A method for detecting calopivir in plasma by HPLC-MS / MS

Abstract

The application discloses a method for detecting calopivir in blood plasma by HPLC-MS / MS. The method is pretreated by a protein precipitation method, and chromatographic conditions are as follows: a Waters CORTECS C8 chromatographic column is used, 2 mM ammonium acetate containing 0.01% formic acid in water and acetonitrile are used as mobile phases, and gradient elution is carried out; mass spectrometry conditions are as follows: an electrospray ionization source (ESI) tandem mass spectrometer is used, and verapamil is used as an internal standard for quantitative detection. The method is simple in operation, fast in analysis speed, high in sensitivity, suitable in linear range, and meets the requirements of large quantities of sample analysis in preclinical and clinical research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for detecting coblopasvir in plasma by HPLC-MS / MS. Background Technology

[0002] Colopavir hydrochloride tablets (research code KW-136, trade name Kailiwei) ® This is an NS5A inhibitor that inhibits hepatitis C virus replication by targeting and binding to the non-structural protein NS5A. It was developed by Beijing Kaiyin Technology Co., Ltd. This product, used in combination with sofosbuvir, is used to treat treatment-naïve or interferon-treated adults with genotypes 1, 2, 3, and 6 of chronic hepatitis C virus (HCV), with or without compensated cirrhosis.

[0003] Therapeutic drug monitoring and generic drug clinical studies require the measurement of blood drug concentrations in subjects or patients to study their pharmacokinetic behavior. To accelerate the clinical application of clopidogrel, a simple, accurate, rapid, and highly sensitive bioanalytical method is needed. Summary of the Invention

[0004] To date, no relevant literature or patents have reported a method for detecting coloprazor in animal and human plasma using HPLC-MS / MS. Given this gap in current analytical methods, this invention develops an analytical method with simple pretreatment procedures that balances sensitivity, accuracy, and analysis speed, suitable for accurate analysis of large batches of samples in preclinical and clinical studies. This analytical method is novel.

[0005] The purpose of this invention is to provide a method for detecting colopamivir in plasma using HPLC-MS / MS.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for detecting clotrimazole in plasma using HPLC-MS / MS includes the following steps:

[0008] (1) Pretreatment: Add acetonitrile to the plasma sample to precipitate the protein, centrifuge, take the supernatant, dilute with water to obtain the test sample;

[0009] (2) The content of clotrimazole in the pretreated plasma samples was determined by HPLC-MS / MS using the internal standard quantification method, wherein:

[0010] Chromatographic conditions:

[0011] Chromatographic column: Waters CORTECS C8 column;

[0012] Mobile phase A: 2 mM ammonium acetate aqueous solution containing 0.01 vol% formic acid; Mobile phase B: acetonitrile.

[0013] Gradient elution program: 0-0.3 min: mobile phase B 50 vol%; 0.3-2.6 min: mobile phase B 80 vol%; 2.6-3.0 min: mobile phase B 50 vol%

[0014] The mobile phase flow rate was 0.55 mL / min;

[0015] The injection volume was 5.00 µL;

[0016] Column temperature: 40℃;

[0017] Mass spectrometry conditions:

[0018] Ion source: electrospray ionization source; positive ion scanning; multiple reaction monitoring; jet voltage: 5500 V; ion source temperature: 550℃; Gas 1 (Gas1): 55 psi, Gas 2 (Gas2): 55 psi, Curtain Gas: 35 psi, collision-induced dissociation: 6 psi, residence time: 250 ms; Colopevir quantitative analysis ion pair: m / z 783.5 → 609.2, collision energy: 56 eV, declustering voltage: 100 V; Verapamil quantitative analysis ion pair: 455.2 → 164.9, collision energy: 30 eV, declustering voltage: 100 V.

[0019] In some embodiments, an internal standard solution is added to the plasma sample, and the sample is processed to obtain a test sample containing the internal standard substance; the internal standard solution is obtained by dissolving verapamil in methanol and then diluting it with acetonitrile-water (50:50, v / v). In some embodiments, the amount of internal standard solution added is 0.5-2 times the volume of the plasma sample, preferably an equal volume.

[0020] In some embodiments, in step (1), the volume of the plasma sample is 10-50 µL, and the amount taken is determined according to the sample volume. In some embodiments, the content of colopidine in the plasma sample is 2.00-5000 ng / mL.

[0021] In some embodiments, in step (1), the content of the internal standard substance in the test sample is 100-400 ng / mL, preferably 200 ng / mL.

[0022] In some embodiments, in step (1), the volume ratio of plasma sample to acetonitrile is 1:(5-20), preferably 1:10.

[0023] In some embodiments, in step (1), the supernatant obtained after centrifugation is diluted with water, which makes the sample less volatile and is beneficial for sample preservation and detection accuracy; the amount of water added is 0.5-2 times the volume of the supernatant, preferably the amount of water added is equal to the volume of the supernatant.

[0024] The beneficial effects of this invention are:

[0025] 1. The method of the present invention has the advantages of simple pretreatment operation, requiring only one extraction and one dilution step for analysis, and the extracted sample is easy to preserve; moreover, the method has a fast analysis speed (analysis time is only 3.00 min), which is suitable for large-scale research sample analysis.

[0026] 2. In preclinical or clinical studies, improved sensitivity of the detection method can reduce the dosage required by subjects and increase the safety of clinical research. The method described in this invention has high sensitivity, low plasma dosage (as low as 10.0 µL), a lower limit of quantification of colopamivir of 2.00 ng / mL, and a calculated limit of detection of 0.500 ng / mL.

[0027] 3. The linear range of this invention is reasonably selected (linear range is 2.00-5000 ng / mL), which can more accurately determine the concentration of low-content drugs.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a mass spectrometry scan of fragment ions for coloprazor.

[0031] Figure 2 This is an ion chromatogram of colopamivir and internal standard verapamil in the lower limit of plasma quantification (2.00 ng / mL) of ICR mice (top: colopamivir, bottom: verapamil).

[0032] Figure 3 This is an ion chromatogram of colopamivir and internal standard verapamil in the upper limit of plasma quantification (5000 ng / mL) of ICR mice (top: colopamivir, bottom: verapamil).

[0033] Figure 4 This is an ion chromatogram of colopamivir and internal standard verapamil after ICR mice were administered 10 mg / kg colopamivir hydrochloride by gavage for 0.5 h (top: colopamivir, bottom: verapamil).

[0034] Figure 5 This is the blood concentration versus time curve of 10 mg / kg colopex hydrochloride administered by gavage to three ICR mice. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0036] The development of methods for detecting drug concentrations in plasma using liquid chromatography-tandem mass spectrometry (LC-MS / MS) typically involves three parts: extraction methods (i.e., pretreatment methods), liquid chromatography methods, and mass spectrometry methods. This invention addresses the shortcomings of existing technologies by establishing analytical methods from these three aspects.

[0037] The method of this invention allows for plasma sample volumes as low as 10.0 µL, making it suitable for preclinical and clinical bioanalysis. The pretreatment method used in this invention is protein precipitation, which exhibits good reproducibility for coloprazepam and is simple to operate, with short extraction time and no time-consuming concentration step. When used with a 96-well plate, it is suitable for high-throughput sample pretreatment in clinical studies.

[0038] Colopevir has a molecular weight of 782 Da and a long-chain linear chemical structure, which easily leads to residue and tailing in liquid chromatography systems, making it unsuitable for establishing rapid, high-throughput liquid chromatography-mass spectrometry (LC-MS) detection methods. Attempts to use columns such as ZORBAX Eclipse XDB-C18, Luna PFP(2), and Gemini C18 failed to obtain sharp, symmetrical peaks; and residue and peak tailing were severe under neutral mobile phase conditions. Therefore, in this embodiment of the invention, a Waters CORTECS C8 column was used for chromatographic separation. This column has good retention of both the analyte and the internal standard, and the peaks are sharp. The mobile phase used in the chromatographic analysis is a 2 mM ammonium acetate aqueous solution containing 0.01% formic acid, which helps maintain pH stability. The added ammonium acetate and formic acid act as modifiers to improve peak shape and increase integration accuracy. In this mode, the instrument has a high throughput, with a chromatographic run time of only 3.00 min, making it suitable for large-scale sample analysis in clinical research.

[0039] In addition, the mass spectrometry conditions were optimized for the detection sensitivity of the compounds. Other methods generally require a plasma volume of 50 µL or more, while in this study, the plasma sample volume can be as low as 10.0 µL, which greatly saves plasma volume and is applicable to situations where only a small amount of blood can be collected, such as in model animals (e.g., pharmacodynamic model mice and rats).

[0040] Abbreviation Explanation

[0041]

[0042] Example 1

[0043] 1. Materials

[0044] 1.1 Instruments

[0045] Chromatograph: LC-30AD rapid liquid chromatography system, Shimadzu Corporation, Japan.

[0046] Mass spectrometer: Model 5500 triple quadrupole tandem mass spectrometer, equipped with an electrospray ionization (ESI) source, Sciex, Canada.

[0047] Data processing was performed using Analyst (version 1.6.3), Sciex Inc., Canada.

[0048] Centrifuge: TGL-16RC benchtop centrifuge, Shandong Baiou Medical Technology Co., Ltd.

[0049] Analytical balance: BCE55I-10CN analytical balance, Beijing Sartorius Instruments Co., Ltd.

[0050] 1.2 Reference Standards and Reagents

[0051] Colopevir hydrochloride was purchased from Maclean's. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich (USA). Ammonium acetate (HPLC grade) was purchased from ROE. Distilled water was prepared by Watson's.

[0052] 2 methods

[0053] 2.1 Preparation of solutions and samples

[0054] Standard series samples: Accurately weigh appropriate amounts of each colopamivir reference standard, dissolve and dilute to volume with methanol to prepare stock solutions of approximately 1.00 mg / mL colopamivir. Accurately pipette appropriate amounts of each stock solution and serially dilute with human blank plasma to obtain a mixed standard series sample, with colopamivir concentrations ranging from 2.00 to 5000 ng / mL.

[0055] Quality control samples: Three concentration levels of colopamivir were prepared using a method similar to that used for the standard series samples. The lower limit of quantitation (LQC) was 2.00 ng / mL, the low quality control (LQC) concentration was 6.00 ng / mL, the medium quality control (MQC) concentration was 100 ng / mL, the high-medium quality control (AMQC) concentration was 1500 ng / mL, and the high quality control (HQC) concentration was 4000 ng / mL.

[0056] Internal standard solution: Accurately weigh verapamil reference standard, dissolve and dilute to volume with methanol to prepare an internal standard stock solution with a concentration of approximately 1.00 mg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution and dilute with acetonitrile:water (50:50, v / v) to obtain an internal standard solution with a verapamil concentration of 200 ng / mL.

[0057] 2.2 Plasma Sample Processing

[0058] 1) Add 10.0 µL of plasma sample, 20.0 µL of internal standard solution (verapamil concentration of 200 ng / mL), and 200 µL of acetonitrile to a 96-well plate;

[0059] 2) Vortex mix and centrifuge for 10 min (4℃, 3900 rpm);

[0060] 3) Transfer 150 µL of the supernatant to another clean 96-well plate;

[0061] 4) Add 150 µL of water and mix thoroughly by vortexing.

[0062] 2.3 Chromatographic and Mass Spectrometric Conditions

[0063] Chromatographic conditions:

[0064]

[0065] Mass spectrometry conditions:

[0066]

[0067] 2.4 Methodological Validation

[0068] Methodological validation of this method was conducted in accordance with the guidelines of Chinese Pharmacopoeia 9012, including stability, selectivity, linearity, accuracy, precision, and recovery.

[0069] Selectivity:

[0070] Blank plasma and separately prepared samples for the lower limit of quantitation (LOQ) were processed and then injected for analysis. The peak area of ​​the chromatographically eluted interfering substances must be less than 20% of the peak area of ​​the analyte at the LOQ and less than 5% of the peak area of ​​the internal standard.

[0071] Standard curve:

[0072] The linear regression equation (weighting factor W=1 / x) is calculated by using the theoretical concentration of the analyte as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis. 2 The method was validated by performing two-sample analysis on each analytical batch of the standard curve samples.

[0073] Precision and accuracy:

[0074] Six samples from each of the four concentration quality control samples were measured. For the limit of quantitation (LOQ), intra-batch precision (calculated as relative standard deviation (RSD)) is acceptable if it is less than 20%, and accuracy (calculated as relative deviation (RE)) is acceptable if it is between -20% and 20%. For the remaining QC samples at all concentration levels, intra-batch and inter-batch precision for each component must be less than 15%, and accuracy must be acceptable if it is between -15% and 15%.

[0075] stability:

[0076] To investigate the stability of each analyte in plasma samples, LQC and HQC were placed in room temperature and ultra-low temperature (below -60°C) environments, and six samples were analyzed after the incubation period. Short-term stability at room temperature for 21 hours was also assessed.

[0077] Recovery rate:

[0078] Take 10.0 μL of blank plasma, extract it (without adding internal standard solution), add the analyte solution and internal standard solution to make the final concentration the same as LQC and HQC, and then inject for analysis. Separately extract 6 aliquots of LQC and HQC, and then inject for analysis. Calculate the extraction recovery rate based on the peak area ratio of the two processing methods.

[0079] 2.5 Pharmacokinetic Studies

[0080] The concentration of clopipvir in plasma samples was analyzed using the method established above for pharmacokinetic studies of clopipvir hydrochloride in mice. Three ICR mice were administered 10 mg / kg clopipvir hydrochloride by gavage at a volume of 10 ml / kg, with DMSO / solutol / Saline (5 / 5 / 90, v / v / v) as the solvent. At different time points before administration (0 h) and within 24 h after administration, 30 μL of venous blood was collected and placed in K2EDTA anticoagulant centrifuge tubes. After centrifugation (8000 g, 4℃) for 5 min, the plasma was separated and stored at -60℃ or below.

[0081] 3 Results and Discussion

[0082] 3.1 Methodological Validation

[0083] Ion confirmation

[0084] like Figure 1 As shown, the ion pair for quantitative analysis of colopidine was confirmed: m / z 783.5 → 609.2.

[0085] Selective

[0086] like Figure 2 ~ Figure 4 As shown, the retention times of colopride and verapamil were approximately 2.45 and 2.56 min, respectively, with no co-eluent interference peaks at the retention times.

[0087] Standard curve

[0088] The linear range for determining colopamivir in plasma samples from clinical studies was 2.00–5000 ng / mL. The typical linear regression equations for the analyte standard curves are as follows:

[0089] Colopevir: y = 0.000101x + 0.0000131 (r = 0.9979);

[0090] Detection limit

[0091] The lower limit of quantitation (LOQ) of colopavir in the sample was 2.00 ng / mL, with a signal-to-noise ratio (SNR) of approximately 20. Based on an SNR of 5, the limit of detection (LOD) was calculated to be 0.500 ng / mL.

[0092] Precision and accuracy

[0093] The precision and accuracy results both met the acceptance criteria, as shown in Table 1.

[0094] Table 1. Precision and accuracy of determining colopavir in mouse plasma

[0095]

[0096] Recovery rate

[0097] LQC and HQC concentration levels: The extraction recoveries of coloprazor were 96.6% and 96.9%, respectively; the recoveries of verapamil were 99.3%, respectively.

[0098] Plasma stability study

[0099] The plasma stability samples of colopamivir at low (6.00 ng / mL) and high (4000 ng / mL) concentrations were prepared, and the differences from the theoretical values ​​were tested after being placed at room temperature for 21 h and at ultra-low temperature (below -60℃). The results show that colopamivir is stable in plasma after being placed at room temperature for 21 h and at ultra-low temperature (below -60℃) for 40 days.

[0100] Table 2. Stability of clotrimazole in mouse plasma at room temperature for 21 hours and at ultra-low temperature for 40 days.

[0101]

[0102] 4. Pharmacokinetic Studies

[0103] The validated method was used to analyze clopavir in plasma to evaluate its pharmacokinetic characteristics. Three ICR mice were administered 10 mg / kg clopavir hydrochloride by gavage. Plasma drug concentration-time curves are shown below. Figure 5 The detection method has high sensitivity and can fully depict the pharmacokinetic characteristics of clopidogrel. Furthermore, the linear range is close to the concentration level of the actual sample, resulting in high accuracy.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting colopamivir in plasma using HPLC-MS / MS, comprising the following steps: (1) Pretreatment: Add internal standard solution to plasma sample, add acetonitrile to plasma sample to precipitate protein, centrifuge and take supernatant, dilute with water to obtain test sample, wherein the internal standard solution is verapamil dissolved in methanol and diluted with acetonitrile-water, 50:50, v / v, the amount of internal standard solution added is 0.5-2 times that of plasma sample, the volume of plasma sample is 10-50 µL; the content of colopidine in plasma sample is 2.00-5000 ng / mL, and the content of internal standard substance in test sample is 100-400 ng / mL; (2) The content of clotrimazole in the pretreated plasma samples was determined by HPLC-MS / MS using the internal standard quantification method, wherein: Chromatographic conditions: Column: Waters CORTECS C8 column; Mobile phase A: 2 mM ammonium acetate aqueous solution containing 0.01 vol% formic acid; Mobile phase B: acetonitrile. Gradient elution program: 0-0.3 min: mobile phase B 50 vol%; 0.3-2.6 min: mobile phase B 80 vol%; 2.6-3.0 min: mobile phase B 50 vol% The mobile phase flow rate was 0.55 mL / min; The injection volume was 5.00 µL; Column temperature: 40℃; Mass spectrometry conditions: Ion source: electrospray ionization source; positive ion scanning; multiple reaction monitoring; jet voltage: 5500 V; ion source temperature: 550℃; gas 1: 55 psi, gas 2: 55 psi, curtain gas: 35 psi, collision-induced dissociation: 6 psi, residence time: 250 ms; clopidogrel quantitative analysis ion pair: m / z 783.5 → 609.2, collision energy: 56 eV, declustering voltage: 100 V; verapamil quantitative analysis ion pair: 455.2 → 164.9, collision energy: 30 eV, declustering voltage: 100 V.

2. The method for detecting clotrimazole in plasma by HPLC-MS / MS according to claim 1, characterized in that, In step (1), the volume ratio of plasma sample to acetonitrile is 1:(5-20).

3. The method for detecting clotrimazole in plasma by HPLC-MS / MS according to claim 1, characterized in that, In step (1), the amount of water added during the dilution process is 0.5-2 times the volume of the supernatant.

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