CN103424496B - Method for determining trace quantity exenatide - Google Patents
Method for determining trace quantity exenatide Download PDFInfo
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- CN103424496B CN103424496B CN201210164281.5A CN201210164281A CN103424496B CN 103424496 B CN103424496 B CN 103424496B CN 201210164281 A CN201210164281 A CN 201210164281A CN 103424496 B CN103424496 B CN 103424496B
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Abstract
The invention relates to a method for determining trace quantity exenatide. Liquid chromatography-mass spectrometry technology is employed to determine trace quantity exenatide in a sample. The method has the advantages of simple operation, accurate result, good singularity and high sensitivity. The invention also relates to the application of the determining method to qualitative or quantitative detection of the exenatide content of a biological sample and to the pharmacokinetics research on an exenatide preparation.
Description
Technical field
The present invention relates to a kind of method adopting liquid chromatography and mass Spectrometry for Determination trace Exenatide.
Background technology
Exenatide is first member of the exendin-4 GLP-1 family of Prof. Du Yucang, secretes the biological pattern of GLP-1 under its simulation human body physiological state, is released into after circulation and can strengthens glucose dependent insulin secretion in intestines.Amino acid sequence and the mankind GLP-1 of Exenatide partly overlap, in vitro display can in conjunction with and activate known mankind GLP-1 acceptor, by comprise cAMP and/or other Cellular Signaling Transduction Mediated mechanism glucose dependent insulin synthesized and beta Cell of islet in vivo excreting insulin increase.When concentration of glucose raises, Exenatide can promote that insulin discharges Exenatide simultaneously and passes through to reduce diabetes B patient empty stomach and postprandial blood sugar concentration from β cell, thus improves glycemic control.
The product that Exenatide has gone on the market mainly contains injection, only for hypodermic injection.Recommend initial dose to be 5 μ g, every day twice, treat after 1 month, according to clinical response, dosage can be increased to 10 μ g.The clinical common adverse events of Exenatide for feeling sick, hypoglycemia, diarrhoea, vomiting, headache and uneasy sense, occurrence frequency is high in patient for treatment and when increasing dosage.Can find out that the dosage of Exenatide has Close relation to clinical adverse, therefore should closely monitor its blood plasma level during clinical treatment.The clinical extremely low consumption of Exenatide, just may cause extremely strong clinical adverse, these situations are all a kind of simple to operate in the urgent need to setting up, good and the high-sensitive trace Exenatide detection technique of specificity, in the biological samples such as current blood plasma, the detection of trace Exenatide utilizes radioimmunology (RIA) or enzyme linked immunological (ELISA) to measure, RIA needs to possess most advanced and sophisticated complicated equipment, and cost is not low yet.Meanwhile, RIA also needs special preventive measure, because it will use radiomaterial.The ELISA method range of linearity is narrow, needs a large amount of Sample Dilution work, and the sample preparation cycle long (at least 6h), require high to human users, kit cost is high simultaneously, and condition of storage is harsh, the problems such as stabilization of kit.LC-MS/MS improves along with technology, has started to be applied to polypeptide and has detected, document " Application of DBS for quantitative assessment of thepeptide Exendin-4; Comparison of plasma and DBS method by UHPLC-MS/MS "--Bioanalysis., 2010,2 (8), 1461-1464, mention and adopt liquid chromatography and mass spectrometric hyphenated technique, detect the plasma concentration of Exenatide, LLOQ and lower limit of quantitation (minimum quantitative limit) are 10ng, but still can not meet the needs that clinical and experimental study detects trace Exenatide.
Summary of the invention
For prior art exist defect, the present inventor furthers investigate, provide a kind of simple to operate, result is accurate, specificity good, the assay method of highly sensitive trace Exenatide.Described method adopts liquid chromatography and mass spectrometric hyphenated technique, and the mobile phase of its efficient liquid phase adopts and carries out gradient elution containing aqueous acid-methanol system, and described acid is formic acid or acetic acid, is preferably formic acid; The concentration expressed in percentage by volume 0.01-2% of acid, is preferably 0.05-0.5%, is more preferably 0.2%.Described concentration expressed in percentage by volume is volume (ml) number of contained acid in 100ml water.
In LC-MS method, the selection of liquid chromatogram mobile phase has very important effect.The organic phase of mobile phase of the present invention selects methyl alcohol, compares other organic solvents such as acetonitrile, and methyl alcohol has higher response and good chromatographic behavior when measuring trace Exenatide; Add the sensitivity that acid can significantly improve method in the aqueous phase of mobile phase, wherein add formic acid, acetic acid has certain advantage than other acid, preferable formic acid; Adopt the trace Exenatide in detection method examination criteria solution example, detectability can reach 0.01ng/ml, and detect the Exenatide in plasma sample, minimum quantitative limit can reach 0.1ng/ml.
Detectability described herein means the minimum flow that in sample, measured object can be detected.Conventional signal to noise ratio (S/N ratio) method, the signal that the signal namely known low concentrations sample measured and blank sample are measured compares, and calculates the least concentration or amount that can be reliably detected.When being generally 3: 1 or 2: 1 with signal to noise ratio (S/N ratio), the amount of respective concentration or injection instrument determines detectability.
LLOQ described herein is lower limit of quantification, i.e. lower limit of quantitation (minimum quantitative limit).FDA specifies the S/N > 5 of LLOQ point and the CV < 20%, S/N of LLOQ point and signal to noise ratio (S/N ratio), and CV is the coefficient of variation, and it equals standard deviation/mean value.
Described gradient elution contains the volume ratio table specific as follows of aqueous acid and methyl alcohol:
The method that the present invention also relates on the other hand described mensuration trace Exenatide is for detecting the mammal content of Exenatide and purposes of change procedure thereof in body after use Exenatide formulation.Successfully can be detected the trace Exenatide in sample by method of the present invention, realize, after use Exenatide formulation, detecting content and the change procedure thereof of Exenatide in its body.
The method of the invention is as a kind of trace Exenatide LC-MS determination method, there is the sensitivity and specificity that are better than existing detection technique, can be used for the detection of trace Exenatide in sample, for the detection of trace Exenatide sample, determine that Exenatide formulation drug metabolism study in vivo provides effective detection means.The method of the invention can be used for the exploitation of Exenatide formulation, measuring the change using Exenatide patient vivo medicine concentration clinically, can provide experimental basis by its dynamic change of Continuous Observation for instructing the reasonable employment of clinical Exenatide.
Accompanying drawing explanation
Figure 10 .01ng/mL Exenatide standard solution is the liquid quality detection collection of illustrative plates of 0.2% aqueous formic acid-methyl alcohol at mobile phase
Figure 20 .1ng/mL Exenatide plasma sample is the liquid quality detection collection of illustrative plates of 0.2% aqueous formic acid-methyl alcohol at mobile phase
The average Drug-time curve collection of illustrative plates of Fig. 3 Exenatide injection in monkey blood plasma
Embodiment
Below by embodiment to the present invention's further instruction in addition, but do not limit the present invention in any form.
Embodiment 1 mobile phase is that 0.2% aqueous formic acid-methyl alcohol detects Exenatide
The preparation precision of standard serial solution sample takes 25mg Exenatide standard items, puts in 25ml volumetric flask, adds methyl alcohol (containing 0.1% formic acid) solubilize, and is diluted to scale, be mixed with the storing solution of 1mg/ml.Get Exenatide storing solution appropriate, use methyl alcohol respectively: water: formic acid (90: 10: 0.1) is diluted to 0.01,0.03,0.1,0.3,1.0,3.0,10.0,30.0ng/ml.
Liquid-phase condition chromatographic column: ZORBAX RRHD Eclipse Plus C
18(1.8 μm, 50 × 2.1mm).Mobile phase: A:0.2% aqueous formic acid, B: methyl alcohol; Flow velocity: 0.5mL/min, column temperature: 40 DEG C, sample size: 10 μ l.Agilent1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 90 | 10 |
| 1.5 | 90 | 10 |
| 2.5 | 10 | 90 |
| 3.0 | 10 | 90 |
| 3.1 | 90 | 10 |
| 5.0 | 90 | 10 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:575 DEG C; Injection electric: 5500V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 1047.4 → 396.3 and m/z1090.7 → 650.3; Separating bunch voltage DP voltage is 240V, 100V, and collision energy CE is respectively 42eV and 48eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.01ng/ml sample, and detection response is 62.5cps, signal to noise ratio (S/N ratio) 5.9, and chromatogram is shown in accompanying drawing 1.
Embodiment 2 mobile phase is that 0.1% aqueous formic acid-methyl alcohol detects Exenatide
The preparation of standard serial solution sample is with embodiment 1
Liquid-phase condition chromatographic column: ZORBAX RRHD Eclipse Plus C
18(1.8 μm, 50 × 2.1mm).Mobile phase: A:0.1% aqueous formic acid, B: methyl alcohol; Flow velocity: 0.5mL/min, column temperature: 40 DEG C, sample size: 10 μ l.Agilent 1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 80 | 20 |
| 1.8 | 80 | 20 |
| 2.5 | 5 | 95 |
| 3.0 | 5 | 95 |
| 3.1 | 80 | 20 |
| 5.0 | 80 | 20 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:575 DEG C; Injection electric: 5500V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 1047.4 → 396.3 and m/z1090.7 → 650.3; Separating bunch voltage DP voltage is 240V, 100V, and collision energy CE is respectively 42eV and 48eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.03ng/ml sample, and detection response is 97cps, and signal to noise ratio (S/N ratio) is 4.1.
Embodiment 3 mobile phase is that 0.5% aqueous formic acid-methyl alcohol detects Exenatide
The preparation of standard serial solution sample is with embodiment 1
Liquid-phase condition chromatographic column: ZORBAX RRHD Eclipse Plus C
18(1.8 μm, 50 × 2.1mm).Mobile phase: A:0.5% aqueous formic acid, B: methyl alcohol; Flow velocity: 0.5mL/min, column temperature: 40 DEG C, sample size: 10 μ l.Agilent 1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 70 | 30 |
| 1.5 | 70 | 30 |
| 2.5 | 20 | 80 |
| 3.5 | 20 | 80 |
| 3.6 | 70 | 30 |
| 5.5 | 70 | 30 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:575 DEG C; Injection electric: 5500V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 1047.4 → 396.3 and m/z 1090.7 → 650.3; Separating bunch voltage DP voltage is 240V, 100V, and collision energy CE is respectively 42eV and 48eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.03ng/ml sample, and detection response is 106cps, and signal to noise ratio (S/N ratio) is 4.7.
Embodiment 4 mobile phase is that 0.2% aqueous formic acid-methyl alcohol detects Exenatide
The preparation of standard serial solution sample is with embodiment 1
Liquid-phase condition chromatographic column: ZORBAX RRHD Eclipse Plus C
18(1.8 μm, 50 × 2.1mm).Mobile phase: A:0.2% aqueous formic acid, B: methyl alcohol; Flow velocity: 0.5mL/min, column temperature: 40 DEG C, sample size: 10 μ l.Agilent 1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 60 | 40 |
| 2 | 60 | 40 |
| 2.5 | 25 | 75 |
| 3.5 | 25 | 75 |
| 3.6 | 60 | 40 |
| 5.5 | 60 | 40 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:575 DEG C; Injection electric: 5500V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 1047.4 → 396.3 and m/z1090.7 → 650.3; Separating bunch voltage DP voltage is 240V, 100V, and collision energy CE is respectively 42eV and 48eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.03ng/ml sample, and detection response is 98cps, signal to noise ratio (S/N ratio) 3.8.
Embodiment 5 mobile phase is that 0.01% aqueous formic acid-methyl alcohol detects Exenatide
Standard serial solution sample, compound method with embodiment 1,
Liquid chromatogram mobile phase: 0.01% aqueous formic acid A-methyl alcohol B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.1ng/ml sample, and detection response is 117cps, signal to noise ratio (S/N ratio) 4.2.
Embodiment 6 mobile phase is that 2% aqueous formic acid-methyl alcohol detects Exenatide
Standard serial solution sample, compound method with embodiment 1,
Liquid chromatogram mobile phase: 2% aqueous formic acid A-methyl alcohol B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.03ng/ml sample, and detection response is 56cps, signal to noise ratio (S/N ratio) 2.9.
Embodiment 7 mobile phase is that 0.2% acetic acid aqueous solution-methyl alcohol detects Exenatide
Standard serial solution sample, compound method with embodiment 1,
Liquid chromatogram mobile phase: 0.2% acetic acid aqueous solution A-methyl alcohol B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.3ng/ml sample, and detection response is 112cps, signal to noise ratio (S/N ratio) 3.8.
Embodiment 8 mobile phase is that 0.2% aqueous formic acid-acetonitrile detects Exenatide
Standard serial solution sample, compound method with embodiment 1,
Liquid chromatogram mobile phase: 0.2% aqueous formic acid A-acetonitrile B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.3ng/ml sample, and detection response is 89cps, signal to noise ratio (S/N ratio) 5.2.
Embodiment 9 mobile phase is that 0.2% fluoroform aqueous acid-methyl alcohol detects Exenatide
Standard serial solution sample, increases concentration 0.5ng/ml sample simultaneously, compound method with embodiment 1,
Liquid chromatogram mobile phase: 0.2% fluoroform aqueous acid A-methyl alcohol B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.5ng/ml sample, and detection response is 52cps, signal to noise ratio (S/N ratio) 5.9.
Test example 1 detects Exenatide in monkey blood plasma
Trial drug: Exenatide injection (
baxter Pharmaceutical Solutions LLC, Indiana, USA, lot number: A812880)
Experimental animal: machin 3, body weight is at 3 ± 0.5kg, male
Test method: 3 male machin subcutaneous administrations, dosage 5 μ g/ is only; Respectively at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h and 24h blood sampling after (0 hour) before administration and administration.
Plasma sample process: in 50 μ L monkey blood+50 μ L, mark (8.5ng/mL bivalirudin)+50 μ L complement amasss, and vortex 30s, 15000g*5min is centrifugal, gets MCX post on supernatant.MCX post methyl alcohol 1mL and water 500 μ L balances; First use 0.5% formic acid water 500 μ L wash-out, discard; Use again, methyl alcohol: 2% formic acid water (96: 4) 1mL wash-out, discards; Collection liquid is acetonitrile: methyl alcohol: water: 25% ammoniacal liquor (4: 1: 1: 1.0) 200 μ L wash-out twice (totally 400 μ L), collects in 10mL plastic centrifuge tube; Add acetonitrile in the most backward collection liquid: methyl alcohol: water: formic acid (6: 5: 1: 0.1) 200 μ L, after mixing, nitrogen 50 DEG C dries up, 50 μ L methyl alcohol: water: formic acid (90: 10: 0.1) redissolves, sample introduction 10 μ L.
The preparation of typical curve: get the eppendorf pipe that 50 μ L monkey blank plasmas are placed in 1.5mL, be configured to respectively be equivalent to 0.1,0.3,1, the plasma solutions of 3,5,10,30ng/mL, add mark in 50 μ L, 50 μ L complements amass, and all the other operations operate by under " plasma sample process " item.
Liquid phase-mass spectrum Series detectors condition is with embodiment 1, and it is minimum is quantitatively limited to 0.1ng/ml
Test findings: sample concentration is 0.1ng/ml, detection response is 290cps, signal to noise ratio (S/N ratio) 13.5, and accompanying drawing 2 is shown in by testing result collection of illustrative plates.
Accompanying drawing 3 is shown in by pharmacokinetics collection of illustrative plates
Result shows: detection method of the present invention, and after use Exenatide formulation, in the whole medication cycle, detect trace Exenatide in blood plasma and have better response, data meet the requirement of LLOQ.
Claims (5)
1. one kind measures the method for trace Exenatide, it is characterized in that method adopts liquid chromatography and mass spectrometric hyphenated technique, the mobile phase of efficient liquid phase adopts and carries out gradient elution containing aqueous acid-methanol system, and described acid is formic acid or acetic acid, and the concentration expressed in percentage by volume of acid is 0.01-2%; The condition that described mass spectrum adopts is ion gun: electron spray ionisation source, CAD:9, gas curtain atmospheric pressure: 35psi, GS1:55, GS2:55, TEM:575 DEG C, injection electric: 5500V, and positive ion mode detects, and scan mode is multiple-reaction monitoring; Described liquid-phase condition: chromatographic column is C
181.8 μm, post, 50 × 2.1mm, flow velocity: 0.5mL/min, column temperature: 40 DEG C; The gradient elution of efficient liquid phase is the volume ratio that first time period contains aqueous acid and methyl alcohol is 60-95: 40-5; Second time period was 40-5: 60-95 containing the volume ratio of aqueous acid and methyl alcohol; 3rd time period was 60-95: 40-5 containing aqueous acid and methyl alcohol volume ratio; Described first time period is 1.5-2.0 minute, and described second time period is 0.5-1 minute, and described 3rd time period is 1.9 minutes; Whole detection time is less than 5.5 minutes.
2. method according to claim 1, is characterized in that the volume ratio that first time period contains aqueous acid and methyl alcohol is 90:10; Second time period was 10: 90 containing the volume ratio of aqueous acid and methyl alcohol; 3rd time period was 90:10 containing the volume ratio of aqueous acid and methyl alcohol.
3. method according to claim 1, is characterized in that the concentration expressed in percentage by volume of acid is 0.05%-0.5%.
4. method according to claim 3, is characterized in that the concentration expressed in percentage by volume of acid is 0.2%.
5., according to the arbitrary described method of claim 1-4, it is characterized in that acid is for formic acid.
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Address after: 264003 Yantai hi tech Zone, Shandong venture Road No. 15 Patentee after: Shandong Luye Pharma Co., Ltd. Address before: 264003 Laishan, Shandong Province Bao Road, No. 9 District, No. Patentee before: Shandong Luye Pharma Co., Ltd. |
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Inventor after: Sha Chunjie Inventor after: Wen Xinjun Inventor after: Dai Xinru Inventor after: Zhang Jinfeng Inventor after: Sun Yu Inventor after: Han Jiangbin Inventor after: Wang Tao Inventor after: Liu Wanhui Inventor before: Sha Chunjie Inventor before: Zhang Jinfeng Inventor before: Sun Yu Inventor before: Han Jiangbin Inventor before: Wang Tao Inventor before: Liu Wanhui |