation in the angiotensin I generated after the incubation. The measured renin activity is low under nonideal pH conditions, and there is a false-positive result when ARR value is calculated by aldosterone/renin activity.
- In some embodiments, the buffer formation solution has a pH value of 5.4-5.6.
- In some embodiments, the pH value of the incubated blood sample is associated with the pH values of the buffer solution, the buffer formation solution, the stop buffer, diluting agent for the preparation of the sample, and the like.
- In some embodiments, preferably, the buffer formation solution has a pH value of 5.5.
- In some embodiments, the step (1) is as follows: adsorbing the blood sample with the magnetic bead that is treated by an activating agent and a balanced solution in order, and then washed with a washing liquid before using an eluent solution to elute the markers from the magnetic bead.
- Further, the activating agent is a solution of 50% ethanol including the magnetic bead.
- Further, the magnetic bead is a magnetic granule which is a core-shell solid phase and has a granularity of 30-50 μm, a specific surface area of 600 m2/g and a pore diameter of 80A.
- Further, the balanced solution is an aqueous solution with 1% formic acid.
- Further, the washing liquid includes a
washing liquid 1 and awashing liquid 2; thewashing liquid 1 is an aqueous solution with 10% methanol and thewashing liquid 2 is isooctane. - Further, the eluent solution in the step (2) is an aqueous solution containing 50% methanol.
- Further, the LC mobile phase in the step (2) includes a mobile phase A and a mobile phase B; the mobile phase A is an aqueous solution containing an additive, and the mobile phase B is a methanol solution containing an additive (with 5% isopropanol); the additive is 1 mM ammonium fluoride.
- In a further aspect, the present invention provides a screening, confirmed and typing diagnosis system for primary aldosteronism; the system includes a marker test module, a data input/output interface and a data analysis module; the marker test module is used for testing a test value of each marker obtained by the above method; the data input/output interface is used for inputting a test value of each marker; the data analysis module is used for analyzing the test value of each marker; the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone; after the analysis of the data analysis module, the data input/output interface is used for outputting a screening, confirmed and typing diagnosis result of primary aldosteronism.
- In the existing diagnostic process of PA, the quantitative determination of aldosterone is throughout the screening, confirmed and typing diagnosis. Aldosterone and renin activity are preferred biochemical indexes to screen PA; the confirmed test mainly replies on the quantitative results of aldosterone, and typing diagnosis relies on the reliable determination of aldosterone and cortisol more. The existing methods are chemiluminescent immunoassay or radioimmunoassay; there is no detection system capable of achieving simultaneous detection under the same standard.
- The system provided by the present invention may be used to simultaneously detect 5 markers by HPLC-tandem mass spectrometry, and then complete the screening, confirmed and typing diagnosis of PA. The whole process is more efficient. Moreover, the system of the present invention has the advantages of high sensitivity, good specificity, quantitative and qualitative determination of interference drugs, and the like during the diagnosis and identification of primary aldosteronism, thus achieving more accurate and reliable screening and typing results.
- Further, the method for analyzing the test value of the marker by the data analysis module is as follows: calculating ARR value based on aldosterone and renin activity and making a judgment in combination with a cut-off value of the ARR and a concentration of a hypertension therapeutic affecting the ARR value; when a positive result is judged, confirmed and typing diagnosis is performed according to the test values of the aldosterone, the angiotensin I, the angiotensin II, the cortisol and the 18-hydrocorticosterone; the renin activity is a yield of the angiotensin I per unit time.
- Further, the ARR has a cut-off value of 20.4.
- The cut-off value of ARR is commonly believed as 30 in the prior art. The cut-off value of ARR needs to be adjusted 20.4 when the screening, confirmed and typing diagnosis system for primary aldosteronism provided in the present invention is applied. Research shows that when the cut-off value of ARR is 20.4, the screening, confirmed and typing result has higher sensitivity and specificity, being 94.4% (95% CI: 72.7-99.9) and 87.2% (95% CI: 72.6-95.7) respectively; Youden index is up to the maximum value (YI=0.82) and area under the curve (AUC) is up to 0.956.
- This is mainly because the ARR value is directly correlated to the detection sensitivity of aldosterone and renin activity. Aldosterone is universally detected by chemiluminescent immunoassay previously, but the test value is higher such that the calculated result of the ARR value is greater than the actual value. Therefore, it needs to set a cut-off value of 30 to judge whether PA is positive or negative accurately more. When the sample pretreatment (extraction by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer) and detection method (HPLC-tandem mass spectrometry) provided by the present invention are applied, the test value may reflect the content of the aldosterone reagent more accurately. Therefore, the previous cut-off value of 30 of ARR has not conformed to the PA screening and typing system provided by the present invention; the cut-off value of ARR needs to be adjusted 20.4 such that the screening and typing result has a higher sensitivity, and the diagnostic result is more accurate.
- In some embodiments, the calculation formula of the ARR is as follows: ARR=concentration of aldosterone/renin activity (production rate of angiotensin I); the renin activity is calculated by detecting the concentration of angiotensin I in the pre-incubation sample and in the post-incubation sample and according to the following formula: production rate of angiotensin I=(concentration of angiotensin I in the post-incubation sample−concentration of angiotensin I in the pre-incubation sample)/incubation time.
- In some embodiments, the concentration of the pre-incubation angiotensin I is very low and thus, may be basically ignored.
- In some embodiments, the binding affects the concentration of the ARR hypertension therapeutic for judgment, which is aimed at eliminating interference from the therapeutic. There are hundreds of drugs for the treatment of hypertension clinically. Drugs which will interfere the screening and diagnosis of PA are angiotensin converting enzyme (ACE) inhibitors (e.g., Perindopril and Captopril), angiotensin II receptor blocking agents (e.g., Losartan and Candesartan), calcium ion antagonists, 3-receptor blocking agents, diuretics and the like. This is solved by the applicant in the patent invention 2021106374412 of the prior application, namely, how to eliminate the false-positive or false-negative drug interference. The PA screening and typing system of PA provided by the present invention needs to be combined with the method provided by the patent invention 2021106374412 to eliminate the drug interference.
- In a further aspect, the present invention provides an application of the magnetic bead in extracting 5 markers in a blood sample simultaneously; the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone; a balanced hydrophilic-lipophilic polymer is bonded on the surface of the magnetic bead (HLB magnetic bead from Biosepur). The magnetic bead is a magnetic granule which is a core-shell solid phase and has a granularity of 30-50 μm, a specific surface area of 600 m2/g and a pore diameter of 80A.
- The screening kit, and the confirmed and typing diagnosis system for primary aldosteronism constructed in this present invention have the following beneficial effects:
-
- 1. The blood sample is treated with a magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof to adsorb 5 markers in the blood sample more fully, thus achieving the accurate extraction and precise detection.
- 2. The extraction process of the magnetic bead is screened and optimized, and the pH value of the blood sample is adjusted; formic acid is added to the eluent solution and LC mobile phase; and suitable activating agent, balanced solution and washing liquid are chosen to greatly improve the enrichment and extraction effects of the magnetic bead and to achieve the efficient extraction of the 5 markers. Therefore, the amount of the markers extracted is closer to the real content of those in the blood sample, thus improving the accuracy of the test results.
- 3. The screening and typing diagnosis system for PA is established; the system has the advantages of high sensitivity, good specificity, quantitative and qualitative determination of interference drugs during the diagnosis and identification of primary aldosteronism, thus achieving more accurate and reliable screening, confirmed and typing results.
- 4. The cut-off value of ARR is adjusted 20.4 such that the screening and typing result has higher sensitivity and specificity, being 94.4% (95% CI: 72.7-99.9) and 87.2% (95% CI: 72.6-95.7) respectively; Youden index is up to the maximum value (YI=0.82) and area under the curve (AUC) is up to 0.956.
- 5. The patients with hypertension secondary to PA can be screened and confirmed in early stage, which provides a reliable laboratory examination basis for clinicians to formulate effective therapeutic interventions.
- (1) Diagnosis or Detection
- Diagnosis or detection here refers to test or assay on a biomarker in a sample, or on a content of a target biomarker, for example, an absolute content or a relative content, and then the presence or the amount of the target marker serves to indicate whether an individual providing the sample has or suffers from a certain disease, or has the possibility of a certain disease. The meaning of the diagnosis or detection here may be exchanged. The result of such detection or diagnosis may not be directly used as a direct result of a disease, but an intermediate result. If it is desired to obtain the direct result, pathological or anatomic or other auxiliary means is required to confirm whether patients suffer from a certain disease. For example, the present invention provides a plurality of novel biomarkers correlated to primary aldosteronism; the change of the content of these markers is directly correlated to the presence of primary aldosteronism.
- (2) Correlation Between Markers or Biomarkers and PA
- Markers or biomarkers have the same meanings in the present invention. The correlation here refers that the presence or content change of a certain biomarker in a sample is directly correlated to a specific disease, for example, the relative increase or decrease of the content indicates that the possibility of suffering from the disease is higher than that for healthy people.
- If the plurality of different markers are present simultaneously or the content changes relatively in a sample, it indicates that the possibility of suffering from the disease is also higher than that for healthy people. That is, for the different types of markers, some markers are strongly correlated to the disease, while some markers are poorly correlated to the disease, or some are not correlated to a specific disease. One or more markers with strong correlation may be used as the markers for the diagnosis of a disease; the markers with weak correlation may be used to diagnose a certain disease in combination with the markers with strong correlation, thus improving the accuracy of the test results.
- Since it has been found in the present invention that the series of markers in blood plasma are closely correlated to PA, and the series of markers can be detected for one time by high performance liquid chromatography (HPLC)-tandem mass spectrometry, which is jointly useful for the screening, confirmed and typing diagnosis of PA.
-
FIG. 1A -FIG. 1E are test chromatogram of 5 markers in a blood sample provided in Example 1 and whereinFIG. 1A is the chromatogram of angiotensin I;FIG. 1B is the chromatogram of angiotensin II;FIG. 1C is the chromatogram of 18OH-Corticosterone;FIG. 1D is the chromatogram of aldosterone;FIG. 1E is the chromatogram of Cortisol. -
FIG. 2 is a ROC curve graph showing that the ARR cut-off value is 20.4 in Example 10. - To describe the present invention more specifically, the technical solutions of the present invention will be described in detail with reference to the accompanying drawings and detailed embodiments. The description merely indicates how the present invention is achieved, but is not construed as limiting the specific scope of the present invention. The scope of the present invention is defined by the claims.
- I. Solution Preparation
- Preparation of the mixed working solution of a calibration product and a quality control product is shown in Table 1:
-
TABLE 1 Preparation of the mixed working solution of a calibration product and a quality control product Concentration Volume Concentration Volume Concentration of the of the of the of the of the primary aqueous secondary aqueous mixed stock solution of stock solution of working solution Volume 50% methanol solution Volume 50% methanol solution Analyte mg/ml μl μl mg/ml μl μl μg/ ml Angiotensin I 1 100 900 100 50 675 5 Angiotensin II 1 10 990 10 25 0.25 Aldosterone 1 10 990 10 25 0.25 Cortisol 1 / / 1000 25 25 18- 0.1 100 900 10 20 0.2 hydrocorticosterone - Preparation and concentration gradient of the calibration product are shown in Table 2:
-
TABLE 2 Preparation of the calibration product Volume Name of of the Volume the working of 18OH- working solution matrix Ang I Ang II Aldosterone Cottisol Corticosterone pg/ml solution μL μl (ng/mL) (pg/mL) (pg/mL) (ng/mL) (pg/mL) LLMI Calibration 15 985 0.3 15 15 1.5 12 product S5 Calibration Calibration 15 985 0.3 15 15 1.5 12 product S1 product S5 Calibration Calibration 37.5 962.5 0.75 37.5 37.5 3.75 30 product S2 product S5 Calibration Calibration 15 985 1.5 75 75 7.5 60 product S3 product S7 Calibration Calibration 50 950 5 250 250 25 200 product S4 product S7 Calibration Calibration 200 800 20 1000 1000 100 800 product S5 product S7 Calibration Mixed 10 990 50 2500 2500 250 2000 product S6 working solution Calibration Mixed 20 980 100 5000 5000 500 4000 product S7 working solution Note: the matrix for the preparation of the calibration product is a PBS (1X) buffer solution containing 4% BSA, stored at 2-8° C. - Preparation of the quality control product is shown in Table 3:
-
TABLE 3 Preparation of the quality control product Volume Name of the of the working Volume of working solution Name of matrix solution μL matrix μL Preparation Quality Calibration 20 Plasma 980 of the control product S5 quality product L control Quality Quality 100 Quality 1100 product control control control product M product H product L Quality Mixed 6.6 Quality 993.4 control working control product H solution product L Note: the matrix for the quality control product is blood plasma which is subpackaged and frozen immediately after being prepared. - Preparation of the internal standard working solution is shown in Table 4:
-
TABLE 4 Preparation of the internal standard working solution Concentration Volume Volume of the Concentration of the Concentration of the mixed of the aqueous of the aqueous internal primary solution secondary solution standard stock with 50% stock with 50% working solution Volume methanol solution Volume methanol solution Analyte mg/ml μl μl mg/ml μl μl ng/ml Angiotensin I-IS 0.1 100 900 10 50 10000 50 Angiotensin II-IS 0.1 10 990 1 25 2.5 Aldosterone-d7 0.1 10 990 1 25 2.5 Cortisol- d4 1 100 900 100 20 200 18- 0.1 100 900 1 25 2.5 hydrocorticosterone- d4 - Preparation of Other Solution:
- Preparation of phenylmethylsulfonyl fluoride (PMSF): 0.174 g PMSF was taken and added to 10 mL methanol and prepared into a 100 mM PMSF methanol solution; storage condition was 2-8° C.
- Buffer formation solution: 12.11 g TRIS and 7.4 g ethylene diamine tetraacetic acid (EDTA) were added to a 100 mL volumetric flask, and added with deionized water to 90 mL, and then subjected to ultrasonic treatment for 30 min to be evenly dissolved. Deionized water was added to the scale line and mixed well. The solution was transferred to a reservoir vessel made of polypropylene. The PH value of the solution was adjusted within 5.45-5.60, and then the solution was stored at −20° C.
- Buffer formation solution A: the solution was prepared at the same day of the detection analysis; 100 μL of 100 mM PMSF (angiotensin converting enzyme inhibitor) solution was added to 10 mL of the buffer formation solution, thus preparing the buffer formation solution A (pH value ranges from 5.4 to 5.6).
- Stop buffer containing internal standard: 1 mL of the internal standard working solution was mixed with 9 mL water and 250 μL formic acid into the stop buffer containing internal standard.
- II. Sample Pretreatment
-
- 1. Sample thawing: a plasma sample to be tested and a quality control sample to be tested were placed into ice water (0° C.) for thawing till melted.
- 2. Sampling: 50 μL of the buffer formation solution A was added to a 96-well plate, and 400 μL of the calibration product, quality control product and quality control sample to be tested were taken and added to two plates prepared in the step (1), and then the remaining sample was immediately frozen.
- 3. Sample incubation: the sample in the
step 2 was sealed with a silicone pad and subjected to vortex treatment for a short period of time, and then put to a 37° C. water bath for 3 h, 3 h later, 400 μL of the stop buffer containing internal standard was added, and the mixed solution was centrifuged for 1 min at 4° C. and 4000 rpm; 800 μL supernatant was taken and added to an automatic magnetic bead extractor, ready for sample extraction. - 4. Magnetic bead extraction: additives of each column in the adaptive 96-well plate of the magnetic bead extractor are shown in Table 5:
-
TABLE 5 Additives of each column in the adaptive 96- well plate of the magnetic bead extractor First Second Second Fourth Fifth Sixth column column column column column column (7) (8) (9) (10) (11) (12) Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution - The magnetic bead is the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A). The activating agent is a solution of 50% ethanol including the magnetic bead; the balanced solution is an aqueous solution containing 1% formic acid; the
washing liquid 1 is an aqueous solution containing 10% methanol; thewashing liquid 2 is isooctane, and the eluent solution is an aqueous solution containing 50% methanol. -
- 5. The sample pretreatment step of the magnetic bead extractor is shown in Table 6; the pretreatment time of each batch of samples is about 10 min.
-
TABLE 6 Sample pretreatment step of the magnetic bead extractor Columns of Mixing Solvent Magnetic the 96- time amount absorption time No. Instruction well plate (S) (μl) (S) 1 Activating 1 (7) 60 300 30 2 Activating 2 (8) 60 300 30 3 Sampling 3 (9) 90 800 30 4 Drip washing 4 (10) 60 300 30 5 Drip washing 5 (11) 60 300 30 6 Eluting 6 (12) 60 100 30 7 Waste 1 (7) 10 100 0 discharge -
- 6. After the extraction was completed by the magnetic bead extractor, the solution to be tested in the columns 6 and 12 of the 96-well plate was transferred to the 96-well loading plate for detection on the machine. The existing magnetic bead extractor may accommodate two 96-well plates for parallel operation for one time. Therefore, the pretreatment flux is 32 samples/batch; a 96-channel magnetic bead extractor may be also available; and the extraction steps are kept same.
- III. Sample Detection
- In the process of the liquid chromatography tandem-mass spectrometry, gradient elution was applied in the liquid chromatography; separation conditions of the object to be detected for the reversed phase chromatography were established as follows: chromatographic column was a Phenomenex C8 chromatographic column; flow rate was 0.4 mL/min; column temperature was 40° C.; where the mobile phase A was an aqueous solution containing 1 mM ammonium fluoride, and the mobile phase B was a methanol solution containing 1 mM ammonium fluoride (with 5% isopropanol); the volume ratio of the mobile phase A to the mobile phase B was 90-5%:10-95%. The gradient elution program is shown in Table 7.
-
TABLE 7 Gradient elution program Time Flow rate Mobile phase A Mobile phase B (min) (mL/min) (%) (%) 0 0.4 80 20 0.3 0.4 80 20 0.6 0.4 50 50 3.3 0.4 50 50 3.4 0.4 5 95 4.2 0.4 5 95 4.3 0.4 80 20 4.8 0.4 80 20 - During the mass spectrometric detection, quantitative detection was performed by a triple quadrupole mass spectrometer with a model of CalQuant-S independently developed and researched by CALIBAR. The mass spectrometry was performed by a positive/negative ion mode (ESI+) of an ESI source and a multi-reaction monitoring MRM mode. The corresponding mass spectrometry is shown in Table 8, and the mass spectrometry conditions are shown in Table 9:
-
TABLE 8 Mass spectrometry Q1 Q3 DWELL ID DP CE CXP 433.1 619.2 30 Angiotensin I-1 139 30 8 433.1 647.4 5 Angiotensin I-2 145 25 7 437.3 660.5 30 Angiotensin I-IS-2 120 24 16 437.3 631.1 5 Angiotensin I-IS-2 130 30 10 523.9 263.4 30 Angiotensin II-1 140 31 7 523.9 784.3 5 Angiotensin II-2 140 28 8 527.3 263.3 30 Angiotensin II- IS-1 140 31 7 527.3 791.4 5 Angiotensin II- IS-2 140 28 7 363.3 121.1 10 18-hydrocorticosterone-1 140 40 10 363.3 269.2 10 18-hydrocorticosterone-2 140 38 10 367.2 121.1 10 18-hydrocorticosterone- 140 40 10 IS-1 367.2 273.2 10 18-hydrocorticosterone- 140 38 10 IS-2 359.2 188.9 25 Aldosterone-NEG-1 −125 −26 −8 359.2 331.3 10 Aldosterone-NEG-2 −125 −23 −8 367.2 194.2 25 Aldosterone-NEG-IS-1 −125 −26 −8 367.2 339.4 10 Aldosterone-NEG-IS-2 −125 −23 −8 363.2 309.2 30 Cortisol-1 175 25 8 363.2 121.2 15 Cortisol-2 175 32 8 367.2 313.3 30 Cortisol-IS-1 175 25 8 367.2 121.2 15 Cortisol-IS-2 175 32 8 -
TABLE 9 Mass spectrometry conditions Mass spectrometry conditions Value Curtain gas CUR 25 psi Atomized gas GS1 55 psi Auxiliary heating gas GS2 55 psi Ion source heating temperature 500° C. Collision gas CAD 10 psi Spray voltage 5500 V/−4500 V - A standard curve was established by the internal standard method. Records of validation on the linear relation is shown in Table 10 with a measuring unit of ng/mL.
-
TABLE 10 Results of validation on the standard curve Coefficient of Clinical Compound Regression equation Weight association r linear range Angiotensin I Y = 0.00957X + 0.000836 1/X2 0.997 0.3-100 ng/mL Angiotensin II Y = 0.0000762593X + 0.00303 1/X2 0.996 15-5000 pg/mL Aldosterone Y = 0.00250X − 0.03029 1/X2 0.997 15-5000 pg/mL Cortisol Y = 0.04826X + 0.13974 1/X2 0.998 1.5-500 ng/mL 18- Y = 0.000956X − 0.07801 1/X2 0.999 12-4000 pg/mL hydrocorticosterone - The standard curve was formulated by the PBS buffer solution matrix containing 4% BSA and subjected to synchronous treatment with the sample to be tested for detection. The test chromatogram is shown in
FIG. 1 , representing angiotensin I (Ang I), angiotensin II (Ang II), 18-hydrocorticosterone (18-OH CORT), aldosterone (Aldo) and Cortisol from top to bottom in order. As can be seen fromFIG. 1 , the method provided by the example may accurately detect the 5 markers simultaneously. - By the validation on accuracy and precision (Table 11), the detection linear relation is good within the scope of concentration.
-
TABLE 11 Accuracy and precision validated by the method Sample Theoretical Measured Compound size concentration value Accuracy Imprecision Angiotensin I 6 1.8 ng/mL 1.79 ng/mL 99.44% 2.50% 4.8 ng/mL 4.81 ng/mL 100.2% 2.08% 34.8 ng/mL 34.5 ng/mL 99.13% 1.58% Angiotensin II 6 50 pg/mL 49.3 pg/mL 98.60% 1.42% 200 pg/mL 198 pg/mL 99.00% 3.08% 1700 pg/mL 1702 pg/mL 100.1% 2.12% Aldosterone 6 50 pg/mL 50.3 pg/mL 100.6% 3.00% 200 pg/mL 198 pg/mL 99.20% 3.47% 1700 pg/mL 1693 pg/mL 90.58% 1.13% Cortisol 6 50 ng/mL 50.7 ng/mL 101.40% 2.46% 65 ng/mL 65.6 ng/mL 100.92% 1.12% 215 ng/mL 214 ng/mL 99.53% 3.46% 18- 6 160 pg/mL 159 ng/mL 99.38% 2.38 % hydrocorticosterone 280 pg/mL 289 pg/mL 104% 2.01% 1480 pg/mL 1489 pg/mL 100.6% 3.20% - The preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. The extraction was performed respectively by the different three methods: 1, extraction by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80A); 2, extraction by PEP 96 Well Microplates of the SPE plate filled with a balanced hydrophilic-lipophilic polymer; 3, extraction by PEP 96 Waters Oasis HLB of the SPE plate filled with a balanced hydrophilic-lipophilic polymer; after elution, the sample was subjected to liquid chromatography tandem-mass spectrometry; the test result of the treated sample was surveyed to measure the peak areas of the 5 markers in the sample S1, as shown in Table 12.
-
TABLE 12 Effects of different treatment methods on the extraction effects of the markers Treatment method HLB PEP 96 Waters Oasis magnetic Well HLB 96 Marker bead Microplates Wellplates Angiotensin I Peak 6460 5876 5031 (theoretical area concentration: 0.3 ng/mL) Angiotensin II Peak 8967 7462 7270 (theoretical area concentration: 15 pg/mL) Aldosterone Peak 4563 3687 3852 (theoretical area concentration: 15 pg/mL) Cortisol Peak 163653 14573 14135 (theoretical area concentration: 1.5 ng/mL) 18- Peak 1019 821 820 Hydrocorticosterone area (theoretical concentration: 12 pg/mL) - As can be seen from Table 12, different sample treatment methods will affect the extraction results of the 5 markers in the sample; compared with the SPE plate embedded the balanced hydrophilic-lipophilic polymer, the magnetic bead has more significant extraction effect on the 5 markers. Meanwhile, the two SPE plates of balanced hydrophilic-lipophilic polymer are compared, and there is a difference in effects to some extent; PEP 96 Well Microplates of the SPE plate are superior to the Waters Oasis HLB 96 Wellplates; compared with the two SPE plates, the HLB magnetic bead has significantly improved extraction effect on the 5 markers.
- Moreover, the solid phase extraction is featured by easy blocking, complex operation and too stronger matrix effect, and the like. Therefore, the method of magnetic bead is applied. The magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is used to adsorb the 5 markers, and then the magnetic bead extractor may extract the magnetic bead which absorbs the markers from the biological sample matrix, thus effectively removing the interference elements in the biological sample. Therefore, after extraction and enrichment of the magnetic bead, compared with the solid phase extraction column, the eluent solution extracted by the magnetic bead has more purified components; the markers have smaller disturbing influences in the detection process; the mass spectrometry ionization efficiency is higher, and better detection sensitivity may be achieved.
- In this example, the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. Based on the search result of the literature on the single and separate detection of the 5 different markers: the mixed anion exchange polymer SPE may be used in the sample pretreatment of the angiotensin detection. Therefore, comparisons were further made on the solid phase extraction column bonded a mixed anion exchange polymer (AgelaCleanert PAX 96 Wellplates) filler, the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A), the magnetic bead bonded a mixed anion exchange polymer (MAX magnetic bead, Biosepur, Art. No.: BNMA1430-SY; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A), and the magnetic bead bonded a mixed cation exchange polymer (MCX magnetic bead, Biosepur, Art. No.: BNMA8300001-0-P; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A) in this example. The 5 markers were adsorbed and extracted in the 4 ways, and after elution, the sample was subjected to liquid chromatography tandem-mass spectrometry; the test result of the treated sample was surveyed to measure the peak areas of the 5 markers in the sample S1, as shown in Table 13.
-
TABLE 13 Effects of the magnetic bead bonded different materials on the extraction effects of the markers Magnetic bead bonding material Cleanert PAX 96 HLB MAX MCX Well- magnetic magnetic magnetic Marker plates bead bead bead Angiotensin I Peak 4961 6460 5261 4884 (theoretical area concentration: 0.3 ng/mL) Angiotensin II Peak 7230 8967 7241 7067 (theoretical area concentration: 15 pg/mL) Aldosterone Peak 2134 4563 4134 3242 (theoretical area concentration: 15 pg/mL) Cortisol Peak 145467 163653 145349 148580 (theoretical area concentration: 1.5 ng/mL) 18- Peak 382 1019 682 666 Hydrocorticosterone area (theoretical concentration: 12 pg/mL) - As can be seen from Table 13, in the same way of being bonded the anionic polymer, the extraction effect of the MAX magnetic bead is obviously superior to that of the AgelaCleanert PAX 96 Wellplates solid phase extraction column; meanwhile, different materials are bonded on the surface of the magnetic bead, which has a large impact on the extraction effects of the 5 markers in the blood sample. Comparisons are made on the magnetic beads bonded the three fillers of the hydrophilic-lipophilic polymer, the mixed anion exchange polymer and the mixed cation exchange polymer; among them, the magnetic bead bonded the hydrophilic-lipophilic polymer may significantly improve the extraction effect on a portion of low-content indexes (aldosterone and 18-hydrocorticosterone) and may achieve better balance on the extraction and enrichment effects of the 5 markers with greater differences in physical and chemical properties. The reason is probably as follows: the balanced hydrophilic-lipophilic polymer may not only achieve the adsorption on high polar compounds, but also may effectively adsorb the low polar compounds; but anionic and cationic polymers have stronger adsorptive selectivity and thus, only selectively achieve the adsorption on high polar anions or cations, and hardly achieve the adsorption on low polar compounds simultaneously, leading to a low extraction efficiency of small molecules such as, aldosterone and 18-hydrocorticosterone, incapable of achieving the sensitivity requirements of clinical test.
- When a biological sample was pretreated by the conventional solid phase extraction column, the 96-well SPE plate (Agela PEP 96 Well Microplates) was firstly extracted by the solid phase extraction column for activation and balance, and then the biological sample solution to be tested was loaded on the SPE plates; the target compound was extracted and adsorbed by a PEP filler selectively; a portion of inorganic salt and other impurities were washed from the SPE column via drip washing, and then the target component was eluted from the extraction column by an eluting agent with stronger binding capacity to the solid phase, afterwards, the eluent solution was blown-dried with nitrogen gas, redissolved and loaded for sample detection. The operation process has more manual steps, is complex and time-consuming; it takes about 2 h to treat a batch of samples. Moreover, the inter-well extraction efficiency of the sample greatly varies from the difference of the biological sample matrix; all the indexes need to be calibrated via isotope internal standards. Further, due to the specificity of the biological sample matrix, partial samples (in especial hemolysis, lipemia and other samples) may cause the blocking of the SPE column, leading to reduced pretreatment efficiency of the total batch of the samples and retest of the blocked sample.
- Additives of each column in the adaptive 96-well plate of the magnetic bead extractor are shown in Table 14 (the same as Table 5 above):
-
TABLE 14 Additives of each column in the adaptive 96- well plate of the magnetic bead extractor First Second Second Fourth Fifth Sixth column column column column column column (7) (8) (9) (10) (11) (12) Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution Activating Balanced Sample Washing Washing Eluent agent solution liquid 1 liquid 2solution - The magnetic bead is the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A). The activating agent is a solution of 50% ethanol including the magnetic bead; the balanced solution is an aqueous solution containing 1% formic acid; the
washing liquid 1 is aqueous solution containing 10% methanol; thewashing liquid 2 is isooctane, and the eluent solution is an aqueous solution containing 50% methanol. - The sample pretreatment step of the magnetic bead extractor is shown in Table 15 (the same as Table 6); the pretreatment time of each batch of samples is about 10 min.
-
TABLE 15 Sample pretreatment step of the magnetic bead extractor Columns of Mixing Solvent Magnetic the 96- time amount absorption time No. Instruction well plate (S) (μl) (S) 1 Activating 1 (7) 60 300 30 2 Activating 2 (8) 60 300 30 3 Sampling 3 (9) 90 800 30 4 Drip 4 (10) 60 300 30 washing 5 Drip 5 (11) 60 300 30 washing 6 Eluting 6 (12) 60 100 30 7 Waste 1 (7) 10 100 0 discharge - After the extraction was completed by the magnetic bead extractor, the solution to be tested in the columns 6 and 12 of the 96-well plate may be transferred to the 96-well loading plate for detection on the machine. The existing magnetic bead extractor may accommodate two 96-well plates for parallel operation for one time. Therefore, the pretreatment flux is 32 samples/batch; a 96-channel magnetic bead extractor may be also available; and the extraction efficiency is kept same. The pretreatment of the sample 96 may be achieved in 10 min. The pretreatment efficiency is greatly improved to achieve the high degree of automation of clinical sample pretreatment.
- During the incubation, angiotensinogen in the plasma sample will be converted into angiotensin I under the catalysis of renin activity, and due to the addition of the angiotensin converting enzyme, the content of the angiotensin I increases. Therefore, the incubation process will directly affect the content of the angiotensin I in the sample. In this example, the preparation, extraction and detection of the low, moderate and high-quality control samples were performed by the method provided in Example 1. The pH values of the buffer formation solution added to the pre-incubation samples were respectively 4.0, 5.0, 5.5, 6.0 and 7.4; the post-incubation samples were absorbed and extracted by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art. No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A); after elution, the samples were subjected to liquid chromatography tandem-mass spectrometry to survey the test results of the post-incubation samples at different pH values, thus measuring the peak areas of the angiotensin I in the low, moderate and high quality control samples after incubation, as shown in Table 16.
-
TABLE 16 Effects of different pH values on the marker (angiotensin I) during the incubation pH Marker 4.0 5.0 5.5 6.0 7.4 Angiotensin Peak area 63920 98144 124610 103072 96902 I in the low Measured 3.21 4.67 6.03 5.25 4.47 quality concentration control (ng/ml) sample after incubation Angiotensin Peak area 122006 142264 179340 157004 109414 I in the Measured 5.97 7.28 8.81 7.54 5.55 moderate concentration quality (ng/ml) control sample after incubation Angiotensin Peak area 206258 247818 291260 248104 206134 I in the high Measured 10.28 12.34 14.99 12.31 10.07 quality concentration control (ng/ml) sample after incubation - As can be seen from Table 16, the pH value of the buffer formation solution must be controlled within a suitable scope during the incubation, or, the catalytic activity of renin will be affected seriously, leading to a larger deviation in the angiotensin I generated after the incubation. The measured renin activity is low under nonideal pH conditions, and when ARR is calculated by aldosterone/renin activity, it is easy to cause a higher value of ARR, leading to a false-positive result.
- In this example, the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. Different additives were added to the eluting agent to prepare different eluting agents, thus eluting the magnetic bead sample; after being eluted, the sample was subjected to liquid chromatography tandem-mass spectrometry to survey the test results of the sample after being eluted by different eluting agents, thus measuring the peak areas of the 5 markers in the sample S1, as shown in Table 17.
-
TABLE 17 Effects of different eluting agents on the extraction effects of the markers Eluent solution Aqueous solution with 0.1% formic acid Aqueous Aqueous Aqueous and aqueous solution solution solution solution with 75% with50% with 25% with 50% Marker Methanol methanol methanol methanol methanol Angiotensin I Peak 4543 5784 6460 5799 6620 (theoretical area concentration: 0.3 ng/mL) Angiotensin II Peak 5932 6161 8967 7246 6876 (theoretical area concentration: 15 pg/mL) Aldosterone Peak 4995 3875 4563 4091 2674 (theoretical area concentration: 15 pg/mL) Cortisol Peak 169965 167501 163651 121041 161041 (theoretical area concentration: 1.5 ng/mL) 18- Peak 707 780 1019 613 732 Hydrocorticosterone area (theoretical concentration: 12 pg/mL) - As can be seen from Table 17, when methanol is directly used as the eluting agent, each index has poor shape of detection peak and there are serious solvent effects; the situation is improved to some extent when the aqueous solution with 75% methanol is used for elution; and the aqueous solution with 50% methanol may effectively improve the peak shape; the elution efficiency is inadequate when 25% methanol is used; after an acid is added to the eluting agent, the ionization efficiency of aldosterone is obviously inhibited and the peak area decreases, thus affecting the detection sensitivity.
- In this example, the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. Mobile phase A was an aqueous solution and mobile phase B was a methanol solution (with 5% isopropanol) as basic mobile phases. Different additives were added to the mobile phase A and mobile phase B to prepare different mobile phases for liquid chromatography tandem-mass spectrometry; the test results of the sample detected under different mobile phases were surveyed, thus measuring the peak areas of the 5 markers in the sample S1, as shown in Table 18.
-
TABLE 18 Effects of different additives on the extraction effects of the markers Mobile phase 1 mM 0.03% Additive- ammonium formic Marker free fluoride acid Angiotensin I Peak 4368 6460 7967 (theoretical area concentration: 0.3 ng/mL) Angiotensin II Peak 8069 8967 8355 (theoretical area concentration: 15 pg/mL) Aldosterone Peak 2668 4563 1656 (theoretical area concentration: 15 pg/mL) Cortisol Peak 139964 163651 147501 (theoretical area concentration: 1.5 ng/mL) 18- Peak 706 1019 739 Hydrocorticosterone area (theoretical concentration: 12 pg/mL) - As can be seen from Table 18, compared with the condition free of an additive, the addition of 1 mM ammonium fluoride both in the mobile phase A and the mobile phase B may effectively improve the detection sensitivity; but the test result of the sample greatly varies from the type of the additives added; compared with the addition of 0.03% formic acid, the addition of 1 mM ammonium fluoride will cause decreased Ang I response to some extent, but may further improve the detection sensitivity of the other 4 indexes, especially for the low-content aldosterone. Therefore, the detection sensitivity of the 5 markers may satisfy the clinical demand more.
- In this example, research shows that during the mass spectrometric detection of the 5 markers, the cationic mode should be chosen to detect angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone, while the anionic mode needs to be chosen to detect aldosterone; this is because when the cationic mode is chosen, there exists a peak diagram of cortisone, an isomer of aldosterone, nearby the detection peak of aldosterone to cause larger interference, and CV % is greater than 15%; but when the anionic mode is applied for detection, the test result is more stable and accurate, and CV % is less than 8.33%.
- In this example, a screening, confirmed and typing diagnosis system for primary aldosteronism is used for the screening, confirmed and typing diagnosis of primary aldosteronism; the specific method is as follows:
-
- (1) A marker test module is used to obtain test values of the 5 markers for one time by the method provided by Example 1.
- (2) ARR is calculated by a data analysis module and according to the test values of aldosterone and angiotensin I; the calculation formula of the ARR is as follows: ARR=concentration of aldosterone/production rate of angiotensin I; the production rate of angiotensin I is calculated by detecting the concentration of angiotensin I in the pre-incubation sample and in the post-incubation sample and according to the following formula: production rate of angiotensin I=(concentration of angiotensin I after incubation−concentration of angiotensin I before incubation)/incubation time, where the concentration of angiotensin I before incubation is very low and thus, may be basically ignored.
- (3) A positive or negative result is judged in combination with a cut-off value 20.4 of the ARR and a concentration of a hypertension therapeutic affecting the ARR (see details in the patent invention 2021106374412 of the prior application). When the test result of ARR is less than 20.4, and if the patient is simultaneously detected to contain the drug capable of reducing ARR in vivo, a false-positive result may be judged, and a confirmed experiment or drug withdrawal needs to be performed for examination. When the test result of ARR is less than 20.4, and if the patient is simultaneously detected to contain the drug capable of increasing ARR in vivo, a positive result may be judged. When the test result of ARR is greater than 20.4, and if the patient is simultaneously detected to contain the drug capable of increasing ARR in vivo, a false-positive result may be judged, and a confirmed experiment or drug withdrawal needs to be performed for examination. When the ARR is greater than 20.4, and if the patient is simultaneously detected to contain the drug capable of reducing ARR in vivo, a positive result may be judged.
- (4) When a positive result is judged, the PA typing is performed according to the test values of aldosterone, angiotensin II, cortisol and 18-hydrocorticosterone; the specific typing diagnosis method is as follows:
- a, adrenal venous sampling (AVS): SI (a ratio of cortisol in adrenal veins to arterio-venous cortisol) 2:1, indicating successful intubation; LI (a ratio of the aldosterone-cortisol ratio at the dominant side to the aldosterone-cortisol ratio at the non-dominant side) 2:1, indicating secretion from the dominant side; CI (a ratio of the aldosterone-cortisol ratio at non-dominant side to the arterio-venous aldosterone-cortisol ratio)<1:1, the contralateral is inhibited;
- b, 18-hydrocorticosterone (18-OHB): the level of 18-OHB in the plasma of aldosteronoma patients at 8:00 a.m. in a lying position is usually >100 ng/dl, while for the patients with idiopathic aldosteronism, the level of 18-OHB is usually <100 ng/dl;
- c, primary and secondary hypertension is subjected to typing diagnosis in combination with the test value of angiotensin II.
- 61 cases of patients receiving secondary hypertension screening were chosen in this example, of which 20 cases were diagnosed with PA.
- 1. Inclusion Criteria
- The patients whose standing position PAC was greater than 15 ng/dL, standing position ARR was greater than 30 and aldosterone inhibition ratio after CCT was less than 30% were brought into the group PA. Before screening, the patients were requested to withdraw diuretics or aldosterone receptor antagonists at least for 4 weeks, and other antihypertensive drugs such as, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor inhibitors (ARB), calcium ion antagonists (CCB) and beta receptor blockers at least for 2 weeks. Before blood sampling, serum potassium should be corrected to the normal range as much as possible. The patients who were grouped into the PA group kept in a standing position at 05:00 a.m. in the following day, and 2 h later, the blood sample was collected in the standing position at 07:00 a.m.
- 2. AVS Judgment Criteria
- Bilateral synchronous blood sampling stimulated by non-adrenocorticotrophic hormone was applied. The content of the 5 markers in the blood sample was detected by the method provided by Example 1. A ratio of cortisol in adrenal veins to arterio-venous cortisol is defined as a selectivity index (SI); SI>2 indicates successful blood sampling. A ratio of the aldosterone-cortisol ratio at the dominant side (a standardized aldosterone value) to the contralateral standardized aldosterone value is defined as a lateralized index (LI); when LI is greater than 2, it is believed that there is unilateral dominant secretion, and when LI is less than 2, it is believed that there is no obvious unilateral dominant secretion.
- 3. Sample Collection: for all the sample collection, the subject was requested to receive the treatment of overnight fasting for 8 h above; sample transfer, centrifugation and separation should be ensured to be completed within 1 h, thus avoiding possible pre-analysis factors. All the samples were kept at −80° C. for test before being analyzed.
- 4. ARR calculation: the content of the 5 markers in the blood sample was detected by the method provided by Example 1. Based on the calculation formula of the ARR: ARR=concentration of aldosterone/renin activity (production rate of angiotensin I); the renin activity is calculated by detecting the concentration of angiotensin I in the pre-incubation sample and in the post-incubation sample and according to the following formula: production rate of angiotensin I=(concentration of angiotensin I after incubation−concentration of angiotensin I before incubation sample)/incubation time, where the concentration of angiotensin I before incubation is very low and thus, may be basically ignored. The screening, confirmed and typing diagnosis system for primary aldosteronism provided in Example 8 was used for the screening, confirmed and typing diagnosis of primary aldosteronism.
- 5. Analysis by a Statistical Method:
- The data was processed by R language software. Based on the analysis of the correlation between the ARR value and the presence of PA patients or not, the analysis result is shown in Table 19.
-
TABLE 19 Comparison of the test result between the ARR value and the presence of PA patients or not Crite- Youden Sensi- Speci- ria index tivity 95% CI ficity 95% CI +LR −LR >13.8 0.6623 94.44 72.7-99.9 71.79 55.1-85.0 3.35 0.077 >20.4 0.8162 94.44 72.7-99.9 87.18 72.6-95.7 7.37 0.064 >29 0.7564 83.33 58.6-96.4 92.31 79.1-98.4 10.83 0.18 >37.2 0.6709 72.22 46.5-90.3 94.87 82.7-99.4 14.08 0.29 >39.1 0.5598 61.11 35.7-82.7 94.87 82.7-99.4 11.92 0.41 - As can be seen from Table 19, when the ARR cut-off value is 20.4, the detection sensitivity, degree of sensitivity and 95% CI are in higher levels and have obvious advantages relative to other cut-off values. The effect of sensitivity is the most crucial index to judge whether ARR is negative or positive by the screening, confirmed and typing diagnosis system for primary aldosteronism provided by the present invention. Missing detection may be prevented effectively only by high sensitivity. Even though it is false-positive, the false-positive result may be further confirmed by PA typing according to the test values of the subsequent aldosterone, angiotensin II, cortisol and 18-hydrocorticosterone. However, if there is lack of sensitivity, originally positive patients are erroneously judged as negative and thus, are not subjected to the subsequent typing diagnosis, which is very easy to cause missing detection. Therefore, 20.4 is applied as the ARR cut-off value, which may effectively prevent missing detection.
- Meanwhile, when LC-MS/MS of the ARR is greater than 20.4, the Youden index is also up to the maximum value (YI=0.82), which indicates that the screening effect of primary aldosteronism is optimal and closest to the real value.
- The ARR cut-off value of 20.4 is applied for analysis; and results are shown in Table 20:
-
TABLE 20 Analysis result of ARR >20.4 ROC area under the curve (AUC) 0.956 95% confidence interval b 0.866-0.993 Significance level P (area = 0.5) <0.0001 Youden index J 0.8162 Relative standard >20.4 Sensitivity 94.44 Specificity 87.18 - As can be seen from Tables 19 and 20, when LC-MS/MS of the ARR is greater than 20.4, the Youden index is also up to the maximum value (YI=0.82); sensitivity and specificity are respectively 94.4% (95% CI: 72.7-99.9) and 87.2% (95% CI: 72.6-95.7); area under the curve (AUC) is up to 0.956 (
FIG. 2 ). - The cut-off value of ARR is commonly believed as 30 in the prior art. The cut-off value of ARR needs to be adjusted 20.4 when the screening, confirmed and typing diagnosis system for primary aldosteronism provided in the present invention is applied. This is mainly because the ARR value is directly correlated to the detection sensitivity of aldosterone and renin activity. Aldosterone is universally detected by chemiluminescent immunoassay previously, but the test value is higher such that the calculated result of the ARR value is greater than the actual value. Therefore, it needs to set a cut-off value of 30 to judge whether PA is positive or negative accurately more. When the sample pretreatment (extraction by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer silica gel) and detection method (HPLC-tandem mass spectrometry) provided by the present invention are applied, the test value may reflect the content of the aldosterone reagent more accurately. Therefore, the previous cut-off value of 30 of ARR has not conformed to the PA screening and typing system provided by the present invention; the cut-off value of ARR needs to be adjusted 20.4 such that the screening and typing result has a higher sensitivity, and the diagnostic result is more accurate.
- Even though the present invention is disclosed above, the present invention is not limited thereto. Any person skilled in art can make various alterations and modifications within the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subjected to the scope defined by the claims.
Claims (4)
1-17. (canceled)
18. A screening, confirmed and typing diagnosis system for primary aldosteronism, wherein the system consists of a marker test module, a data input/output interface and a data analysis module; wherein the markers test module is configured to obtain test values of markers by a method of detecting the markers in blood, the data input/output interface is configured to input the test values of five markers, the data analysis module is configured to analyze the test values of the markers, the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone; and, wherein after analysis by the data analysis module, the data input/output interface is configured to output a screening, confirmed and typing diagnosis result of primary aldosteronism,
wherein the method of detecting the markers in the blood comprises the following steps:
(1) treating a blood sample with a magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on a surface of the magnetic bead to absorb the markers in the blood sample; and
(2) using an eluent solution to eluent the markers from the magnetic bead, and using a liquid chromatography tandem-mass spectrometry to test contents of the markers in the blood sample, wherein the markers are aldosterone, angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone.
19. The system of claim 18 , wherein a method for analyzing the test values of the markers by the data analysis module is as follows: calculating an aldosterone/renin activity ratio (ARR) value based on aldosterone and renin activity and making a judgment in combination with a cut-off value, which is 20.4, of the ARR value and a concentration of a hypertension therapeutic affecting the ARR value; when a positive result is judged, confirmed and typing diagnosis is performed according to the test values of the aldosterone, the angiotensin I, the angiotensin II, the cortisol and the 18-hydrocorticosterone; wherein the rennin activity is a yield of the angiotensin I per unit time.
20. (canceled)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/526,887 US20240110929A1 (en) | 2022-07-13 | 2023-12-01 | Screening kit and diagnosis system for primary aldosteronism |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210819638.2 | 2022-07-13 | ||
CN202210819638.2A CN114895043B (en) | 2022-07-13 | 2022-07-13 | Primary aldosteronism screening kit and definite diagnosis and typing diagnosis system |
CN2022108196382 | 2022-07-13 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US18/526,887 Continuation-In-Part US20240110929A1 (en) | 2022-07-13 | 2023-12-01 | Screening kit and diagnosis system for primary aldosteronism |
Publications (2)
Publication Number | Publication Date |
---|---|
US11860171B1 US11860171B1 (en) | 2024-01-02 |
US20240019449A1 true US20240019449A1 (en) | 2024-01-18 |
Family
ID=82730297
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US18/304,915 Active US11860171B1 (en) | 2022-07-13 | 2023-04-21 | Screening kit and diagnosis system for primary aldosteronism |
Country Status (2)
Country | Link |
---|---|
US (1) | US11860171B1 (en) |
CN (2) | CN117491639A (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116106466B (en) * | 2023-01-09 | 2023-10-20 | 泽达精准(杭州)生物医药有限公司 | Kit for simultaneously determining multiple causative markers of endocrine hypertension |
CN117074698B (en) * | 2023-10-11 | 2024-03-15 | 湖南凯莱谱生物科技有限公司 | Marker combination, kit, system and application for early diagnosis of acute myocardial infarction |
CN117554603B (en) * | 2023-11-28 | 2024-05-10 | 中国医学科学院阜外医院 | Marker combination for primary aldosteronism prescreening and application thereof |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8426214B2 (en) * | 2009-06-12 | 2013-04-23 | University Of Washington | System and method for magnetically concentrating and detecting biomarkers |
DE102014005993A1 (en) * | 2014-04-28 | 2015-10-29 | Magnamedics Gmbh | Method for enriching trace components from a liquid biological sample |
WO2018194152A1 (en) * | 2017-04-21 | 2018-10-25 | 株式会社ハプロファーマ | Method for detecting aldosterone and renin |
WO2020045497A1 (en) * | 2018-08-29 | 2020-03-05 | 国立大学法人宮崎大学 | Testing method and agent for diagnostic tolerance testing for distinguishing aldosterone-producing adenoma and idiopathic aldosteronism |
CN112014509A (en) * | 2020-09-03 | 2020-12-01 | 复旦大学附属中山医院 | Method for synchronously determining angiotensin I and aldosterone in sample |
CN114354806A (en) * | 2021-12-31 | 2022-04-15 | 上海药明奥测医疗科技有限公司 | Human plasma renin activity liquid chromatography-mass spectrometry combined detection method |
CN114544836A (en) * | 2022-03-11 | 2022-05-27 | 天津国科医工科技发展有限公司 | Pretreatment method and detection method for detecting trace estrogen, 17-hydroxypregnanolone, aldosterone and dehydroepiandrosterone sulfate |
CN114487210B (en) * | 2022-03-11 | 2024-05-10 | 天津国科医疗科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of steroid hormone based on magnetic solid phase extraction |
-
2022
- 2022-07-13 CN CN202211268276.9A patent/CN117491639A/en active Pending
- 2022-07-13 CN CN202210819638.2A patent/CN114895043B/en active Active
-
2023
- 2023-04-21 US US18/304,915 patent/US11860171B1/en active Active
Also Published As
Publication number | Publication date |
---|---|
US11860171B1 (en) | 2024-01-02 |
CN114895043B (en) | 2022-12-13 |
CN117491639A (en) | 2024-02-02 |
CN114895043A (en) | 2022-08-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11860171B1 (en) | Screening kit and diagnosis system for primary aldosteronism | |
JP2009500641A (en) | Diagnostic method for multiple sclerosis | |
US10247736B2 (en) | Identification and quantification of biomarkers for evaluating the risk of preterm birth | |
US10338076B2 (en) | Methods and compositions for the diagnosis of ovarian cancer | |
CN112782328B (en) | Method for detecting catecholamine and metabolites thereof in urine, kit and application of kit | |
CN113533552B (en) | Method for synchronously detecting ARR (acute respiratory syndrome) drugs in process of detecting renin activity by liquid chromatography-tandem mass spectrometry | |
CN115453012B (en) | Reversed-phase HPLC method for simultaneously measuring multiple positional isomers in voathixetine hydrobromide | |
Bahlmann et al. | Cetirizine as pH-dependent cross-reactant in a carbamazepine-specific immunoassay | |
JP2008508502A (en) | TIMP-2 as a target / marker of beta cell failure | |
US20120149041A1 (en) | Identification and quantification of biomarkers for evaluating the risk of preterm birth | |
Davies et al. | Liquid chromatography tandem mass spectrometry for plasma metadrenalines | |
Wang et al. | A simple and precise LC-MS/MS method for the simultaneous determination of serum 25-hydroxyvitamin D 3 and D 2 without interference from the C 3 epimer | |
US20240110929A1 (en) | Screening kit and diagnosis system for primary aldosteronism | |
CN112362780A (en) | High performance liquid detection method of propranolol hydrochloride | |
AU2016273230B2 (en) | Biomarkers for a combination therapy comprising lenvatinib and everolimus | |
Kirby et al. | Sensitive and specific LC‐MS assay for quantification of digoxin in human plasma and urine | |
CN108120838B (en) | Dipeptide derivative for detecting angiotensin II and preparation method and application thereof | |
EP3861347A1 (en) | Biomarkers for a combination therapy comprising lenvatinib and everolimus | |
US20110247404A1 (en) | Identifying and quantifying biomarkers associated with preeclampsia | |
JPS63212861A (en) | Method and apparatus for simultaneous analysis of vanillylmandelic acid, homovanillic acid and creatinine | |
Wei et al. | Evaluation of Bio-Rad D100 for HbA1c testing of dried blood spot samples | |
Rogers et al. | The use oe the steroid analyser in conjunction with a semi-automatic gas chromatograph in the routine analysis of urinary pregnanediol | |
CN117007696A (en) | Method and kit for monitoring citalopram blood concentration | |
AU2015200775A1 (en) | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
AS | Assignment |
Owner name: HANGZHOU CALIBRA DIAGNOSTICS CO., LTD., CHINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LIU, PENGYUN;YUAN, XIAOFEN;MA, JINFEI;AND OTHERS;REEL/FRAME:064532/0280 Effective date: 20230320 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |