Method for determining concentration of indobufen in human plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology

Abstract

The invention provides a method for determining the concentration of indobufen in human plasma based on a liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology. The specific operation of the method comprises the following steps: adding an internal standard solution and an organic solvent into a plasma sample, and ensuring full extraction of indobufen through vortex mixing. And separating by using a centrifugal technology, and collecting supernate as a pretreated sample to be detected. An Eclipse Plus C18 chromatographic column is adopted for isocratic elution, effective separation of indobufen is achieved, and then a sample is subjected to mass spectrometric detection. And drawing a standard curve based on the mass spectrum peak area ratio, obtaining a regression equation, and calculating to obtain the concentration of the indobufen in the sample to be detected. The method has the characteristics of simplicity and convenience in pretreatment operation, low plasma consumption and short analysis time, and is suitable for detection of indobufen in plasma and clinical pharmacokinetic research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for determining the concentration of indobufen, an antiplatelet aggregation drug, in human plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology. Background Art

[0002] In the treatment of cardiovascular and cerebrovascular diseases, aspirin, as a classic antiplatelet drug, is widely used in clinical practice. However, long-term use of aspirin also causes a series of adverse reactions. At the same time, some patients are allergic or intolerant to aspirin, which often leads to the need for desensitization therapy or exploration of alternative treatment options to maintain the patient's treatment needs and safety.

[0003] Indobufen is an isoindolinylphenylbutyric acid derivative, a nonsteroidal anti-inflammatory drug, with a chemical name of 2-[4-(1-oxo-2-isoindolyl)-phenyl]butyric acid. It was developed by Farmfalia Carlo Erba in Italy and first marketed in Italy, and later in my country. It is currently the only reversible selective multi-target antithrombotic drug that reversibly inhibits platelet cyclooxygenase (COX) and reduces the production of thromboxane A2 (TXA2), thereby blocking platelet aggregation, and has less gastrointestinal reactions and a lower risk of bleeding. Indobufen is recommended as an alternative treatment for patients with aspirin intolerance who have a high risk of bleeding and gastric ulcers.

[0004] In order to advance the research and development of indobufen generic drugs, it is necessary to monitor its concentration level in human plasma to obtain the pharmacokinetic characteristics and then evaluate the bioequivalence of the original drug and the generic drug.

[0005] The analytical methods for indobufen in plasma currently reported in the literature mostly use chiral separation methods, with an analysis time of up to 20 minutes per injection, and mainly rely on liquid chromatography-ultraviolet detection (LC-UV) technology, which has relatively poor selectivity and is easily interfered by endogenous substances in plasma.

[0006] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology combines the advantages of liquid chromatography's efficient separation performance with tandem mass spectrometry's high sensitivity and high specificity, and has now become the most recommended quantitative analysis technology in the biopharmaceutical field. To date, there are no relevant literature or patent reports on detailed methodological studies on the determination of indobufen in human plasma based on LC-MS / MS technology. Summary of the invention

[0007] In order to fill the gap in the prior art, the object of the present invention is to provide an analytical method for determining the concentration of indobufen in human plasma based on LC-MS / MS technology.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In one aspect, the present invention provides a method for determining the concentration of indobufen in human plasma based on LC-MS / MS technology, comprising the following steps:

[0010] Internal standard solution and organic solvent were added to the plasma samples, and vortex mixing was performed to ensure sufficient extraction of indobufen. The samples were separated by centrifugation and the supernatant was collected as the pre-treated sample to be tested.

[0011] An Eclipse Plus C18 column was used for isocratic elution to achieve effective separation of indobufen, and then the sample was tested by mass spectrometry. A standard curve was drawn based on the mass spectrometry peak area ratio, and the regression equation was obtained to calculate the concentration of indobufen in the sample to be tested.

[0012] In the above method, the internal standard solution is an indobufen structural analog or a stable isotope-labeled indobufen. Preferably, the internal standard solution is indobufen-d5. More preferably, the concentration of the internal standard solution is 2.00 μg / mL.

[0013] In the above method, the organic solution is methanol or acetonitrile or a combination of the two. Preferably, the organic solvent is acetonitrile. More preferably, the organic solvent is added in a ratio of 3-5 times the volume of plasma.

[0014] In the above method, preferably, the centrifugation time is 10 min, the centrifugation temperature is 4° C., and the centrifugation speed is 3900 rpm.

[0015] In the above method, preferably, the chromatographic column used for liquid chromatography separation is: Eclipse Plus C18 chromatographic column, 3.5μm, 4.6×100mm; the mobile phase used is: phase A: aqueous solution containing 0.1% formic acid and 5mM ammonium acetate, phase B: acetonitrile.

[0016] In the above method, preferably, the liquid chromatography separation and elution conditions are:

[0017] Isocratic elution: Phase A: Phase B = 35%: 65%

[0018] Elution time: 3.00min;

[0019] Flow rate: 0.650 mL / min;

[0020] Injection volume: 5.00 μL;

[0021] Autosampler temperature: 4 °C;

[0022] Column temperature: 40℃.

[0023] In the above method, preferably, the mass spectrometry conditions for mass spectrometry detection are:

[0024] Ion source: electrospray ionization (ESI);

[0025] Injection voltage: -4500V;

[0026] Spray gas (Gas1): 55psi;

[0027] Auxiliary gas (Gas2): 55psi;

[0028] Detection method: negative ion;

[0029] Ion source temperature: 500°C;

[0030] Collision-induced dissociation (CAD): 9;

[0031] Curtain Gas: 40psi;

[0032] Dwell time: 200ms.

[0033] In the above method, preferably, the mass spectrometry detection is performed using a quantitative analysis ion pair, and the quantitative analysis ion pair is:

[0034] Indobufen m / z 294.1→250.0, collision energy (CE) -11 eV, declustering potential (DP) -40 V;

[0035] Indobufen-d5 m / z 299.0→255.2, collision energy (CE) -12eV, declustering potential (DP) -40V.

[0036] In the above method, preferably, the standard curve is specifically prepared as follows:

[0037] The linear regression equation was obtained by performing regression analysis with the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to be tested to the internal standard as the ordinate.

[0038] On the other hand, the present invention also provides the use of the above method in analyzing the concentration of indobufen in a plasma sample; the indobufen is used to treat ischemic cardiovascular disease, ischemic cerebrovascular disease and venous thrombosis caused by arteriosclerosis.

[0039] Beneficial effects of the present invention:

[0040] (1) Currently, there is no relevant literature or patent report on detailed methodological research on the determination of indobufen in human plasma based on LC-MS / MS technology. Therefore, the LC-MS / MS method established in the present invention is original and meets all the indicators of methodological validation.

[0041] (2) The present invention has the characteristics of simple pretreatment operation, and only requires one step of extraction for analysis. The sample after extraction is easy to preserve, and the analysis speed is fast, with the analysis time being only 3 minutes. Therefore, the present invention meets the requirements of high-throughput and large-volume biological sample detection.

[0042] (3) The method of the present invention has good accuracy, high precision, and good reproducibility, and can more accurately analyze the concentration of the drug.

[0043] (4) The LC-MS / MS method established in the present invention is used to determine the concentration of indobufen in human plasma, which can accelerate the progress of the bioequivalence evaluation of the generic drug.

[0044] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 is the product ion scanning mass spectrum of indobufen; Figure 2 MRM chromatograms of indobufen (upper) and internal standard indobufen-d5 (lower) in blank plasma samples; Figure 3 The MRM chromatograms of indobufen (upper) and internal standard indobufen-d5 (lower) in the lower limit of quantification sample; Figure 4 MRM chromatograms of indobufen (upper) and internal standard indobufen-d5 (lower) in the quantitative upper limit sample; Figure 5 This is the drug concentration-time curve of indobufen after a single oral dose of 0.2 g indobufen tablets in a healthy subject. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not particularly limit the contents.

[0051] Example

[0052] The method for rapidly determining the concentration of indobufen in human plasma provided by the present invention can generally be divided into three parts, namely, extraction method (i.e., pretreatment method), liquid chromatography method and mass spectrometry method. The present invention aims at the shortcomings of the prior art and establishes the analysis method from the above three aspects.

[0053] 1. Preprocessing:

[0054] The amount of plasma used in the present invention is only 50.0 μL, which is suitable for clinical research bioanalysis. In terms of extraction method, the present invention adopts a protein precipitation method, which shows a high recovery rate for indobufen, is simple and quick to operate, does not require a lengthy concentration step, and greatly shortens the extraction time. In addition, the method is used in conjunction with a 96-well plate, which is very suitable for high-throughput sample pretreatment requirements in clinical research.

[0055] The specific pre-processing steps are as follows:

[0056] 1. Add 50.0 μL of plasma sample, 50.0 μL of internal standard solution (indobufen-d5 concentration is 2.00 μg / mL), and 250 μL of acetonitrile to a 96-well plate;

[0057] 2. Vortex mix and centrifuge for 10 min (4°C, 3900 rpm);

[0058] 3. Take 200 μL of supernatant into another clean 96-well plate;

[0059] 4. The injection volume is 5.0 μL.

[0060] 2. Chromatographic analysis:

[0061] Chromatography: The samples to be tested were separated by liquid chromatography using an Eclipse Plus C18 column and isocratic elution [35% phase A: 65% phase B]. Mobile phase A was an aqueous solution containing 0.1% formic acid and 5 mM ammonium acetate, and mobile phase B was acetonitrile.

[0062] Indobufen is susceptible to interference from other endogenous substances in plasma due to its small molecular weight. For this reason, the present invention uses an Eclipse Plus C18 chromatographic column in chromatographic separation, which shows good retention effects on both the target detection object and the internal standard. At the same time, 0.1% formic acid and ammonium acetate are added to the mobile phase as modifiers, which helps to optimize the peak shape and improve the symmetry of the peak shape. Under this condition, the analytical throughput of the instrument is high, the chromatographic running time is shortened to 3 minutes, and the detection speed is fast, which is very suitable for large-scale sample analysis in clinical research.

[0063] 3. Mass spectrometry analysis:

[0064] An electrospray ion source was used, with negative ion detection, spray voltage of -4500V, gas 1 (Gas1) 55psi, gas 2 (Gas2) 55psi, curtain gas (Curtain Gas) 40psi, ion source temperature of 500℃, collision induced dissociation 9, dwell time of 200ms, indobufen quantitative analysis ion pair m / z 294.1→250.0, collision energy (CE) -11eV, declustering voltage (DP) -40V, and internal standard indobufen-d5 quantitative analysis ion pair m / z 299.0→255.2, collision energy (CE) -12eV, declustering voltage (DP) -40V.

[0065] The present invention is described in detail below through specific embodiments:

[0066] Embodiment 1:

[0067] Abbreviation Description:

[0068]

[0069]

[0070] 1 Materials

[0071] 1.1 Instrument

[0072] Chromatograph: LC-30AD fast liquid chromatography system, Shimadzu Corporation, Japan.

[0073] Mass spectrometer: Model 6500+ triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (Turbo Ion Spray) from Sciex, Canada.

[0074] Data processing software used: Analyst (version 1.6.3), Sciex, Canada.

[0075] Centrifuge: 5810R multifunctional desktop centrifuge, Eppendorf, Germany.

[0076] Analytical balance: CPA225D analytical balance, Beijing Sartorius Instrument Co., Ltd.

[0077] 1.2 Reference substances and reagents

[0078] Indobufen (content 99.9%) was purchased from China Food and Drug Administration, and indobufen-d5 (purity 100%) was purchased from Dadao Biotechnology (Shaoxing) Co., Ltd. Methanol (HPLC grade) was purchased from Sigma, USA. Formic acid (HPLC grade) was purchased from TCI. Ammonium acetate (HPLC grade) was purchased from ROE. Deionized water (18.2 mΩ, TOC ≤ 50 ppb) was prepared by a Milli-Q ultrapure water system.

[0079] 2 Methods

[0080] 2.1 Preparation of solutions and samples

[0081] Standard series samples: Accurately weigh an appropriate amount of reference substance, dissolve it in methanol and make up to volume to prepare a stock solution with an indobufen concentration of about 1.00 mg / mL. Accurately pipette an appropriate amount of each stock solution and dilute it stepwise with human blank plasma to obtain a mixed standard series sample with an indobufen concentration range of 0.200-50.0 μg / mL.

[0082] Quality control samples: Indobufen mixed quality control samples at four concentration levels were prepared in a similar manner to the standard series samples. The concentration of low quality control (LQC) was 0.600 μg / mL, the concentration of accessory middle quality control (AMQC) was 3.00 μg / mL, the concentration of medium quality control (MQC) was 15.0 μg / mL, and the concentration of high quality control (HQC) was 37.5 μg / mL.

[0083] Internal standard solution: Accurately weigh the indobufen-d5 reference substance, dissolve it in methanol and make up to volume to prepare an internal standard stock solution with a concentration of about 0.100 mg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution, dilute it with methanol, and obtain an internal standard solution with an indobufen-d5 concentration of 2.00 μg / mL.

[0084] 2.2 Plasma sample processing

[0085]

[0086] 2.3 Chromatographic and mass spectrometry conditions

[0087] Chromatographic conditions:

[0088]

[0089] Mass spectrometry conditions:

[0090]

[0091]

[0092] 3. Verification of the Method in This Example

[0093] The LC-MS / MS method validation of indobufen provided in this example includes selectivity, standard curve and lower limit of quantification, precision and accuracy, matrix effect, recovery, residue and stability, etc., which meets the requirements of the guidelines.

[0094] [Selectivity]

[0095] The peak area of ​​the chromatographic coeluting interferent at the retention time of each analyte and internal standard must be less than 20% of the LLOQ peak area (analyte) and less than 5% of the peak area (internal standard) by analyzing blank plasma and LLOQ samples from 6 different sources.

[0096]

Standard curve and lower limit of quantitation

[0097] Theoretical concentration of the analyte is taken as the horizontal axis (x), and the peak area ratio of the analyte to the internal standard is taken as the vertical axis (y). The linear regression operation is performed using the weighted (W=1 / x2) least square method, and the linear regression equation obtained is the standard curve. At least 75% of the standard curve samples and at least 6 concentration levels should meet the accuracy deviation of the calculated concentration within ±15.0% (within ±20.0% of the lower limit of quantification concentration level) to be accepted.

[0098] Accuracy and precision

[0099] At least 4 concentrations of quality control samples (low, sub-medium, medium, and high concentrations) are performed for each analysis batch, and 6 samples are analyzed for each concentration. Inter-batch accuracy and precision need to be evaluated on at least 3 analysis batches. The intra-batch and inter-batch accuracy of the quantitative lower limit should be within ±20.0%, and the precision should be within 20% to be acceptable. The intra-batch and inter-batch accuracy of the remaining concentration levels of quality control samples should be within ±15%, and the precision should be within 15.0%.

[0100]

Recovery rate

[0101] The treatment recovery was calculated by comparing the peak area ratios of the two treatment methods obtained by adding the quality control sample working solution before and after extraction.

[0102]

Matrix effect

[0103] Take blank plasma from 6 different sources, extract it (without adding internal standard solution), add the analyte solution and internal standard solution of the same concentration as LQC and HQC, mix by vortex and measure. Take deionized water instead of plasma and treat it according to the above method. Calculate the matrix factor by the peak area ratio obtained by the two methods, and evaluate the matrix effect by the RSD of the matrix factor normalized by the internal standard, which is less than 15% to be acceptable.

[0104]

Residue

[0105] After the ULOQ sample analysis, the blank plasma sample was analyzed immediately. The chromatographic peak areas at the retention times of the analyte and internal standard in the blank plasma sample and the LLOQ sample were compared to assess whether the carryover affected the accuracy of the analysis.

[0106]

stability

[0107] When investigating the stability of the analyte in plasma samples, the plasma samples at LQC and HQC concentration levels were placed in different temperatures and environments. A total of four placement conditions were investigated, namely:

[0108] 1) Short-term stability: Place at room temperature for 19 hours,

[0109] 2) Place in the autosampler at 4°C for 117 hours,

[0110] 3) undergo 5 freeze-thaw cycles (from -80°C to room temperature),

[0111] 4) Place at -80℃ for 54 days.

[0112] Stability samples at each concentration level under each storage condition were considered stable if the accuracy RE% did not exceed ±15.0%.

[0113] 4 Results of the method verification of this embodiment

[0114] Selectivity of method

[0115] The product ion scanning mass spectrum of indobufen is shown in Figure 1 As shown; the retention times of indobufen and internal standard indobufen-d5 are approximately 2.16min and 2.15min, respectively. Figures 2 to 4 As shown, no interference was observed at any retention time.

[0116] Standard curve and limit of quantitation

[0117] The linear range of indobufen in the plasma samples of the clinical study of indobufen was 0.200–50.0 μg / mL, and the typical linear regression equation of the standard curve of the analyte was:

[0118] Indobufen: y = 0.000431x + 0.00107 (r 2=0.9980)

[0119] The concentration of indobufen in the lower limit of quantitation sample was 2.00 μg / mL. The intra- and inter-batch accuracy of the lower limit of quantitation was between -4.4 and 2.9%, and the intra- and inter-batch precision was within 4.9%, both meeting the acceptance criteria.

[0120] Precision and accuracy of the method

[0121] The intra-assay and inter-assay accuracy of indobufen was between -7.3% and -1.8%, and the intra-assay and inter-assay precision was within 8.8%, both meeting the acceptance criteria. The results are shown in Table 1.

[0122] Table 1: Precision and accuracy of indobufen in human plasma

[0123]

[0124] Treatment recovery rate

[0125] At the concentration levels of LQC, MQC and HQC, the extraction recoveries of indobufen were 97.6%, 101.0% and 95.1%, respectively, and the recovery of the internal standard indobufen-d5 was 92.1%, which met the requirements of analytical testing.

[0126] Matrix Effects

[0127] The matrix factors of the internal standard normalized for indobufen at the LQC and HQC concentration levels were 117% and 109%, and the RSDs were 13.8% and 2.1%, respectively. The above results indicate that the matrix effect does not interfere with the accuracy of the analysis of the analyte.

[0128] Residue

[0129] After the ULOQ sample analysis, the blank plasma sample was analyzed immediately. The peak areas of the analyte and internal standard retention time in the blank plasma sample were less than 20% of the LLOQ analyte peak area and less than 5% of the internal standard peak area, indicating that there was no residual effect under this method.

[0130] Plasma stability study

[0131] The results of the plasma stability test are shown in Table 2, which show that indobufen is stable under the conditions investigated.

[0132] Table 2 Stability of indobufen in human plasma under different conditions

[0133]

[0134] Example 2: Application of the method of Example 1 in human pharmacokinetic studies

[0135] The validated method was used to analyze indobufen in human plasma to evaluate the pharmacokinetic characteristics of indobufen. A healthy subject took 0.2 g of indobufen tablets orally after a single meal. The plasma drug concentration-time curve is shown in Figure 5 The sensitivity of the detection method can fully describe the pharmacokinetic properties of indobufen, and the method has good reproducibility and high measurement accuracy.

[0136] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A method for determining the concentration of indobufen in human plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology, comprising the following steps: Internal standard solution and organic solvent were added to the plasma samples, and vortex mixing was performed to ensure sufficient extraction of indobufen. The samples were separated by centrifugation and the supernatant was collected as the pre-treated sample to be tested. An Eclipse Plus C18 column was used for isocratic elution to achieve effective separation of indobufen, and then the sample was tested by mass spectrometry. A standard curve was drawn based on the mass spectrometry peak area ratio, and the regression equation was obtained to calculate the concentration of indobufen in the sample to be tested.

2. The method according to claim 1, wherein: The internal standard solution is an indobufen structural analog or a stable isotope-labeled indobufen. Preferably, the internal standard solution is indobufen-d5. More preferably, the concentration of the internal standard solution is 2.00 μg / mL.

3. The method according to claim 1, wherein: The organic solution is methanol or acetonitrile or a combination of the two. Preferably, the organic solvent is acetonitrile. More preferably, the organic solvent is added in a ratio of 3-5 times the volume of the plasma.

4. The method according to claim 1, wherein the centrifugation time is 10 min, the centrifugation temperature is 4°C, and the centrifugation speed is 3900 rpm.

5. The method according to claim 1, wherein: The chromatographic column used for liquid chromatography separation is: EclipsePlus C18 chromatographic column, 3.5 μm, 4.6×100 mm; the mobile phases used are: Phase A: an aqueous solution containing 0.1% formic acid and 5 mM ammonium acetate, Phase B: acetonitrile.

6. The method according to claim 5, wherein: The elution conditions for liquid chromatography separation are: Isocratic elution: Phase A: Phase B = 35%: 65% Elution time: 3.00min; Flow rate: 0.650 mL / min; Injection volume: 5.00 μL; Autosampler temperature: 4 °C; Column temperature: 40℃.

7. The method according to claim 1, wherein: The mass spectrometry conditions for mass spectrometry detection were: Ion source: electrospray ionization (ESI); Injection voltage: -4500V; Spray gas (Gas1): 55psi; Auxiliary gas (Gas2): 55psi; Detection method: negative ion; Ion source temperature: 500°C; Collision-induced dissociation (CAD): 9; Curtain Gas: 40psi; Dwell time: 200ms.

8. The method according to claim 1 or 6, wherein: The mass spectrometry detection was performed using a quantitative analysis ion pair, wherein the quantitative analysis ion pair is: Indobufen m / z 294.1→250.0, collision energy (CE) -11 eV, declustering potential (DP) -40 V; Indobufen-d5 m / z 299.0→255.2, collision energy (CE) -12eV, declustering potential (DP) -40V.

9. The method according to claim 1, wherein: The standard curve is specifically made as follows: The linear regression equation was obtained by performing regression analysis with the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to be tested to the internal standard as the ordinate.

10. Use of the method according to any one of claims 1 to 9 in analyzing the concentration of indobufen in a plasma sample; the indobufen is used to treat ischemic cardiovascular disease, ischemic cerebrovascular disease and venous thrombosis caused by arteriosclerosis.

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