HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method for detecting arenecycline

Abstract

The invention discloses a method for detecting arenecycline in plasma by an HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method. According to the method, a protein precipitation method is adopted for pretreatment; an Agela Venusil MP C18 (2) chromatographic column is adopted as the chromatographic condition, and an aqueous solution of 1% FA and methanol of 1% FA are used as mobile phases for gradient elution; an electrospray ionization source (ESI) tandem mass spectrum is adopted as a mass spectrum condition, and verapamil is used as an internal standard for quantitative detection. The method is easy and convenient to operate, high in analysis speed, high in sensitivity and suitable in linear range, and meets the requirement for large-batch sample analysis in clinical research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for detecting sarecycline in plasma by HPLC-MS / MS. Background Art

[0002] Sarrecycline is a novel narrow-spectrum tetracycline that was approved by the U.S. Food and Drug Administration (FDA) in October 2018 for the treatment of inflammatory non-nodular moderate to severe acne vulgaris. Sarrecycline has antimicrobial activity against important skin / soft tissue pathogens, has targeted activity against P. acnes, and also has the same anti-inflammatory effects as other tetracyclines used to treat acne vulgaris. It also shows a lower tendency to drug resistance compared to other tetracyclines and is also active against tetracycline-resistant Staphylococcus aureus and erythromycin- and clindamycin-resistant P. acnes strains. A large number of Phase I, Phase II, and 3 Phase III studies have been successfully conducted, determining that it is a well-tolerated oral medication that can be used once daily as a tablet to treat patients 9 years of age or older.

[0003] In drug clinical monitoring and generic drug clinical research, it is necessary to measure the blood drug concentration of subjects or patients to study their pharmacokinetic behavior. In order to accelerate the clinical application of sarecycline, a simple, accurate, rapid and sensitive bioanalytical method is needed. Up to now, there are no relevant literature and patents reporting the HPLC-MS / MS method for detecting sarecycline in human or animal plasma, so the analytical method of this study is original. In view of the current gap in analytical methods, it is necessary to develop an analytical method with simple pretreatment operation and taking into account sensitivity, accuracy and analysis speed, so as to be suitable for the accurate analysis of large batches of samples in clinical research. Summary of the invention

[0004] In order to solve the above technical problems, the object of the present invention is to provide a method for detecting sarecycline in plasma by HPLC-MS / MS.

[0005] To achieve the above object, the present invention adopts the following technical solution: A method for detecting sarecycline in plasma by HPLC-MS / MS method comprises the following steps: (1) Pretreatment: Add methanol to the plasma sample to precipitate protein, take the supernatant after centrifugation, and dilute it with water to obtain the test sample; (2) The pretreated plasma samples were tested by HPLC-MS / MS, and the content of sarecycline therein was determined by internal standard quantitative method, wherein: Chromatographic conditions: Chromatographic column: Agela Venusil MP C18(2) column; Mobile phase A: 1% FA in water, mobile phase B: 1% FA in methanol; Gradient elution program: 0-0.5 min: mobile phase B 65%; 0.5-1.0 min: mobile phase B 65% 90%; 1.0-2.5 min: mobile phase B is 90%; 2.5-2.6 min: mobile phase B is 90% 65%; 2.6-3.0min: mobile phase B is 65%; Column temperature: 35~45℃; Mass spectrometry conditions: Ion source: electrospray ion source; positive ion scanning; multiple reaction monitoring; spray voltage was 5500 V; ion source temperature was 550 °C.

[0006] In some embodiments, an internal standard solution is added to the plasma sample, and the sample containing the internal standard substance is obtained after treatment; the internal standard solution is obtained by dissolving verapamil in methanol and diluting it with methanol-water (50: 50, v / v). In some embodiments, the amount of the internal standard solution added is 0.5-2 times that of the plasma sample, preferably an equal volume.

[0007] In some embodiments, in step (1), the volume of the plasma sample is 30-100 µL, preferably 40-60 µL, for example 50 µL. In some embodiments, the content of sarecycline in the plasma sample is 5.00-5000 ng / mL.

[0008] In some embodiments, in step (1), the content of the internal standard substance in the test sample is 25-100 ng / mL, preferably 100 ng / mL.

[0009] In some embodiments, in step (1), the volume ratio of the plasma sample to methanol is 1: (3-7), preferably 1: (4-6).

[0010] In some embodiments, in step (1), after the supernatant obtained after centrifugation is diluted with water, the sample is not easy to volatilize, which is beneficial to the preservation of the sample and the accuracy of the detection; the amount of water added is 0.5-2 times the volume of the supernatant, and preferably the amount of water added is equal to the volume of the supernatant.

[0011] In some embodiments, in step (2), the column temperature is 40°C; the mobile phase flow rate is 0.7 mL / min; and the injection volume is 2 µL.

[0012] In some embodiments, in the mass spectrometry conditions of step (2), gas 1 (Gas1): 55 psi, gas 2 (Gas2): 55 psi, curtain gas (Curtain Gas): 35 psi, collision induced dissociation: 6 psi, dwell time: 100 ms.

[0013] In some embodiments, in the mass spectrometry conditions of step (2), the ion pair for quantitative analysis of sarecycline is: m / z 488.1 410.2, collision energy: 30 eV, declustering voltage: 100 V; Verapamil quantitative analysis ion pair: m / z 455.2 164.9, collision energy: 40 eV, declustering voltage: 100 V.

[0014] The beneficial effects of the present invention are: 1. The method of the present invention has the characteristics of simple pretreatment operation, and only requires one step of extraction and one step of dilution for analysis. The sample after extraction is easy to preserve. In addition, the method has a fast analysis speed (analysis time is only 3 min), which is suitable for large-scale clinical research sample analysis.

[0015] 2. In clinical research, the improvement of the sensitivity of the detection method can reduce the dosage of the subjects and increase the safety of clinical research. The method of the present invention has high sensitivity and low plasma dosage (the plasma dosage can be only 50.0 µL). The lower limit of quantification of sarecycline is 5.00 ng / mL, the detection limit is calculated to be 0.700 ng / mL, and the amount of the analyte on the detection limit column is calculated to be 0.0875 pg according to the dilution factor of 16 and the injection volume of 2 µL.

[0016] 3. The linear range of the present invention is reasonably selected (linear range is 5.00 – 5000 ng / mL), which can more accurately determine the concentration of low-content drugs.

[0017] 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

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1It is a scanning mass spectrum of the product ion of sarecycline in the method of the present invention.

[0020] Figure 2 This is the ion current chromatogram of sarecycline and internal standard verapamil in human blank plasma (upper: verapamil, lower: sarecycline).

[0021] Figure 3 This is the ion current chromatogram of sarecycline and internal standard verapamil in human plasma at the lower limit of quantification (5.00 ng / mL) (upper: verapamil, lower: sarecycline).

[0022] Figure 4 Ion current chromatograms of sarecycline and internal standard verapamil in the upper limit of quantitation (5000 ng / mL) of human plasma (upper: verapamil, lower: sarecycline).

[0023] Figure 5 This is the ion current chromatogram of sarecycline and internal standard verapamil 1.5 hours after healthy subjects took sarecycline (upper: verapamil, lower: sarecycline).

[0024] Figure 6 This is the blood concentration and time curve of a healthy subject after taking 60 mg of sarecycline. DETAILED DESCRIPTION

[0025] The present invention is further described in detail in conjunction with the following specific examples and drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. 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 specifically limit the content.

[0026] The development of a method for detecting drug concentration in plasma by liquid chromatography-tandem mass spectrometry 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 an analytical method from the above three aspects.

[0027] The plasma dosage in the method of the present invention can be only 50.0 µL, which is suitable for clinical research bioanalysis; the pretreatment method of the present invention uses protein precipitation method, which has good reproducibility for sarecycline, simple operation, short extraction time, and no time-consuming concentration step. It is used with 96-well plates and is suitable for high-throughput sample pretreatment in clinical research.

[0028] In the embodiment of the present invention, the chromatographic separation adopts Agela Venusil MP C18 (2) chromatographic column, which has good retention for both the analyte and the internal standard, and has sharp peak shape. The mobile phase used in the chromatographic analysis is 1% FA in water and 1% FA in methanol, the purpose of which is to solve the problem of the residue of sarecycline on the chromatographic column and improve the chromatographic peak shape. In this mode, the instrument analysis throughput is high, the chromatographic running time is only 3.0 min, the detection is fast, and it is suitable for large-scale sample analysis in clinical research.

[0029] Abbreviation

[0030] Example 1 1 Materials 1.1 Instrument Chromatograph: LC-30AD fast liquid chromatography system, Shimadzu Corporation, Japan.

[0031] Mass spectrometer: Model 5500 triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) source (Sciex, Canada).

[0032] Data processing software used: Analyst (version 1.6.3), Sciex, Canada.

[0033] Centrifuge: TGL-16RC desktop centrifuge, Shandong Bio Medical Technology Co., Ltd.

[0034] Analytical balance: BCE55I-10CN analytical balance, Beijing Sartorius Instrument Co., Ltd.

[0035] 1.2 Reference substances and reagents Sarrecycline was purchased from Panphy Chemical Corporation, and verapamil was purchased from Aladdin. Methanol (HPLC grade) was purchased from Sigma, USA. Formic acid (FA) (HPLC grade) was purchased from TCI. Distilled water was prepared by Watsons.

[0036] 2 Methods 2.1 Preparation of solutions and samples Standard series samples: Accurately weigh an appropriate amount of each sarecycline reference substance, dissolve and dilute to volume with methanol to prepare a sarecycline stock solution 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 of samples, with sarecycline concentrations ranging from 5.00 to 5000 ng / mL.

[0037] Quality control samples: Five concentration levels of Sha Rui Huan quality control samples were prepared in a similar manner to the standard series samples. The lower limit of quantification concentration was 5.00 ng / mL, the low quality control (LQC) concentration was 15.0 ng / mL, the accessorial middle quality control (AMQC) concentration was 150 ng / mL, the medium quality control (MQC) concentration was 1500 ng / mL, and the high quality control (HQC) concentration was 3750 ng / mL.

[0038] Internal standard solution: Accurately weigh the verapamil reference substance, dissolve it in methanol and make up to volume to prepare an internal standard stock solution with a concentration of about 1.00 mg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution, add methanol: water (50: 50, v / v) to dilute it to obtain an internal standard solution with a verapamil concentration of 100 ng / mL.

[0039] 2.2 Plasma sample processing 1) Add 50.0 µL of plasma sample, 50.0 µL of internal standard solution (verapamil concentration is 100 ng / mL), and 300 µL of methanol to a 96-well plate; 2) Vortex mix and centrifuge for 10 min (4°C, 3900 rpm); 3) Take 150 µL of supernatant into another clean 96-well plate; 4) Add 150 µL of water and vortex to mix.

[0040] 2.3 Chromatographic and mass spectrometry conditions Chromatographic conditions:

[0041] Mass spectrometry conditions:

[0042] The scanning mass spectrum of sarecycline product ions is shown in Figure 1 .

[0043] 2.4 Methodological validation The method was validated according to the guidelines of Chinese Pharmacopoeia 9012, including stability, selectivity, linearity, accuracy, precision, recovery, matrix effect, etc.

[0044] Optional: Six blank plasma samples from different sources and their respective prepared lower limit of quantitation samples were processed and then injected for analysis. The peak area of ​​the chromatographic co-eluting interference must be less than 20% of the peak area of ​​the lower limit of quantitation analyte and less than 5% of the peak area of ​​the internal standard.

[0045] Standard curve: The linear regression equation (weight factor W = 1 / x) is calculated by taking the theoretical concentration of the analyte as the horizontal axis (x) and the peak area ratio of the analyte to the internal standard as the vertical axis (y). 2 ). For method validation, two samples of the standard curve were analyzed for each analytical batch.

[0046] Precision and Accuracy: Measure six samples of each of the five concentrations of quality control samples. The intra-batch and inter-batch precision of the quantitative lower limit calculated by relative standard deviation (RSD) is acceptable only when it is less than 20%, and the accuracy calculated by relative deviation (RE) is acceptable only when it is between -20% and 20%. The intra-batch and inter-batch precision of each component of the QC samples at other concentration levels must be less than 15% to be acceptable, and the accuracy must be between -15% and 15% to be acceptable.

[0047] stability: When investigating the stability of each analyte in plasma samples, LQC and HQC were placed in room temperature and ultra-low temperature (below -60°C), and six samples were analyzed after the placement. The short-term stability at room temperature for 10 h was investigated.

[0048] Recovery rate: Take 50.0 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, MQC and HQC, and inject it for determination. Extract 6 portions of LQC, MQC and HQC respectively, and inject them for determination. Calculate the extraction recovery rate by the peak area ratio of the two treatment methods.

[0049] Matrix Effects: Six normal blank plasmas from different sources were used to prepare two quality control plasma samples with low and high concentrations, and three samples were analyzed for each concentration. The standard curve prepared with normal plasma was used for determination.

[0050] 2.5 Clinical studies The above established method was used to analyze the concentration of sarecycline in clinical study plasma samples for the pharmacokinetics study of sarecycline in humans. A healthy subject was given 60 mg of sarecycline tablets, and 3-4 mL of venous blood was collected before administration (0 h) and at different time points within 12 h after administration. The blood was placed in a K2EDTA anticoagulant centrifuge tube and centrifuged (2000 g, 4 ºC) for 10 min to separate the plasma, which was then stored in an ultra-low temperature refrigerator at -60 °C or below.

[0051] 3 Results and discussion 3.1 Methodological validation Selectivity like Figure 3-Figure 5 As shown, the retention times of sarecycline and verapamil were approximately 2.16 and 2.13 min, and there were no co-eluting interfering peaks at the retention times.

[0052] Standard curve The linear range of the determination of sarecycline in plasma samples from clinical studies of sarecycline is 5.00 - 5000 ng / mL. The typical linear regression equations of the standard curves of the analytes are: Serrecycline: y = 0.000833x-0.00015 (r = 0.9992); Detection limit The concentration of sarecycline in the sample with the lower limit of quantification was 5.00 ng / mL, and the signal-to-noise ratio was about 22. The detection limit was calculated to be 0.700 ng / mL based on the signal-to-noise ratio of 3. The amount of the analyte on the column was calculated to be 0.0875 pg based on the dilution factor of 16 and the injection volume of 2 µL.

[0053] Precision and Accuracy The precision and accuracy results all met the acceptance criteria and are shown in Table 1.

[0054] Table 1. Precision and accuracy of determination of sarecycline in human plasma

[0055] Recovery rate At LQC, MQC and HQC concentration levels: the extraction recoveries of sareccycline were 90.6%, 102.0% and 93.5%, respectively; and the recovery of verapamil was 90.5%.

[0056] Matrix Effects The CV% of the measured values ​​of each QC sample of each concentration from each source at the LQC and HQC concentration levels was ≤15.0%; the average RE% of the measured values ​​of each QC sample of each concentration from each source did not exceed ±15.0%. The above results showed that the matrix effect did not interfere with the accuracy of the analysis of the analytes.

[0057] Plasma stability study Low (15.0 ng / mL) and high concentration (3750 ng / mL) sarecycline plasma stability samples were prepared and placed at room temperature for 10 hours and at ultra-low temperatures (below -60°C) to test the differences with the theoretical values. The results are shown in Table 2. The results show that sarecycline is stable in plasma at room temperature for 10 hours and at ultra-low temperatures (below -60°C) for 30 days.

[0058] Table 2 Stability of sarecycline in human plasma at room temperature for 10 h and at ultra-low temperature for 20 days

[0059] 4 Human pharmacokinetics studies The validated method was used to analyze sarecycline in plasma to evaluate the pharmacokinetic characteristics of sarecycline. A healthy subject took a single oral dose of 60 mg sarecycline tablets. The plasma drug concentration-time curve is shown in Figure 6 The sensitivity of the detection method can fully describe the pharmacokinetic properties of sareccycline, and the selection of the linear range is close to the concentration level of the actual sample, and the determination accuracy is high.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting sacrocycline in plasma by HPLC-MS / MS, comprising the following steps: (1) Pretreatment: adding an internal standard solution to a plasma sample, adding methanol to the plasma sample to precipitate protein, taking the supernatant after centrifugation, and diluting with water to obtain a test sample, wherein the internal standard solution is obtained by dissolving verapamil in methanol and diluting with methanol-water, 50:50, v / v, the amount of the internal standard solution added is 0.5-2 times that of the plasma sample, and the volume of the plasma sample is 30-100 μL; the content of sarecycline in the plasma sample is 5.00-5000 ng / mL, and the content of the internal standard substance in the test sample is 25.0-100 ng / mL; (2) The pretreated plasma samples were tested by HPLC-MS / MS, and the content of sarecycline therein was determined by internal standard quantitative method, wherein: Chromatographic conditions: Chromatographic column: Agela Venusil MP C18(2) column; Mobile phase A: 1% FA in water, mobile phase B: 1% FA in methanol; Gradient elution program: 0-0.5 min: mobile phase B 65%; 0.5-1.0 min: mobile phase B 65% 90%; 1.0-2.5min: mobile phase B is 90%; 2.5-2.6 min: mobile phase B is 90% 65%; 2.6-3.0min: mobile phase B is 65%; The mobile phase flow rate was 0.7 mL / min; The injection volume was 2.0 µL; Column temperature: 40°C; Mass spectrometry conditions: Ion source: electrospray ion source; positive ion scanning; multiple reaction monitoring; spray voltage: 5500 V; ion source temperature: 550°C; gas 1: 55 psi, gas 2: 55 psi, curtain gas: 35 psi, collision-induced dissociation: 6 psi, dwell time: 100 ms; ion pair for quantitative analysis of sarecycline: m / z 488.1 410.2, collision energy: 30 eV, declustering voltage: 100 V; ion pair for verapamil quantitative analysis: 455.2 164.9, collision energy: 40 eV, declustering voltage: 100 V.

2. The method for detecting sarecycline in plasma by HPLC-MS / MS according to claim 1, characterized in that In step (1), the volume ratio of the plasma sample to acetonitrile is 1:(3-6).

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

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