CN105527357B - A kind of method of antioxidant BHT in measure insulin glargine injecta - Google Patents
A kind of method of antioxidant BHT in measure insulin glargine injecta Download PDFInfo
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- CN105527357B CN105527357B CN201610077845.XA CN201610077845A CN105527357B CN 105527357 B CN105527357 B CN 105527357B CN 201610077845 A CN201610077845 A CN 201610077845A CN 105527357 B CN105527357 B CN 105527357B
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- Prior art keywords
- insulin glargine
- antioxidant bht
- bht
- injecta
- glargine injecta
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- 108010057186 Insulin Glargine Proteins 0.000 title claims abstract description 35
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 35
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 35
- COCFEDIXXNGUNL-RFKWWTKHSA-N Insulin glargine Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(=O)NCC(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 COCFEDIXXNGUNL-RFKWWTKHSA-N 0.000 title claims abstract description 34
- 229960002869 insulin glargine Drugs 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 11
- 238000002203 pretreatment Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 108090001061 Insulin Proteins 0.000 claims description 4
- 102000004877 Insulin Human genes 0.000 claims description 4
- YAJCHEVQCOHZDC-QMMNLEPNSA-N actrapid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3N=CNC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@H](C)CC)[C@H](C)CC)[C@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C(N)=O)C1=CNC=N1 YAJCHEVQCOHZDC-QMMNLEPNSA-N 0.000 claims description 3
- 235000009508 confectionery Nutrition 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- DJFBJKSMACBYBD-UHFFFAOYSA-N phosphane;hydrate Chemical compound O.P DJFBJKSMACBYBD-UHFFFAOYSA-N 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- CRKADHVTAQCXRA-UHFFFAOYSA-K trisodium;phosphate;dihydrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]P([O-])([O-])=O CRKADHVTAQCXRA-UHFFFAOYSA-K 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 12
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 abstract description 9
- 229940100630 metacresol Drugs 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 7
- 238000012856 packing Methods 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 6
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000002960 lipid emulsion Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical class N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920005549 butyl rubber Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000003182 parenteral nutrition solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- -1 BHT Chemical class 0.000 description 1
- 244000043261 Hevea brasiliensis Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 239000006067 antibiotic powder Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 229920003052 natural elastomer Polymers 0.000 description 1
- 229920001194 natural rubber Polymers 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- CKRORYDHXIRZCH-UHFFFAOYSA-N phosphoric acid;dihydrate Chemical compound O.O.OP(O)(O)=O CKRORYDHXIRZCH-UHFFFAOYSA-N 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- 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)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a kind of method for determining antioxidant BHT in insulin glargine injecta, belong to Key works Drug packing and drug compatibility field, specially detected using high performance liquid chromatography, mobile phase is acetonitrile：Water：Phosphate buffer, the pre-treatment means of complexity are avoided, successfully antioxidant BHT chromatographic peak is separated with insulin glargine and metacresol by the present invention, the generation of conditions of streaking is avoided, accurately detects the content of antioxidant BHT in sample.
Description
Technical field
The present invention relates in Key works Drug packing and drug compatibility field, more particularly to a kind of measure insulin glargine injecta
The method of antioxidant BHT.
Background technology
Drug packing material ceases to ensureing that the stability of medicine plays vital effect with the drug safety of people
It is related.Pharmaceutical packing material selection is improper to cause the migration of active constituents of medicine, absorption even to chemically react, and make medicine
Thing fails, and some can also produce serious toxic side effect.
Medicinal butyl rubber stopper is compared with natural rubber, due to good air-tightness, stability, resistance to ag(e)ing and low precipitation
Property, have become one of widely used material in medical packaging.Because the comparison of ingredients of plug is complicated, comprising chemical combination
The species of thing is also relatively more, as pharmaceutical packing material is directly contacted, even if the chemical stability of butyl rubber plug is fine, its
In compound be also possible to move in decoction, there is a possibility that influence drug effect or pollute medicine.Some antibiotic
Powder ampoule agent for injection, occur the exceeded phenomenon of clarity in storage process, have document report relevant with plug used.
Antioxidant 2,6- di-tert-butyl-4-methy phenols, i.e. BHT, be it is currently known be present in it is volatilizable in plug
Property one of composition, research shows that BHT has different degrees of toxic action to the liver and spleen stomach of human body.BHT in research plug is moved
The migration amount size moved on in decoction, plays an important role to the Study on Compatibility of plug and medicine.It is directed on conventional art
The most of method for using GC-MS of detection of BHT migration amounts, but requirement of this method to instrument is higher, it is difficult to popularize.
Insulin glargine injecta is insulin analog, and clinic is primarily adapted for use in the diabetes that need to use insulin therapy,
BHT in plug equally exists the possibility moved in insulin glargine injecta, and conventional art can not detect sweet well
BHT in smart regular iletin, it is therefore necessary to be improved for this.
《Today pharmacy》The periodical 05 interim " antioxidant in high performance liquid chromatography detection fat emulsion injection in 2015
The method that BHT in fat emulsion injection is detected using high performance liquid chromatography is described in the texts of BHT " one, this method is to Fat Emulsion
Parenteral solution has preferable Detection results, but needs to carry out complicated pretreatment process before its detection, by this method application
During into insulin glargine injecta, it is impossible to obtain preferable result.It is therefore desirable to in insulin glargine injecta
A kind of new detection method is developed in BHT detections.
The content of the invention
In view of this, it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of simple to operate and can be effective
The method for determining antioxidant BHT in insulin glargine injecta.
In order to solve the above-mentioned technical problem, the present invention is realized using following scheme：
The method of antioxidant BHT, is detected using high performance liquid chromatography in a kind of measure insulin glargine injecta,
And mobile phase is acetonitrile：Water：Phosphate buffer.
The side that " antioxidant BHT in high performance liquid chromatography detection fat emulsion injection " in background technology is mentioned to
Method is applied in insulin glargine injecta, can not be done the trick, and inventor is had found by studying, insulin glargine
Insulin glargine and bacteriostatic agent metacresol in parenteral solution occur chromatographic peak and trailed under the chromatographic condition, so as to shadow
The chromatographic peak of antioxidant BHT is rung, based on this, inventor further has found, before can using liquid-liquid extraction etc. in the prior art
Processing means remove insulin glargine and metacresol from sample, but this pre-treatment means are extremely complex and efficiency is low,
Experimental error is big, and organic reagent dosage is big, and cost is higher.Inventor has used acetonitrile by research and development：Water：Phosphate buffer
It is used as mobile phase, antioxidant BHT chromatographic peak is separated with insulin glargine and metacresol chromatographic peak, so as to accurate to realize
Antioxidant BHT lays the first stone in measure insulin glargine injecta.
For sodium dihydrogen phosphate dihydrate and sodium chloride are dissolved in the water, adjustment pH to 2.5 ~ 4 makes the phosphate buffer
It is standby to form so that in the mobile phase finally mixed, the concentration of sodium dihydrogen phosphate dihydrate is 0.0031 ~ 0.0052g/ml, sodium chloride
Concentration be 0 ~ 0.0032g/ml.By further optimizing the dosage of sodium dihydrogen phosphate dihydrate and sodium chloride, salt is avoided
Separate out, sodium dihydrogen phosphate dihydrate ensure that the pH of mobile phase, insulin glargine and metacresol can be caused to shift to an earlier date appearance, avoid shadow
Ring the chromatographic peak of test substance antioxidant BHT；Sodium chloride can modify BHT peak type so that the parameter at peak is more easy to read.
Further, volume ratio is acetonitrile between mobile phase composition：Water：Phosphate buffer=65 ~ 75:10:15~25.It is logical
The optimization to three's proportioning is crossed, on the premise of three peaks are successfully separated, avoid and separates out salt and cause the damage of instrument.
Further, high performance liquid chromatography also includes following condition：
Chromatographic column：C18 filler chromatographic columns；
Column temperature：30~40℃；
Flow velocity：1~1.5ml/min.
Before sample introduction, insulin glargine injecta sample needs to carry out pre-treatment, specially using methanol or acetonitrile and sweet essence
Regular iletin is mixed.First sample is diluted, it is molten in insulin glargine injecta that antioxidant BHT can be increased
Xie Du, and dilute the concentration of insulin glargine and metacresol.
Compared with prior art, the present invention has the advantages that：
1st, pre-treatment of the present invention is simple, it is only necessary to which with methanol or dilution in acetonitrile sample, the used time is few, organic reagent dosage
It is few, high-efficiency environment friendly, operating error is reduced, result of the test is accurately and reliably；
2nd, the present invention is avoided that insulin glargine, the metacresol even presence of its structure similar substance to antioxidant BHT color
The influence of spectral peak, so as to accurately detect the content of antioxidant BHT.
Brief description of the drawings
Fig. 1 is insulin glargine injecta mark-on chromatogram in embodiment 1；
Fig. 2 is the standard curve of antioxidant BHT in embodiment 1；
Fig. 3 is the sample mark-on chromatogram of comparative example 1；
Fig. 4 is the sample mark-on chromatogram of comparative example 2；
Fig. 5 is the sample mark-on chromatogram of comparative example 3；
Fig. 6 is the sample mark-on chromatogram of comparative example 4.
Embodiment
In order to allow those skilled in the art to more fully understand technical scheme, below in conjunction with the accompanying drawings to the present invention
It is further elaborated.
Embodiment 1
Detection object：Antioxidant BHT；
Detect matrix：Insulin glargine injecta, to be administered to Types of Medicine solution；
Detection method：High performance liquid chromatography, C18 filler chromatographic columns, UV-detector, mobile phase is acetonitrile：Water：Phosphoric acid
Salt buffer, three's volume ratio are 65:10:25,35 DEG C, flow velocity 1ml/min, Detection wavelength 280nm of column temperature；
The phosphate buffer is formulated as follows：Sodium dihydrogen phosphate dihydrate 20.7g is taken, adds water 800ml to dissolve, uses phosphoric acid
PH to 2.5 is adjusted, sodium chloride 12.8g is added, adds water to 1000ml.
1. specificity is tested
5ml insulin glargine injectas are taken to add 1ml BHT standard mother liquors, with methanol constant volume to 10ml, according to above-mentioned chromatogram
Condition enters the μ l of high performance liquid chromatograph 20.As shown in figure 1, the retention time of antioxidant BHT is 23.1min.
2. linear test
Antioxidant BHT control storing solution is diluted to 0.4364,1.091,2.182,5.455,10.91,21.82 μ g/ml,
The μ l of sample introduction 20 respectively, carry out HPLC measure.Each concentration is surveyed 3 times, is averaged.As shown in Fig. 2 linear equation is C=4.846
×10-5A-8.603 × 10-2, R2=0.9996, show that antioxidant BHT is linear good in 0.4364 ~ 21.82 μ g/ml.
3. precision test
Take concentration to compare storing solution for 0.4364 μ g/ml, 2.182 μ g/ml, 10.91 μ g/ml antioxidant BHT, inject liquid
Chromatography, the μ l of sample introduction 20.The pin of continuous sample introduction 3, record chromatographic peak, relative standard's difference of peak area（RSD）Such as following table：
The Precision test result of table 1
4. stability test
Concentration is taken to compare each 20 μ l of storing solution for 0.2182 μ g/ml, 1.091 μ g/ml, 5.455 μ g/ml antioxidant BHT,
Respectively at 0h and 24h injection high performance liquid chromatography, chromatogram is recorded, the relative standard deviation value (RSD) of peak area see the table below.
The stability test result of table 2
5. minimum detection limit and minimum quantitative limit
It is 3 that BHT control storing solutions are constantly diluted into signal to noise ratio, obtains instrument detection and is limited to 0.1 μ g/ml, sample detection limit
For 0.21 μ g/ml.It is 10 that BHT control storing solutions are constantly diluted into signal to noise ratio, obtains instrument quantitative and is limited to 0.3 μ g/ml, sample is fixed
Amount is limited to 0.6 μ g/ml.
6. recovery test in insulin glargine injecta
5ml insulin glargine injectas are taken to add 50 μ l, 0.25ml, 1ml BHT standard mother liquors, with methanol constant volume extremely
10ml.With liquid chromatograph is injected after 0.45 μm of membrane filtration, the μ l of sample introduction 20, chromatogram is recorded, calculate the rate of recovery.
The recovery test result of table 3
To sum up, for insulin glargine injecta after dilution, this method can accurately detect antioxidant BHT.
Embodiment 2
Except column temperature is 40 DEG C, flow velocity 1.5ml/min, acetonitrile：Water：Phosphate buffer=75:10:15, phosphate delays
Fliud flushing is outer using 12.4g sodium dihydrogen phosphate dihydrate and 0g sodium chloride preparation, and other conditions are the same as embodiment 1.
After tested, during linear test, BHT is linear good in 0.4364 ~ 21.82 μ g/ml, R2=0.9997；Precision
During experiment, RSD is between 0.21 ~ 0.76%；During stability test, RSD is between 0.59 ~ 1.54%；Insulin glargine injecta
In middle recovery test, average recovery rate is between 98.12 ~ 109.31%, and RSD is between 0.13 ~ 0.54%.To sum up, the method
Antioxidant BHT can accurately be detected.
Comparative example 1
During except preparing mobile phase, pH to 2.0 is adjusted, taking 2.535 μ g/ml antioxidant BHT to compare outside storing solution sample introduction,
Other chromatographic conditions are with embodiment 1, as shown in figure 3, under the conditions of the mobile phase of pH=2, baseline rises before antioxidant BHT appearance
Substantially.
Comparative example 2
Except mobile phase is methanol：Water=80：20, take insulin glargine injecta to add antioxidant BHT standard mother liquor to anti-
Oxygen agent BHT concentration is 2.7275 μ g/ml, and outside the μ l of sample introduction 20, other chromatographic conditions are with embodiment 1, as shown in figure 4, flowing herein
Under conditions of dynamic phase, the rate of recovery of antioxidant BHT is poor, substantially not appearance.
Comparative example 3
Except mobile phase is acetonitrile：Water：Phosphate buffer=63：12：25, take insulin glargine injecta to add antioxygen
The concentration of agent BHT standards mother liquor to antioxidant BHT is 2.7275 μ g/ml, outside the μ l of sample introduction 20, other chromatographic conditions with embodiment 1,
As shown in figure 5, being matched between changing the component of mobile phase, antioxidant BHT non-appearance, detection time in 50min is long.
Comparative example 4
During except preparing phosphate buffer, dihydrogen phosphate dihydrate is that other chromatographic conditions are the same as embodiment 1 outside 11g.Such as figure
Shown in 6, although successfully BHT chromatographic peaks can be separated with insulin glargine and metacresol, metacresol and insulin glargine peak
Shape is poor, and BHT peak types are asymmetric.
Claims (2)
- A kind of 1. method for determining antioxidant BHT in insulin glargine injecta, it is characterised in that use high performance liquid chromatography Detected, high performance liquid chromatography includes following condition：Detector：UV-detector；Chromatographic column：C18 filler chromatographic columns；Mobile phase is acetonitrile：Water：Phosphate buffer=65 ~ 75:10:15~25；The phosphate buffer is by phosphate dihydrate Sodium dihydrogen and sodium chloride are dissolved in the water, and adjustment pH to 2.5 ~ 4 is prepared so that in the mobile phase finally mixed, two water phosphorus The concentration of acid dihydride sodium is 0.0031 ~ 0.0052g/ml, and the concentration of sodium chloride is 0 ~ 0.0032g/ml；Column temperature：30~40℃；Flow velocity：1~1.5ml/min.
- 2. the method for antioxidant BHT in measure insulin glargine injecta according to claim 1, it is characterised in that sweet Smart regular iletin sample needs to carry out pre-treatment, is specially mixed using methanol or acetonitrile with insulin glargine injecta Close.
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