CN115326957A - Method for measuring blood concentration of folic acid and 5-methyl tetrahydrofolic acid in serum - Google Patents
Method for measuring blood concentration of folic acid and 5-methyl tetrahydrofolic acid in serum Download PDFInfo
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- CN115326957A CN115326957A CN202210950069.5A CN202210950069A CN115326957A CN 115326957 A CN115326957 A CN 115326957A CN 202210950069 A CN202210950069 A CN 202210950069A CN 115326957 A CN115326957 A CN 115326957A
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- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 128
- 210000002966 serum Anatomy 0.000 title claims abstract description 69
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 64
- 239000011724 folic acid Substances 0.000 title claims abstract description 64
- 229960000304 folic acid Drugs 0.000 title claims abstract description 64
- ZNOVTXRBGFNYRX-ABLWVSNPSA-N levomefolic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 ZNOVTXRBGFNYRX-ABLWVSNPSA-N 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 54
- 210000004369 blood Anatomy 0.000 title claims abstract description 30
- 239000008280 blood Substances 0.000 title claims abstract description 30
- 229940105150 5-methyltetrahydrofolic acid Drugs 0.000 title claims description 48
- 239000000243 solution Substances 0.000 claims abstract description 59
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 50
- 244000309466 calf Species 0.000 claims abstract description 36
- 238000002156 mixing Methods 0.000 claims abstract description 34
- 108010071390 Serum Albumin Proteins 0.000 claims abstract description 30
- 102000007562 Serum Albumin Human genes 0.000 claims abstract description 30
- 235000007635 levomefolic acid Nutrition 0.000 claims abstract description 9
- 239000011578 levomefolic acid Substances 0.000 claims abstract description 9
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 8
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 7
- 238000001819 mass spectrum Methods 0.000 claims abstract description 6
- 239000012224 working solution Substances 0.000 claims description 134
- 239000000523 sample Substances 0.000 claims description 106
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 69
- 238000003908 quality control method Methods 0.000 claims description 67
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 52
- 239000007864 aqueous solution Substances 0.000 claims description 48
- 239000000126 substance Substances 0.000 claims description 39
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 33
- 239000011550 stock solution Substances 0.000 claims description 31
- 238000010790 dilution Methods 0.000 claims description 30
- 239000012895 dilution Substances 0.000 claims description 30
- 239000013062 quality control Sample Substances 0.000 claims description 30
- 238000004458 analytical method Methods 0.000 claims description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 27
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 26
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 26
- 229930003268 Vitamin C Natural products 0.000 claims description 26
- 235000019154 vitamin C Nutrition 0.000 claims description 26
- 239000011718 vitamin C Substances 0.000 claims description 26
- 229960000485 methotrexate Drugs 0.000 claims description 24
- 239000012496 blank sample Substances 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 21
- 238000007865 diluting Methods 0.000 claims description 18
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000012545 processing Methods 0.000 claims description 17
- 239000007924 injection Substances 0.000 claims description 16
- 238000002347 injection Methods 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 238000003672 processing method Methods 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 13
- 239000011159 matrix material Substances 0.000 claims description 12
- 230000004044 response Effects 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 12
- 239000012498 ultrapure water Substances 0.000 claims description 12
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- 239000012452 mother liquor Substances 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 8
- 238000002474 experimental method Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 4
- 238000000861 blow drying Methods 0.000 claims description 3
- JMNIIIQOMSQWJN-ACGFUFEJSA-L calcium;(4s)-4-[[4-[(2-amino-5-methyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl)methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate Chemical compound [Ca+2].C1NC=2N=C(N)NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1.C1NC=2N=C(N)NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1 JMNIIIQOMSQWJN-ACGFUFEJSA-L 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000012937 correction Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000011835 investigation Methods 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 230000007774 longterm Effects 0.000 claims description 3
- 239000010413 mother solution Substances 0.000 claims description 3
- 239000012716 precipitator Substances 0.000 claims description 3
- 230000003595 spectral effect Effects 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 230000036765 blood level Effects 0.000 claims 7
- 210000003462 vein Anatomy 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
Classifications
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- 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
- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of blood concentration determination methods, and discloses a blood concentration determination method of folic acid and 5-methyltetrahydrofolate in serum, which comprises the following steps of adopting a sample, mixing 2% of calf serum albumin physiological saline solution with folic acid and a 5-methyltetrahydrofolate She Suanbiao product, and highly simulating a clinical real sample. The method for measuring the blood concentration of folic acid and 5-methyltetrahydrofolate in serum is used for measuring the blood concentration of folic acid and 5-methyltetrahydrofolate in serum of a patient by establishing a rapid, accurate and high-sensitivity liquid chromatography-mass spectrometry method, adopts 2% calf serum albumin normal saline solution to mix with folic acid and a 5-methyltetrahydro She Suanbiao product, highly simulates a clinical real sample, and has high chromatographic resolution, high mass spectrum selectivity and high sensitivity, and the two methods are effectively combined to rapidly and accurately quantify folic acid and 5-methyltetrahydrofolate.
Description
Technical Field
The invention relates to the technical field of blood concentration determination methods, in particular to a blood concentration determination method of folic acid and 5-methyltetrahydrofolic acid in serum.
Background
The blood concentration refers to the total concentration of the drug in plasma after absorption, including the drug bound to plasma proteins or free in plasma, and sometimes also refers to the concentration of the drug in whole blood, the intensity of the drug action is proportional to the concentration of the drug in plasma, and the concentration of the drug in the body varies with time.
At present, when the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum of a patient is measured, the process is complex, the accuracy is low, and the accuracy of measured data is influenced.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum, which has the advantages of rapidness, accuracy and high sensitivity, is a liquid chromatography-mass spectrometry method, is used for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in the serum of a patient, and the like, and solves the problems of slow measurement and low accuracy of the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme:
the blood concentration measuring method of folic acid and 5-methyl tetrahydrofolic acid in serum comprises the following steps:
s1, sample adoption
2% calf serum albumin normal saline solution is mixed with folic acid and 5-methyltetrahydro She Suanbiao products to highly simulate clinical real samples;
s2, performance parameters
Limit of detection (LOD): folic acid: 1ng/mL;
5-methyltetrahydrofolic acid: 1ng/mL;
lower limit of assay (LOQ): folic acid: 2ng/mL;
5-methyltetrahydrofolic acid: 2ng/mL;
linear range: folic acid: 2ng/mL-100ng/mL;
5-methyltetrahydrofolic acid: 2ng/mL-100ng/mL;
when the method is verified, the requirements of recovery rate and precision are as follows: the recovery rate is 85-115%, and the precision (precision in day, precision in day and precision in instrument) is RSD <15%;
stability: (long-term stability of stock solution, stability of the simulated sample after being placed at room temperature for 4h before being placed, stability of the simulated sample after being placed at 4 ℃ for 24h after being placed, stability of the simulated sample after being repeatedly frozen and thawed for 3 times, and stability of the simulated sample after being stored at-80 ℃ for a long time);
s3, original sample system
The sample type was serum, and the sample collection was performed by collecting venous whole blood with a blood collection needle, whole blood centrifugation (3500 r/min,10 min), collecting supernatant to obtain serum, and storing the sample in a storage container at-80 deg.C;
s4, source of blank serum
Because folic acid and 5-methyltetrahydrofolic acid are endogenous substances, the experiment adopts a substitute matrix as a standard curve and a quality control sample, the substitute matrix is a normal saline solution of 2% calf serum albumin, and the normal saline is prepared by the substitute matrix;
s5, required reagent
S5.1, water: ultrapure water, made by laboratory Poll ultrapure water meter;
acetonitrile, methanol, ethanol, formic acid, 25mol/L sodium hydroxide: mass spectral grade, fisher corporation;
the solutions were as follows:
mobile phase: 0.05% aqueous formic acid (A) -acetonitrile (B);
s5.2, standard substance and various solutions
Preparation of the used solution: precisely weighing 2.822mg of She Suanbiao product, multiplying by 92.3% of purity coefficient, preparing folic acid stock solution with concentration of 0.5mg/mL by using 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, storing in a refrigerator at-20 ℃ for later use, precisely weighing 2.260mg of 5-methyltetrahydrofolate calcium standard product, dividing by a correction factor, preparing 5-methyltetrahydrofolate stock solution with concentration of 0.5mg/mL by using 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, and storing in a refrigerator at-20 ℃ for later use;
preparing an internal standard mother solution and a working solution:
precisely weighing a methotrexate standard 2.147mg, preparing a methotrexate stock solution with the concentration of 0.5mg/mL by using a 5-percent NaOH (0.1 mol/L) 20% ethanol aqueous solution, and storing the methotrexate stock solution in a refrigerator at the temperature of-20 ℃ for later use;
precisely transferring 0.02mL of the prepared methotrexate mother liquor, placing the methotrexate mother liquor into a 1.5mL centrifuge tube, adding 0.98mL of methanol: diluting with water (1; and precisely transferring 0.01mL of methotrexate internal standard solution 1 again, placing the solution in a 1.5mL centrifuge tube, and adding 0.99mL of methanol: diluting with water (1;
an internal standard working solution, namely a precipitator, is a methanol solution (0.1 mg/mL VC) with methotrexate concentration of 2ng/mL;
adding 10 μ L vitamin C stock solution and 20 μ L methotrexate internal standard solution 2 (100 ng/mL) into 970 μ L methanol to obtain precipitant;
preparing a vitamin C solution:
precisely weighing a certain amount of vitamin C, preparing a vitamin C stock solution with the concentration of 10mg/mL by using ultrapure water, storing the vitamin C stock solution in a refrigerator at the temperature of-20 ℃ for later use, adding 10 mu L of the vitamin C stock solution into 990 mu L of water, and performing vortex to obtain 0.1mg/mL of vitamin C working solution;
preparation of 9% sodium chloride aqueous solution:
precisely weighing a certain amount of sodium chloride, preparing a sodium chloride aqueous solution with the concentration of 0.9% by using ultrapure water, and storing the sodium chloride aqueous solution in a refrigerator at 4 ℃ for later use;
standard Curve working dilutions and redissolves (10% acetonitrile in water (containing 0.1mg/mL VC):
adding 10 mu L of vitamin C stock solution and 100 mu L of pure acetonitrile into 890 mu L of water, and performing vortex to obtain a standard curve working diluent and a redissolution;
s5.3, preparation of working solution of quality control sample
Precisely transferring 0.02mL to 1.5mL of prepared folic acid and 5-methyltetrahydrofolic acid mother liquor into a centrifuge tube respectively, and adding 0.96mL of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 1 with the concentration of 10000 ng/mL;
HQC working fluid: precisely transferring the prepared quality control working solution 1,0.160mL to 1.5mL into a centrifuge tube, and adding 0.840mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 2 with the concentration of 1600ng/mL, wherein the solution is high-concentration quality control working solution;
MQC working solution: precisely transferring prepared quality control working solution 2,0.5mL to 1.5mL into a centrifuge tube, and adding 0.5mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain mixed quality control working solution 3 with the concentration of 800ng/mL, wherein the solution is medium-concentration quality control working solution;
precisely transferring the prepared quality control working solution 3,0.5mL to 1.5mL into a centrifuge tube, and adding 0.5mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 4 with the concentration of 400 ng/mL;
LQC working solution: precisely transferring prepared quality control working solution 4,0.3mL to 1.5mL in a centrifuge tube, and adding 0.7mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain mixed quality control working solution 5 with the concentration of 120ng/mL, wherein the solution is low-concentration quality control working solution;
storing the prepared quality control working solution in a refrigerator at the temperature of-20 ℃ for later use;
s6, preparation of standard curve working solution
Precisely transferring 0.02mL of the prepared folic acid and 5-methyltetrahydrofolic acid, putting the folic acid and 5-methyltetrahydrofolic acid into a 1.5mL centrifuge tube, adding 0.960mL of standard curve diluent for dilution, and uniformly mixing by vortex to obtain a mixed working solution 1 with the concentration of 10000 ng/mL;
standard curve L1 point: precisely transferring 0.2mL of prepared working solution 1, putting the working solution into a 1.5mL centrifuge tube, adding 0.8mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing by vortex to obtain mixed working solution 2 with the concentration of 2000 ng/mL;
standard curve L2 point: precisely transferring 0.5mL of prepared work 2, putting the work 2 into a 1.5mL centrifuge tube, adding 0.5mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing in a vortex manner to obtain a mixed work liquid 3 with the concentration of 1000 ng/mL;
standard curve L3 point: precisely transferring 0.4mL of prepared work 3, putting the work 3 into a 1.5mL centrifuge tube, adding 0.6mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing by vortex to obtain a mixed working solution 4 with the concentration of 400 ng/mL;
standard curve L4 point: precisely transferring 0.5mL of prepared working solution 4, putting the working solution 4 into a 1.5mL centrifuge tube, adding 0.5mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing in a vortex manner to obtain a mixed working solution 5 with the concentration of 200 ng/mL;
standard curve L5 point: precisely transferring 0.2mL of prepared work 4, putting the work 4 into a 1.5mL centrifuge tube, adding 0.3mL10% acetonitrile water (VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain a mixed working solution 6 with the concentration of 160 ng/mL;
standard curve L6 point: precisely transferring 0.2mL of prepared work 6, putting the work 6 into a 1.5mL centrifuge tube, adding 0.2mL10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain a mixed working solution 7 with the concentration of 80 ng/mL;
standard curve L7 point: precisely transferring 0.2mL of prepared working solution 7, placing the working solution in a 1.5mL centrifuge tube, adding 0.2mL10% acetonitrile water (the concentration of VC is 0.1 mg/mL), diluting, and uniformly mixing in a vortex manner to obtain mixed working solution 8 with the concentration of 40 ng/mL;
placing the working solution of each point of the marked yeast in a refrigerator for storage at-20 ℃ for later use;
s7, program step
S7.1, preparation of Standard Curve sample
Respectively taking 50 mu L of blank 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of standard curve series concentration mixed working solution to prepare a standard curve sample with the concentration range of 2-100ng/mL, then processing according to the processing method of the serum sample, analyzing and determining, and freshly preparing each time of determination;
s7.2, preparation of quality control sample
Respectively taking 50 mu L of 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of prepared mixed quality control working solution of 3-concentration folic acid and 5-methyl tetrahydrofolic acid, and uniformly mixing for 5min by vortex to prepare an HQC quality control sample with the concentration of 80ng/mL, an MQC quality control sample with the concentration of 40ng/mL and an LQC quality control sample with the concentration of 6 ng/mL. Then, the treatment is carried out according to the treatment method of the serum sample, the analysis and the determination are carried out, the quality control sample is prepared once, the treatment and the determination of 7 batches of samples can be continuously carried out according to the treatment method of the serum sample, the related stability experiment is investigated, and the next batch of quality control sample is prepared after the quality control sample prepared in the previous batch is used up;
s7.3 and method for processing serum sample
Taking a serum sample from 50 mu L to 1.5mL, adding 5 mu L of 0.1mg/mL VC aqueous solution, 2.5 mu L of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL), 200 mu L of methotrexate methanol solution (the VC concentration is 0.1 mg/mL) with the concentration position of 2ng/mL, 3500rpm, whirling and mixing for 5min, centrifuging at 4 ℃ and 15000r/min for 10min, taking 150 mu L of supernatant to 1.5mL of a middle chamber Wen Danqi for blow-drying, adding 80 mu L of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL), whirling for 5min at 3500rpm, centrifuging at 4 ℃ and 15000r/min for 5min, and taking 70 mu L of supernatant for sample injection analysis;
s7.4, method for processing substitute matrix blank sample
Taking 50 mu L of 2% calf serum albumin physiological saline solution, adding 200 mu L of blank methanol, 3500rpm, uniformly mixing by vortex for 5min, centrifuging for 10min at 4 ℃ and 15000r/min, taking 150 mu L of supernatant to a 1.5mL centrifuge tube middle chamber Wen Danqi for drying, adding 80 mu L of 10% acetonitrile aqueous solution (VC concentration is 0.1 mg/mL) to vortex for 5min at 3500rpm, centrifuging for 5min at 4 ℃ and 15000r/min, and taking 70 mu L of supernatant for sample injection analysis;
s7.5, compiling of sample injection table Worklist
Double Blank 2% calf serum sample (3 needles) + standard curve (7 concentration points) + high, medium and low quality control samples (1 sample per concentration) + real clinical samples (25 samples) + Double Blank 2% calf serum sample (3 needles) + real clinical samples (25 samples) + high, medium and low quality control samples (1 sample per concentration), the samples are arranged in sequence;
s7.6, LC/MS analysis conditions
S7.6.1, LC analysis conditions
A chromatographic column: waters, ACQULTY UPLC, BEH C18 (2.1X 50mm,1.7 μm);
column temperature: 25 ℃;
mobile phase: 0.05% aqueous formic acid (A) -acetonitrile (B);
flow rate: 0.22mL/min;
sample introduction amount: 5 mu L of the solution;
an elution mode: gradient elution;
s7.6.2 MS analysis conditions
S7.6.3, operation according to Shimadzu LC-MS/MS 8050SOP to turn on MS and LC, respectively; setting liquid phase and mass spectrum conditions according to experimental requirements, balancing the system for 10min, starting batch sample injection analysis, wherein software calculation numerical values are report results, and performing result analysis;
s8, quality control program
S8.1, selectivity/specificity
Comparing the chromatogram of the blank sample with the chromatogram of LLOQ (standard curve minimum concentration point), wherein the response value of the substance to be detected in the blank sample should not exceed 20% of the substance to be detected in the LLOQ.
The interference of the internal standard substance in the blank sample on the substance to be detected in the LLOQ is not more than 20 percent of the LLOQ;
the interference of the internal standard substance in the blank sample on the internal standard substance in the LLOQ is not more than 5% of the response of the internal standard substance;
s8.2, residual investigation
Respectively transferring 50 mu L of 2% calf serum albumin normal saline solution, placing the solution into a 1.5mL centrifuge tube, respectively adding 2.5 mu L of prepared standard curve working solution 1 (2000 ng/mL) and 2.5 mu L of prepared standard curve working solution 7 (40 ng/mL), processing samples according to a processing method of serum samples, and carrying out sample injection analysis and determination, wherein the sample injection sequence of the samples is as follows: 3 needles of L7 spot +20 needles of L1 spot +3 needles of blank serum sample;
after the sample sequence is completed, the response value of the residual substance to be detected in the blank sample should not exceed 20% of the substance to be detected in the LLOQ, and the response value of the residual internal standard substance in the blank sample should not exceed 5% of the internal standard substance in the LLOQ;
s8.3, marking of yeast and quality control
There were at least 7 calibration concentration points per analysis batch standard curve, and the accuracy of each concentration point should be 85-115%. In all test points of the standard curve, 75% of the points meet the standard (the points which do not meet the standard can not be the same concentration point), regression calculation is carried out on the points which meet the standard, the correlation coefficient R and R2 values of the regression equation are more than 0.99, and the accuracy of quality control of each concentration point is 85-115%;
s8.4, precision and accuracy
The daily and diurnal precision (RSD) is less than 15%, the daily and diurnal accuracy (RE) is within + -15% of the theoretical value, at least 2/3 of the QC samples meet the above standard, and at least 50% of the same concentration meets the standard.
Preferably, in the step S2, at an intra-day precision, 50 μ L of a blank 2% calf serum albumin physiological saline solution is taken, 5 μ L of 0.1mg/mL VC aqueous solution is added, 2.5 μ L of a mixed quality control working solution with three high, medium and low concentration points is added, 6 parts of each concentration point are prepared in parallel, processing is performed according to a "serum sample processing method", two batches (one batch in the morning and one batch in the afternoon) are continuously examined within one day, and a follow-up standard curve is prepared.
Preferably, in the step S2, blank 2% bovine serum albumin physiological saline solution 50 μ L is taken for daily precision, 5 μ L of 0.1mg/mL VC aqueous solution is added, 2.5 μ L of mixed quality control working solution with high, medium and low concentration points is added, 6 parts of each concentration point are prepared in parallel, three days are examined continuously according to the "serum sample processing method", and the accompanying standard curve is prepared.
Preferably, in the step S2, the precision of the apparatus is measured by taking 50 μ L of blank 2% bovine serum albumin physiological saline solution, adding 5 μ L of 0.1mg/mL VC aqueous solution, adding 2.5 μ L of low, medium and high quality control mixed working solution, preparing 6 parts per concentration, processing according to the "processing method of serum samples", continuously injecting 2 samples per sample, preparing a standard curve according to the line, and examining the precision of the apparatus according to the measured value.
Preferably, all the steps from step S1 to step S8 are performed under the condition of keeping out light.
Preferably, the processed sample is detected within 48h in the step S1 due to the instability of folic acid.
Preferably, the sample in the same Worklist in the step S7 is prepared by using the precipitant in the same tube, and preparing a plurality of samples according to the required amount before the sample is measured.
Preferably, in the step S5, because the amount of the working fluid added is small, the operation process needs to pay attention to accurate suction, and the gun head needs to be moistened when the working fluid is sucked, so as to reduce errors.
(III) advantageous effects
Compared with the prior art, the invention provides a method for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum, which has the following beneficial effects:
the method for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum is used for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in the serum of a patient by establishing a rapid, accurate and high-sensitivity liquid chromatography-mass spectrometry method, adopts 2% calf serum albumin normal saline solution to mix with folic acid and a 5-methyltetrahydro She Suanbiao product, highly simulates a clinical real sample, has high chromatographic resolution and high mass spectrum selectivity and sensitivity, and effectively combines the two methods to rapidly and accurately quantify folic acid and 5-methyltetrahydrofolic acid.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The blood concentration measuring method of folic acid and 5-methyl tetrahydrofolic acid in serum comprises the following steps:
s1, sample adoption
2 percent of calf serum albumin normal saline solution is mixed with folic acid and 5-methyltetrahydro She Suanbiao products, and clinical real samples are highly simulated.
S2, performance parameters
Limit of detection (LOD): folic acid: 1ng/mL.
5-methyltetrahydrofolic acid: 1ng/mL.
Determination lower Limit (LOQ): folic acid: 2ng/mL.
5-methyltetrahydrofolic acid: 2ng/mL.
Linear range: folic acid: 2ng/mL-100ng/mL.
5-methyltetrahydrofolic acid: 2ng/mL-100ng/mL.
When the method is verified, the requirements of recovery rate and precision are as follows: the recovery rate is 85-115%, the precision (intra-day precision, inter-day precision, instrument precision) is RSD <15%.
In-day precision, blank 2% calf serum albumin normal saline solution 50 μ L is taken, VC aqueous solution of 5 μ L0.1mg/mL is added, 2.5 μ L of mixed quality control working solution with high, medium and low concentration points is respectively added, 6 parts are prepared in parallel at each concentration point, the treatment is carried out according to a serum sample treatment method, two batches (one batch in the morning and afternoon) are continuously examined within one day, and random standard koji is prepared.
The day precision is to take 50 mu L of blank 2% calf serum albumin normal saline solution, add 5 mu L of 0.1mg/mL VC aqueous solution, respectively add 2.5 mu L of mixed quality control working solution with high, medium and low concentration points, prepare 6 parts of each concentration point in parallel, continuously inspect for three days according to the 'method for processing serum samples', and prepare follow-up standard curve.
The precision of the instrument is to take 50 mu L of blank 2% calf serum albumin normal saline solution, add 5 mu L of 0.1mg/mL VC aqueous solution, respectively add 2.5 mu L of low, medium and high quality control mixed working solution, prepare 6 parts for each concentration, process according to the 'processing method of serum samples', continuously sample 2 needles for each sample, prepare random standard, and examine the precision of the instrument according to the measured value.
Stability: (long-term stability of stock solution, stability of the simulated sample after being placed at room temperature for 4h before being placed, stability of the simulated sample after being placed at 4 ℃ for 24h after being placed, stability of the simulated sample after being repeatedly frozen and thawed for 3 times, and stability of the simulated sample after being stored at-80 ℃ for a long time).
S3, original sample system
The sample type was serum, and the sample collection was performed by collecting venous whole blood with a blood collection needle, whole blood centrifugation (3500 r/min,10 min), collecting supernatant to obtain serum, and storing the sample in a storage container at-80 deg.C.
S4, source of blank serum
As folic acid and 5-methyltetrahydrofolic acid are endogenous substances, the experiment adopts a substitute matrix as a standard curve and a quality control sample, the substitute matrix is a normal saline solution of 2% calf serum albumin, and the normal saline is prepared by the substitute matrix.
S5, required reagent
S5.1, water: ultrapure water, manufactured by the laboratory Poll ultrapure water meter.
Acetonitrile, methanol, ethanol, formic acid, 25mol/L sodium hydroxide: mass spectral grade, fisher corporation.
The solutions were as follows:
mobile phase: 0.05% aqueous formic acid (A) -acetonitrile (B).
S5.2, standard substance and various solutions
Standard article
Standard article information
Preparation of the used solution: 2.822mg of She Suanbiao was weighed precisely, multiplied by 92.3% purity factor, and prepared into a stock solution of folic acid with a concentration of 0.5mg/mL using a 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, and stored in a refrigerator at-20 ℃ for use, 2.260mg of calcium 5-methyltetrahydrofolate was weighed precisely, divided by a correction factor, and prepared into a stock solution of 5-methyltetrahydrofolate with a concentration of 0.5mg/mL using a 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, and stored in a refrigerator at-20 ℃ for use.
Preparing an internal standard mother solution and a working solution:
methotrexate standard 2.147mg was weighed precisely, and prepared into methotrexate stock solution with concentration of 0.5mg/mL using 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, and stored in a refrigerator at-20 deg.C for further use.
Precisely transferring 0.02mL of the prepared methotrexate mother liquor, placing the methotrexate mother liquor into a 1.5mL centrifuge tube, adding 0.98mL of methanol: and (3) diluting with water (1. V. And precisely transferring 0.01mL of methotrexate internal standard solution 1 again, placing the solution in a 1.5mL centrifuge tube, adding 0.99mL of methanol: and (3) diluting with water (1. V.
The internal standard working solution, namely the precipitator, is a methanol solution (0.1 mg/mL VC) with methotrexate concentration of 2ng/mL.
mu.L of vitamin C stock solution and 20. Mu.L of methotrexate internal standard solution 2 (100 ng/mL) were added to 970. Mu.L of methanol, i.e., the precipitant.
Preparing a vitamin C solution:
precisely weighing a certain amount of vitamin C, preparing a vitamin C stock solution with the concentration of 10mg/mL by using ultrapure water, storing the vitamin C stock solution in a refrigerator at the temperature of-20 ℃ for later use, adding 10 mu L of the vitamin C stock solution into 990 mu L of water, and performing vortex to obtain 0.1mg/mL of vitamin C working solution.
Preparation of 9% sodium chloride aqueous solution:
precisely weighing a certain amount of sodium chloride, preparing a sodium chloride aqueous solution with the concentration of 0.9% by using ultrapure water, and storing the sodium chloride aqueous solution in a refrigerator at 4 ℃ for later use.
Standard Curve working dilutions and redissolves (10% acetonitrile in water (containing 0.1mg/mL VC):
and adding 10 mu L of vitamin C stock solution and 100 mu L of pure acetonitrile into 890 mu L of water, and performing vortex to obtain a standard curve working diluent and a redissolution.
S5.3, preparation of quality control sample working solution
Precisely transferring 0.02mL to 1.5mL of the prepared folic acid and 5-methyltetrahydrofolic acid mother liquor into a centrifuge tube respectively, and adding 0.96mL of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 1 with the concentration of 10000 ng/mL.
HQC working fluid: precisely transferring the prepared quality control working solution 1,0.160mL to 1.5mL into a centrifuge tube, and adding 0.840mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 2 with the concentration of 1600ng/mL, wherein the solution is high-concentration quality control working solution.
MQC working solution: precisely transferring the prepared quality control working solution 2,0.5mL to 1.5mL into a centrifuge tube, and adding 0.5mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 3 with the concentration of 800ng/mL, wherein the solution is medium-concentration quality control working solution.
Precisely transferring the prepared quality control working solution 3,0.5mL to 1.5mL into a centrifuge tube, and adding 0.5mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 4 with the concentration of 400 ng/mL.
LQC working solution: precisely transferring prepared quality control working solution 4,0.3mL to 1.5mL in a centrifuge tube, and adding 0.7mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain mixed quality control working solution 5 with the concentration of 120ng/mL, wherein the solution is low-concentration quality control working solution.
The prepared quality control working solution is stored in a refrigerator at the temperature of minus 20 ℃ for later use.
S6, preparation of standard curve working solution
Precisely transferring 0.02mL of the prepared folic acid and 5-methyltetrahydrofolic acid, putting the folic acid and the 5-methyltetrahydrofolic acid into a 1.5mL centrifuge tube, adding 0.960mL of standard curve diluent for dilution, and uniformly mixing by vortex to obtain a mixed working solution 1 with the concentration of 10000 ng/mL.
Standard curve L1 point: precisely transferring 0.2mL of prepared working solution 1, putting the working solution into a 1.5mL centrifuge tube, adding 0.8mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing by vortex to obtain mixed working solution 2 with the concentration of 2000 ng/mL.
Standard curve L2 point: precisely transferring 0.5mL of prepared working solution 2, placing the working solution in a 1.5mL centrifuge tube, adding 0.5mL of 10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing in a vortex manner to obtain a mixed working solution 3 with the concentration of 1000 ng/mL.
Standard curve L3 point: precisely transferring 0.4mL of prepared working solution 3, placing the working solution in a 1.5mL centrifuge tube, adding 0.6mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain mixed working solution 4 with the concentration of 400 ng/mL.
Standard curve L4 point: precisely transferring 0.5mL of prepared working solution 4, putting the working solution into a 1.5mL centrifuge tube, adding 0.5mL of 10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain a mixed working solution 5 with the concentration of 200 ng/mL.
Standard curve L5 point: precisely transferring 0.2mL of prepared working solution 4, putting the working solution into a 1.5mL centrifuge tube, adding 0.3mL of 10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain mixed working solution 6 with the concentration of 160 ng/mL.
Standard curve L6 point: precisely transferring 0.2mL of prepared working solution 6, placing the working solution in a 1.5mL centrifuge tube, adding 0.2mL10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain mixed working solution 7 with the concentration of 80 ng/mL.
Standard curve L7 point: precisely transferring 0.2mL of prepared working solution 7, putting the working solution into a 1.5mL centrifuge tube, adding 0.2mL10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain a mixed working solution 8 with the concentration of 40 ng/mL.
And placing the working solution of each point of the standard yeast in a refrigerator for storage at-20 ℃ for later use.
S7, program step
S7.1, preparation of Standard Curve sample
Respectively taking 50 mu L of blank 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of standard curve series concentration mixed working solution to prepare a standard curve sample with the concentration range of 2-100ng/mL, then processing according to the processing method of a serum sample, analyzing and determining, and freshly preparing when determining each time, wherein the preparation method is shown in the following table 1:
TABLE 1 preparation of standard curve samples
S7.2, preparation of quality control sample
Respectively taking 50 mu L of 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of prepared mixed quality control working solution of 3-concentration folic acid and 5-methyl tetrahydrofolic acid, and uniformly mixing for 5min by vortex to prepare an HQC quality control sample with the concentration of 80ng/mL, an MQC quality control sample with the concentration of 40ng/mL and an LQC quality control sample with the concentration of 6 ng/mL. Then, the treatment is carried out according to the treatment method of the serum sample, the analysis and the measurement are carried out, the quality control sample is prepared once, the treatment and the measurement of 7 batches of samples can be continuously carried out according to the treatment method of the serum sample, the related stability experiment is considered, and the next batch of quality control sample is prepared after the quality control sample prepared in the previous batch is used up. The formulation method is shown in table 2 below:
TABLE 2 preparation of quality control samples
S7.3 and method for processing serum sample
Taking a serum sample from 50 mu L to 1.5mL, adding 5 mu L of 0.1mg/mL VC aqueous solution, 2.5 mu L of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL), 200 mu L of methotrexate methanol solution (the VC concentration is 0.1 mg/mL) with the concentration of 2ng/mL, 3500rpm, whirling and mixing for 5min, centrifuging at 4 ℃ and 15000r/min for 10min, taking 150 mu L of supernatant to 1.5mL of a middle chamber Wen Danqi for blow drying, adding 80 mu L of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL), whirling for 5min at 3500rpm, centrifuging at 4 ℃ and 15000r/min for 5min, and taking 70 mu L of supernatant for sample injection analysis.
S7.4, method for processing substitute matrix blank sample
Taking 50 mu L of 2% calf serum albumin physiological saline solution, adding 200 mu L of blank methanol, 3500rpm, uniformly mixing by vortex for 5min, centrifuging at 4 ℃ and 15000r/min for 10min, taking 150 mu L of supernatant to a 1.5mL centrifuge tube middle chamber Wen Danqi for drying, adding 80 mu L of 10% acetonitrile aqueous solution (VC concentration is 0.1 mg/mL) to vortex for 5min at 3500rpm, centrifuging at 4 ℃ and 15000r/min for 5min, and taking 70 mu L of supernatant for sample injection analysis.
S7.5, compiling of sample injection table Worklist
Double Blank 2% calf serum sample (3 needles) + standard curve (7 concentration points) + high, medium and low quality control samples (1 sample per concentration) + real clinical samples (25 samples) + Double Blank 2% calf serum sample (3 needles) + real clinical samples (25 samples) + high, medium and low quality control samples (1 sample per concentration), the samples being arranged in sequence.
S7.6, LC/MS analysis conditions
S7.6.1, LC analysis conditions
A chromatographic column: waters, ACQULTY UPLC, BEH C18 (2.1X 50mm,1.7 μm).
Column temperature: at 25 ℃.
Mobile phase: 0.05% aqueous formic acid (A) -acetonitrile (B).
Flow rate: 0.22mL/min.
Sample introduction amount: 5 μ L.
And (3) an elution mode: gradient elution, elution procedure is shown in table 3 below:
TABLE 3 elution procedure
S7.6.2, MS analysis conditions
The mass spectrometry conditions and parameters are shown in table 4 below:
TABLE 4 Mass Spectrometry conditions and parameters
S7.6.3 Shimadzu LC-MS/MS 8050SOP, according to the procedure, turns on MS and LC, respectively. And (3) setting conditions of liquid phase and mass spectrum according to experimental requirements, balancing the system for 10min, starting batch sample injection analysis, and carrying out result analysis after software calculation numerical values are report results.
S8, quality control program
S8.1, selectivity/specificity
Comparing the chromatogram of the blank sample with the chromatogram of LLOQ (standard curve minimum concentration point), wherein the response value of the substance to be detected in the blank sample should not exceed 20% of the substance to be detected in the LLOQ.
The interference of the internal standard substance in the blank sample on the substance to be detected in the LLOQ should not exceed 20 percent of the LLOQ.
The interference of the internal standard substance in the blank sample on the internal standard substance in the LLOQ should not exceed 5% of the response of the internal standard substance.
S8.2, residual investigation
Respectively transferring 50 mu L of 2% calf serum albumin physiological saline solution, placing the solution in a 1.5mL centrifuge tube, respectively adding 2.5 mu L of prepared standard curve working solution 1 (2000 ng/mL) and 2.5 mu L of prepared standard curve working solution 7 (40 ng/mL), processing a sample according to a serum sample processing method, and analyzing and determining sample introduction, wherein the sample introduction sequence is as follows: 3 pins L7 spots +20 pins L1 spots +3 pins blank serum samples.
After the sample sequence is completed, the response value of the residual substance to be detected in the blank sample should not exceed 20% of the substance to be detected in the LLOQ, and the response value of the residual internal standard substance in the blank sample should not exceed 5% of the internal standard substance in the LLOQ.
S8.3, marking of yeast and quality control
There were at least 7 calibration concentration points per analysis batch standard curve, and the accuracy of each concentration point should be 85-115%. In all test points of the standard curve, 75% of the points meet the standard (the points which do not meet the standard can not be the same concentration point), regression calculation is carried out on the points which meet the standard, the correlation coefficient R and R2 values of the regression equation are greater than 0.99, and the accuracy of quality control on each concentration point is 85-115%.
S8.4, precision and accuracy
The daily and diurnal precision (RSD) is less than 15%, the daily and diurnal accuracy (RE) is within + -15% of the theoretical value, at least 2/3 of the QC samples meet the above standard, and at least 50% of the same concentration meets the standard.
Among these, it is noted that:
all the operation steps of the experiment need to be carried out under the condition of avoiding light.
Due to the instability of folic acid, the detection of the treated sample is completed within 48 h.
Samples from the same Worklist were run with the same tube containing the precipitant. Before the sample measurement, more samples are prepared according to the required quantity.
Care should be taken for static conditions when weighing calf serum albumin.
Because the working solution addition amount is less, the operation process needs to pay attention to the accurate absorption, and the gun head needs to be moistened when the working solution is absorbed, so that the error is reduced.
The samples were thawed and then processed as soon as possible, e.g., they could not be processed for a short period of time in a refrigerator at 4 ℃ for no more than 4 hours.
In view of the instability of vitamin C, a stock solution of vitamin C (10 mg/mL) was weighed out once a week for formulation.
In view of the instability of 5-methyltetrahydrofolic acid in freezing and thawing at-20 ℃, the stock solution is divided into a plurality of portions after being prepared for the first time, and each portion can be used once.
The invention has the beneficial effects that: the rapid, accurate and high-sensitivity liquid chromatography-mass spectrometry method is established and used for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum of a patient, 2% calf serum albumin normal saline solution is mixed with folic acid and a 5-methyltetrahydrofolate She Suanbiao product, clinical real samples are highly simulated, the LC-MS/MS method has high chromatographic resolution and high mass spectrum selectivity and sensitivity, and the two methods are effectively combined to rapidly and accurately quantify the folic acid and the 5-methyltetrahydrofolic acid.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The method for measuring the blood concentration of folic acid and 5-methyltetrahydrofolic acid in serum is characterized by comprising the following steps:
s1, sample adoption
2% calf serum albumin normal saline solution is mixed with folic acid and 5-methyltetrahydro She Suanbiao products to highly simulate clinical real samples;
s2, performance parameters
Limit of detection (LOD): folic acid: 1ng/mL;
5-methyltetrahydrofolic acid: 1ng/mL;
lower limit of assay (LOQ): folic acid: 2ng/mL;
5-methyltetrahydrofolic acid: 2ng/mL;
linear range: folic acid: 2ng/mL-100ng/mL;
5-methyltetrahydrofolic acid: 2ng/mL-100ng/mL;
when the method is verified, the requirements of recovery rate and precision are as follows: the recovery rate is 85-115%, and the precision (precision in day, precision in day and precision in instrument) is RSD <15%;
stability: (long-term stability of stock solution, stability of the simulated sample after being placed at room temperature for 4h before being placed, stability of the simulated sample after being placed at 4 ℃ for 24h after being placed, stability of the simulated sample after being repeatedly frozen and thawed for 3 times, and stability of the simulated sample after being stored at-80 ℃ for a long time);
s3, original sample system
The sample type is serum, the sample is collected by collecting vein whole blood through a blood collecting needle, the whole blood is centrifuged (3500 r/min,10 min), the supernatant is taken to obtain the serum, and the sample is stored in a storage container at the temperature of-80 ℃;
s4, source of blank serum
Because folic acid and 5-methyltetrahydrofolic acid are endogenous substances, the experiment adopts a substitute matrix as a standard curve and a quality control sample, the substitute matrix is a normal saline solution of 2% calf serum albumin, and the normal saline is prepared by the substitute matrix;
s5, required reagent
S5.1, water: ultrapure water, made by laboratory Poll ultrapure water meter;
acetonitrile, methanol, ethanol, formic acid, 25mol/L sodium hydroxide: mass spectral grade, fisher corporation;
the solutions were as follows:
mobile phase: 0.05% aqueous formic acid (A) -acetonitrile (B);
s5.2, standard substance and various solutions
Preparation of the used solution: precisely weighing 2.822mg of She Suanbiao product, multiplying by 92.3% of purity coefficient, preparing folic acid stock solution with concentration of 0.5mg/mL by using 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, storing in a refrigerator at-20 ℃ for later use, precisely weighing 2.260mg of 5-methyltetrahydrofolate calcium standard product, dividing by a correction factor, preparing 5-methyltetrahydrofolate stock solution with concentration of 0.5mg/mL by using 5% NaOH (0.1 mol/L) 20% ethanol aqueous solution, and storing in a refrigerator at-20 ℃ for later use;
preparing an internal standard mother solution and a working solution:
precisely weighing a methotrexate standard 2.147mg, preparing a methotrexate stock solution with the concentration of 0.5mg/mL by using a 5-percent NaOH (0.1 mol/L) 20% ethanol aqueous solution, and storing the methotrexate stock solution in a refrigerator at the temperature of-20 ℃ for later use;
precisely transferring 0.02mL of the prepared methotrexate mother liquor, placing the methotrexate mother liquor into a 1.5mL centrifuge tube, adding 0.98mL of methanol: diluting with water (1; and precisely transferring 0.01mL of methotrexate internal standard solution 1 again, placing the solution in a 1.5mL centrifuge tube, and adding 0.99mL of methanol: diluting with water (1;
an internal standard working solution, namely a precipitator, is a methanol solution (0.1 mg/mL VC) with methotrexate concentration of 2ng/mL;
adding 10 μ L vitamin C stock solution and 20 μ L methotrexate internal standard solution 2 (100 ng/mL) into 970 μ L methanol to obtain precipitant;
preparing a vitamin C solution:
precisely weighing a certain amount of vitamin C, preparing a vitamin C stock solution with the concentration of 10mg/mL by using ultrapure water, storing the vitamin C stock solution in a refrigerator at the temperature of-20 ℃ for later use, adding 10 mu L of the vitamin C stock solution into 990 mu L of water, and performing vortex to obtain 0.1mg/mL of vitamin C working solution;
preparation of 9% sodium chloride aqueous solution:
precisely weighing a certain amount of sodium chloride, preparing a sodium chloride aqueous solution with the concentration of 0.9% by using ultrapure water, and storing the sodium chloride aqueous solution in a refrigerator at the temperature of 4 ℃ for later use;
standard Curve working dilutions and redissolves (10% acetonitrile in water (containing 0.1mg/mL VC):
adding 10 mu L of vitamin C stock solution and 100 mu L of pure acetonitrile into 890 mu L of water, and performing vortex to obtain a standard curve working diluent and a redissolution;
s5.3, preparation of working solution of quality control sample
Precisely transferring 0.02mL to 1.5mL of prepared folic acid and 5-methyltetrahydrofolic acid mother liquor into a centrifuge tube respectively, and adding 0.96mL of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 1 with the concentration of 10000 ng/mL;
HQC working fluid: precisely transferring the prepared quality control working solution 1,0.160mL to 1.5mL into a centrifuge tube, and adding 0.840mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 2 with the concentration of 1600ng/mL, wherein the solution is high-concentration quality control working solution;
MQC working solution: precisely transferring prepared quality control working solution 2,0.5mL to 1.5mL into a centrifuge tube, and adding 0.5mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain mixed quality control working solution 3 with the concentration of 800ng/mL, wherein the solution is medium-concentration quality control working solution;
precisely transferring the prepared quality control working solution 3,0.5mL to 1.5mL into a centrifuge tube, and adding 0.5mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain a mixed quality control working solution 4 with the concentration of 400 ng/mL;
LQC working solution: precisely transferring prepared quality control working solution 4,0.3mL to 1.5mL in a centrifuge tube, and adding 0.7mL of 10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution to obtain mixed quality control working solution 5 with the concentration of 120ng/mL, wherein the solution is low-concentration quality control working solution;
storing the prepared quality control working solution in a refrigerator at the temperature of-20 ℃ for later use;
s6, preparation of standard curve working solution
Precisely transferring 0.02mL of the prepared folic acid and 5-methyltetrahydrofolic acid, putting the folic acid and 5-methyltetrahydrofolic acid into a 1.5mL centrifuge tube, adding 0.960mL of standard curve diluent for dilution, and uniformly mixing by vortex to obtain a mixed working solution 1 with the concentration of 10000 ng/mL;
standard curve L1 point: precisely transferring 0.2mL of prepared working solution 1, putting the working solution into a 1.5mL centrifuge tube, adding 0.8mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing by vortex to obtain mixed working solution 2 with the concentration of 2000 ng/mL;
standard curve L2 point: precisely transferring 0.5mL of prepared work 2, putting the work 2 into a 1.5mL centrifuge tube, adding 0.5mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing in a vortex manner to obtain a mixed work liquid 3 with the concentration of 1000 ng/mL;
standard curve L3 point: precisely transferring 0.4mL of prepared work 3, putting the work 3 into a 1.5mL centrifuge tube, adding 0.6mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing by vortex to obtain a mixed working solution 4 with the concentration of 400 ng/mL;
standard curve L4 point: precisely transferring 0.5mL of prepared working solution 4, putting the working solution 4 into a 1.5mL centrifuge tube, adding 0.5mL10% acetonitrile water (VC concentration is 0.1 mg/mL) for dilution, and uniformly mixing in a vortex manner to obtain a mixed working solution 5 with the concentration of 200 ng/mL;
standard curve L5 point: precisely transferring 0.2mL of prepared work 4, putting the work 4 into a 1.5mL centrifuge tube, adding 0.3mL10% acetonitrile water (VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain a mixed working solution 6 with the concentration of 160 ng/mL;
standard curve L6 point: precisely transferring 0.2mL of prepared work 6, putting the work 6 into a 1.5mL centrifuge tube, adding 0.2mL10% acetonitrile water (the VC concentration is 0.1 mg/mL), diluting, and uniformly mixing by vortex to obtain a mixed working solution 7 with the concentration of 80 ng/mL;
standard curve L7 point: precisely transferring 0.2mL of prepared working solution 7, placing the working solution in a 1.5mL centrifuge tube, adding 0.2mL10% acetonitrile water (the concentration of VC is 0.1 mg/mL), diluting, and uniformly mixing in a vortex manner to obtain mixed working solution 8 with the concentration of 40 ng/mL;
placing the working solution of each point of the marked yeast in a refrigerator for storage at-20 ℃ for later use;
s7, program step
S7.1, preparation of Standard Curve sample
Respectively taking 50 mu L of blank 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of standard curve series concentration mixed working solution to prepare a standard curve sample with the concentration range of 2-100ng/mL, then processing according to the processing method of a serum sample, analyzing and determining, and freshly preparing during each determination;
s7.2, preparation of quality control sample
Respectively taking 50 mu L of 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of prepared mixed quality control working solution of 3-concentration folic acid and 5-methyl tetrahydrofolic acid, and uniformly mixing for 5min by vortex to prepare an HQC quality control sample, an MQC quality control sample and an LQC quality control sample, wherein the concentrations of the HQC quality control sample, the MQC quality control sample and the LQC quality control sample are respectively 80ng/mL, 40ng/mL and 6 ng/mL. Then, the treatment is carried out according to the treatment method of the serum sample, the analysis and the determination are carried out, the quality control sample is prepared once, the treatment and the determination of 7 batches of samples can be continuously carried out according to the treatment method of the serum sample, the related stability experiment is investigated, and the next batch of quality control sample is prepared after the quality control sample prepared in the previous batch is used up;
s7.3 and method for processing serum sample
Taking a serum sample from 50 mu L to 1.5mL, adding 5 mu L of 0.1mg/mL VC aqueous solution, 2.5 mu L of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL), 200 mu L of methotrexate methanol solution (the VC concentration is 0.1 mg/mL) with the concentration position of 2ng/mL, 3500rpm, whirling and mixing for 5min, centrifuging at 4 ℃ and 15000r/min for 10min, taking 150 mu L of supernatant to 1.5mL of a middle chamber Wen Danqi for blow-drying, adding 80 mu L of 10% acetonitrile aqueous solution (the VC concentration is 0.1 mg/mL), whirling for 5min at 3500rpm, centrifuging at 4 ℃ and 15000r/min for 5min, and taking 70 mu L of supernatant for sample injection analysis;
s7.4, method for processing substitute matrix blank sample
Taking 50 mu L of 2% calf serum albumin physiological saline solution, adding 200 mu L of blank methanol, 3500rpm, vortex mixing uniformly for 5min, centrifuging for 10min at 4 ℃, 15000r/min, taking 150 mu L of supernatant liquid to a 1.5mL centrifuge tube middle chamber Wen Danqi for drying, adding 80 mu L of 10% acetonitrile aqueous solution (VC concentration is 0.1 mg/mL) to vortex for 5min at 3500rpm, centrifuging for 5min at 4 ℃, 15000r/min, and taking 70 mu L of supernatant liquid for sample injection analysis;
s7.5, compiling of sample injection table Worklist
Double Blank 2% calf serum sample (3 needles) + standard curve (7 concentration points) + high, medium and low quality control samples (1 sample per concentration) + real clinical samples (25 samples) + Double Blank 2% calf serum sample (3 needles) + real clinical samples (25 samples) + high, medium and low quality control samples (1 sample per concentration), the samples are arranged in sequence;
s7.6, LC/MS analysis conditions
S7.6.1, LC analysis conditions
A chromatographic column: waters, ACQULTY UPLC, BEH C18 (2.1X 50mm,1.7 μm);
column temperature: 25 ℃;
mobile phase: 0.05% aqueous formic acid (A) -acetonitrile (B);
flow rate: 0.22mL/min;
sample introduction amount: 5 mu L of the solution;
and (3) an elution mode: gradient elution;
s7.6.2, MS analysis conditions
S7.6.3, operation according to Shimadzu LC-MS/MS 8050SOP to turn on MS and LC, respectively; setting liquid phase and mass spectrum conditions according to experimental requirements, balancing the system for 10min, starting batch sample injection analysis, wherein software calculation numerical values are report results, and performing result analysis;
s8, quality control program
S8.1, selectivity/specificity
Comparing the chromatogram of the blank sample with the chromatogram of LLOQ (standard curve minimum concentration point), wherein the response value of the substance to be detected in the blank sample should not exceed 20% of the substance to be detected in the LLOQ.
The interference of the internal standard substance in the blank sample on the substance to be detected in the LLOQ should not exceed 20 percent of the LLOQ;
the interference of the internal standard substance in the blank sample on the internal standard substance in the LLOQ should not exceed 5% of the response of the internal standard substance;
s8.2, residual investigation
Respectively transferring 50 mu L of 2% calf serum albumin normal saline solution, placing the solution into a 1.5mL centrifuge tube, respectively adding 2.5 mu L of prepared standard curve working solution 1 (2000 ng/mL) and 2.5 mu L of prepared standard curve working solution 7 (40 ng/mL), processing samples according to a processing method of serum samples, and carrying out sample injection analysis and determination, wherein the sample injection sequence of the samples is as follows: 3 needles of L7 spot +20 needles of L1 spot +3 needles of blank serum sample;
after the sample sequence is completed, the response value of the residual substance to be detected in the blank sample should not exceed 20% of the substance to be detected in the LLOQ, and the response value of the residual internal standard substance in the blank sample should not exceed 5% of the internal standard substance in the LLOQ;
s8.3, marking of yeast and quality control
There were at least 7 calibration concentration points per analysis batch standard curve, and the accuracy of each concentration point should be 85-115%. In all test points of the standard curve, 75% of the points meet the standard (the points which do not meet the standard can not be the same concentration point), regression calculation is carried out on the points which meet the standard, the values of the correlation coefficient R and R2 of the regression equation are more than 0.99, and the accuracy of quality control of each concentration point is 85-115%;
s8.4, precision and accuracy
The daily and diurnal precision (RSD) is less than 15%, the daily and diurnal accuracy (RE) is within + -15% of the theoretical value, at least 2/3 of the QC samples meet the above standard, and at least 50% of the same concentration meets the standard.
2. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the step S2, within-day precision is realized by taking 50 mu L of blank 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of mixed quality control working solution with three concentration points of high, medium and low, preparing 6 parts in parallel at each concentration point, processing according to a 'serum sample processing method', continuously inspecting two batches (one batch in the morning and afternoon) within one day, and preparing follow-up standard koji.
3. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the step S2, blank 2% calf serum albumin normal saline solution 50 mu L is taken for daily precision, 5 mu L of 0.1mg/mL VC aqueous solution is added, 2.5 mu L of mixed quality control working solution with high, medium and low concentration points is respectively added, 6 parts of each concentration point are prepared in parallel, three days are continuously examined according to a serum sample processing method, and the follow-up standard curve is prepared.
4. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the step S2, the precision of the instrument is measured by taking 50 mu L of blank 2% calf serum albumin normal saline solution, adding 5 mu L of 0.1mg/mL VC aqueous solution, respectively adding 2.5 mu L of low, medium and high quality control mixed working solution, preparing 6 parts for each concentration, processing according to a 'processing method of serum samples', continuously injecting 2 needles for each sample, preparing a random standard curve, and observing the precision of the instrument according to a measured value.
5. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the steps S1 to S8, all the operation steps need to be performed under a dark condition.
6. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the step S1, the detection of the processed sample is completed within 48h in view of the instability of folic acid.
7. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the step S7, the same precipitant in the same tube is used for the sample in the same Worklist, and more precipitant is prepared according to the required amount before the sample is measured.
8. The method for measuring blood levels of folic acid and 5-methyltetrahydrofolic acid in serum according to claim 1, which comprises: in the step S5, the amount of the working fluid added is small, so that the operation process needs to pay attention to accurate suction, and the gun head needs to be moistened when the working fluid is sucked, so as to reduce errors.
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