A LC-MS / MS detection method for determining abaparatide in plasma

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to an LC-MS / MS detection method for determining abaparatide in plasma, comprising the following steps: (1) pre-treatment of plasma samples; (2) detection using a liquid chromatography-mass spectrometer, wherein the liquid chromatography conditions are: a chromatographic column: C 18 Column; mobile phase A is 0.3% (v / v) formic acid aqueous solution, mobile phase B is acetonitrile; gradient elution; autosampler temperature is 10°C; analysis time is 7.5min; mass spectrometry conditions are set as ion source using electrospray ionization source ESI, positive ion detection; ionization voltage is 3500V, ion source temperature is 350°C; scanning mode is multiple reaction monitoring scanning MRM. The method of the present invention has high sensitivity, and the lowest concentration that can be accurately quantified can reach 0.02 ng / mL, and can quickly, sensitively and accurately determine abaloparatide in plasma.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an LC-MS / MS detection method for determining abaparatide in plasma. Background Art

[0002] Abaloparatide is a newly synthesized human parathyroid hormone-related protein (PTHrP) analogue, which has 76% homology with PTHrP 1-34 and 41% homology with parathyroid hormone PTH 1-34. It is used to treat postmenopausal osteoporosis in women. Compared with teriparatide, abaloparatide can not only significantly increase lumbar bone mineral density (BMD), but also increase hip and femoral BMD, significantly reduce the risk of vertebral and non-vertebral fractures, and reduce the incidence of hypercalcemia. At present, the clinical application research of abaloparatide is limited to the comparison of pharmacodynamic treatment, and there are few reports on its pharmacokinetics. It is imperative to establish a highly applicable and fully validated biological sample analysis method for its pharmacokinetic study.

[0003] Common biological sample analysis methods for peptide drugs mainly include ligand binding analysis (LBA) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). As the most commonly used analysis method for peptide drugs, LBA mainly includes radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence (MSD), etc. It is based on the specific recognition of antibodies to analytes / antigens for quantitative analysis of target peptides. However, due to the small molecular weight and relatively simple structure of peptides, the number of peptide epitopes is limited, and they often have hapten-like properties. Therefore, the preparation of specific reagent antibodies required for the LBA method is difficult and expensive, and the interference of endogenous analogs and their metabolites cannot be ruled out. At present, the LC-MS / MS method has become one of the main technologies for analyzing peptide drugs due to its good selectivity, high sensitivity, large throughput and high quantitative accuracy. Summary of the invention

[0004] The present invention provides an LC-MS / MS detection method for determining abaparatide in plasma. The detection method has strong selectivity and high sensitivity, and the lowest concentration that can be quantified can reach 0.02 ng / mL, thereby realizing rapid, accurate and sensitive determination of abaparatide in plasma.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A LC-MS / MS detection method for determining abaparatide in plasma comprises the following steps:

[0007] (1) Pretreatment of plasma samples:

[0008] Take the plasma sample containing abaparatide to be tested, thaw it at 4-8°C, and place it in an ice bath. Add a solution containing internal standard teriparatide and methanol containing 0.1% (v / v) formic acid (i.e., the methanol contains 0.1% (v / v) formic acid) and vortex to precipitate. Centrifuge, take the supernatant and put it in another centrifuge tube, blow dry the supernatant with a nitrogen blower, re-dissolve the residue with 20% (v / v) methanol, and take the supernatant after centrifugation as the sample solution to be tested;

[0009] (2) Detection by liquid chromatography-mass spectrometry:

[0010] HPLC conditions: Column: Waters peptide CSH C 18 Column (2.1×150mm, 130Å, 1.7µm); mobile phase A: 0.3% (v / v) formic acid in water; mobile phase B: acetonitrile; gradient elution; autosampler temperature was 10°C; analysis time was 7.5min;

[0011] The mass spectrometry conditions were set as follows: the ion source used an electrospray ionization source (ESI), positive ion detection, an ionization voltage of 3500 V, and an ion source temperature of 350°C; the scanning mode was multiple reaction monitoring (MRM).

[0012] Furthermore, in step (1), the volume ratio of the methanol (containing 0.1% (v / v) formic acid) solution containing the internal standard teriparatide to the plasma sample to be tested is 3-5:1.

[0013] Furthermore, in step (1), the concentration of the internal standard teriparatide is 1-2 ng / mL.

[0014] Furthermore, in step (1), the rotation speed of the vortex is 3000 rpm and the time is 1 min; the centrifugal conditions are a rotation speed of 12000 rpm, a temperature of 4° C., and a time of 5 min.

[0015] Furthermore, in step (1), the setting temperature of the nitrogen blowdown device is 40-50°C.

[0016] Furthermore, in step (2), the conditions of the gradient elution are as follows:

[0017]

[0018] Furthermore, in step (2), the liquid chromatography conditions are: column temperature 40-50°C; injection volume 30-50 μL; flow rate 0.2-0.4 mL / min.

[0019] Furthermore, in step (2), the liquid chromatography conditions are: column temperature 45°C; injection volume 30 μL; flow rate 0.3 mL / min.

[0020] Furthermore, in step (2), the mass spectrometry conditions are: sheath gas N 2 35Arb, auxiliary gas N 2 It is 15Arb.

[0021] Furthermore, in step (2), the parameters of the multiple reaction monitoring scan MRM are as follows:

[0022]

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Peptide drugs are administered in small doses and have low concentrations in the body, often at the ng / mL level. The present invention provides a liquid chromatography-tandem mass spectrometry method for determining abaloparatide in biological samples based on the characteristics of abaloparatide. The method is rapid, accurate, and sensitive, and the lowest concentration that can be quantified can reach 0.02 ng / mL.

[0025] (2) The present invention uses methanol to precipitate plasma samples, which is simple and convenient to operate, has a short analysis time and good reproducibility, meets the requirements of in vivo drug analysis methods, and is suitable for in vivo pharmacokinetic studies of abaparatide. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The mass spectrometry scans of the parent ion (1-1) and the product ion (1-2) of abaparatide;

[0027] Figure 2 The typical chromatograms of abaparatide (A) and internal standard teriparatide (B) in blank plasma samples;

[0028] Figure 3 The typical chromatograms of abaparatide (A) and internal standard teriparatide (B) in plasma samples at 0 point;

[0029] Figure 4 Typical chromatograms of abaparatide (A) and internal standard teriparatide (B) in LLOQ quality control samples;

[0030] Figure 5 The typical chromatograms of abaparatide (A) and internal standard teriparatide (B) in actual plasma samples;

[0031] Figure 6 Rats were injected subcutaneously with 24 μg kg -1 Mean plasma concentration-time curves after abaparatide. DETAILED DESCRIPTION

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] Unless otherwise specified, the test materials used in the following examples are conventional test materials purchased from commercial channels.

[0034] Instruments, materials and experimental animals

[0035] Materials: Abaparatide (batch number: P2023110601, purity ≥98.0%) API and teriparatide (batch number: P2024022210, purity ≥98.0%) API were purchased from Zhejiang Paipeptide Biotechnology Co., Ltd.; chromatography-grade methanol and acetonitrile, and mass spectrometry-grade formic acid were purchased from Thermo Fisher Scientific, USA; deionized water was prepared by a Milli-Q Reference ultrapure water instrument.

[0036] Instruments: Ultimate 3000 ultra-high performance liquid chromatograph coupled with TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific, USA); 5425R high-speed refrigerated centrifuge (Eppendorf, Germany); XSR105 1 / 100,000 analytical balance (Mettler-Toledo, Switzerland); VX-II multifunctional vortex mixer (Beijing Tajinyi Technology Co., Ltd.); Millipore water purifier (Millipore Corporation).

[0037] Animals: SPF-grade SD female rats, weighing 220-250 g, were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number: SCK (Lu) 20220006.

[0038] Example 1: Determination of Abaparatide in Rat Plasma

[0039] 1. Preparation of standard curve series samples and quality control samples

[0040] (1) Abaparatide stock solution: Accurately weigh 2 portions of abaparatide standard and dissolve them in 20% (v / v) methanol aqueous solution (containing 0.1% (v / v) formic acid) to prepare an abaparatide stock solution with a concentration of 100 μg / mL. One portion is used for the preparation of the standard series solutions and the other portion is used for the preparation of the quality control solution. Store them in brown glass bottles at -20°C.

[0041] (2) Accurately measure one portion of abaparatide stock solution and dilute it with 20% (v / v) methanol solution to a standard working solution with a concentration of 200 ng / mL. Dilute the standard working solution with rat blank plasma to obtain a series of standard curve samples with abaparatide concentrations of 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 4 and 8 ng / mL in plasma, respectively. Accurately measure another portion of abaparatide stock solution and dilute it with 20% (v / v) methanol solution to a quality control working solution with a concentration of 200 ng / mL. Dilute the quality control working solution with rat blank plasma to obtain four quality control samples with abaparatide concentrations of 0.02 ng / mL (LLOQ), 0.06 ng / mL (LQC), 0.6 ng / mL (MQC) and 6 ng / mL (HQC) in plasma, respectively.

[0042] 2. Preparation of internal standard solution

[0043] Accurately weigh the teriparatide standard into a 5 mL volumetric flask, add methanol to dissolve and prepare an internal standard stock solution with a solubility of 100 μg / mL. Accurately measure the internal standard stock solution and add methanol (containing 0.1% (v / v) formic acid) solution to dilute it to an internal standard solution with a concentration of 2 ng / mL.

[0044] 3. Plasma sample pretreatment

[0045] Take 150 μL of plasma sample, place it in an ice bath, add 600 μL of methanol (containing 0.1% (v / v) formic acid) solution containing internal standard teriparatide (2 ng / mL), vortex at 3000 rpm for 1 min, centrifuge at 12000 rpm for 5 min at 4°C, take the supernatant and put it in another centrifuge tube, blow dry the supernatant with a nitrogen blower (set temperature at 40~50°C), re-dissolve the residue with 20% (v / v) methanol, centrifuge at 4°C at 12000 rpm for 5 min, and take the supernatant as the sample solution to be tested.

[0046] 4. LC-MS / MS Analysis

[0047] 4.1 Chromatographic conditions

[0048] Using Waters peptide CSH C 18(2.1×150mm, 130Å, 1.7µm) chromatographic column, column temperature 45℃, flow rate 0.3 mL / min, injection volume 30μL, autosampler temperature 10℃. Mobile phase A and mobile phase B were used for gradient elution. Mobile phase A was 0.3% (v / v) formic acid in water, and mobile phase B was acetonitrile. The gradient elution program was: 0.0~0.2min, 15% (v / v) B; 0.2~4.0min, 15~80% (v / v) B; 4.0~4.5min, 80~90% (v / v) B; 4.5~5.5min, 90% (v / v) B; 5.5~6.0min, 90~15% (v / v) B; 6.0~7.5min, 15% (v / v) B.

[0049] 4.2 Mass spectrometry conditions

[0050] The ion source used was electrospray ionization (ESI), positive ion detection, ionization voltage 3500 V, ion source temperature 350 °C, sheath gas N 2 35Arb, auxiliary gas N 2 The scanning mode is multiple reaction monitoring scanning MRM; the ion pair used for quantitative analysis of abaparatide is: 566.5→632.4, RF voltage 92V, collision energy 13.6V; the ion pair used for quantitative analysis of internal standard teriparatide is 687.0→787.2, RF voltage 120V, collision energy 18.0V. (See Figure 1 )

[0051] Example 2: Methodological Validation

[0052] 1. Selectivity

[0053] 150 μL of blank plasma samples (n=6) from rats of different sources were taken, and except for not adding the internal standard, the rest was processed according to the plasma sample pretreatment method in Example 1, and the blank plasma sample graph was obtained by sampling and analysis; rat blank plasma (n=6) was taken, only the internal standard solution was added, and the plasma sample pretreatment method in Example 1 was followed, and the 0-point plasma sample graph was obtained by sampling and analysis; rat blank plasma (n=6) was taken, and the abaparatide lower limit of quantitation (LLOQ) concentration sample was prepared, and the plasma sample pretreatment method in Example 1 was followed, and the quality control plasma sample graph was obtained by sampling and analysis; plasma samples after subcutaneous injection of abaparatide were taken from rats, and the plasma sample pretreatment method in Example 1 was followed, and the actual plasma sample graph was obtained to evaluate the selectivity of the method.

[0054] The results showed that the peak times of abaparatide and internal standard were 2.92 min and 3.20 min, respectively. Endogenous substances in blank plasma did not interfere with the determination of internal standard and abaparatide. The typical chromatogram is shown in Figure 2-Figure 5 .

[0055] 2. Standard curve

[0056] The concentration of abaparatide in plasma (ng / mL) is used as the horizontal axis ( x ), the peak area ratio of abaparatide to the internal standard was the ordinate (y), and linear regression was performed using the weighted least squares method (weight coefficient w = 1 / x 2 )calculate.

[0057] The results showed that abaparatide had a good linear relationship in the range of 0.02-8 ng / mL, and the linear regression equation was y=-0.002509+1.08086 x (r 2 =0.9920).

[0058] 3. Precision and Accuracy

[0059] Three analytical batches were used for method validation. Each analytical batch tested four quality control (QC) samples at the lower limit of quantification (LLOQ: 0.02 ng / mL), low (LQC: 0.06 ng / mL), medium (MQC: 0.6 ng / mL), and high (HQC: 6 ng / mL) concentrations of abaloparatide. Six samples were analyzed at each concentration, and intra-day and inter-day precision and accuracy were calculated. The results are shown in Table 1:

[0060] Table 1 Intra-day and inter-day precision and accuracy results

[0061]

[0062] The results showed that the accuracy of this method for determining abaparatide was between 93.0% and 108%, and the intra-day and inter-day precisions were both <15%, which met the requirements. The minimum limit of quantification of abaparatide was 0.02 ng / mL.

[0063] 4. Matrix Effects

[0064] Low- and high-concentration quality control samples (3 each) prepared from blank plasma from 6 different sources were analyzed, and the precision and accuracy were calculated.

[0065] The results showed that the concentration analysis was not affected by the matrix effect. The accuracy (%) was 97.3±6.78 (LQC) and 100±9.40 (HQC), the precision of the single-source matrix did not exceed 9.38%, and the overall precision did not exceed 8.15%.

[0066] 5. Extraction recovery rate

[0067] Six low-, medium-, and high-concentration quality control samples, as well as six low-, medium-, and high-concentration samples spiked with blank matrix treatment, were analyzed, and the extraction recoveries of abaparatide and the internal standard were calculated.

[0068] The results showed that the extraction recoveries of abaparatide and the internal standard at each concentration level (%) were 103±13.9 (LQC), 101±8.32 (MQC), 88.7±1.95 (HQC), 96.9±9.73 (overall), and 102±8.10 (IS), respectively. The coefficients of variation of the extraction recoveries were 13.6% (LQC), 8.21% (MQC), 2.19% (HQC), 10.0% (overall), and 7.95% (IS), respectively, all of which were less than 15%, meeting the requirements.

[0069] 6. Stability

[0070] Abaloparatide quality control samples of low, medium and high concentrations were prepared using blank plasma to investigate the stability under the following conditions: (1) Plasma samples were placed at 4°C for 2 hours; (2) Plasma samples were placed in an autosampler for 17 hours after processing; (3) Plasma samples were subjected to three freeze-thaw cycles; (4) Plasma samples were stored at -80°C for 50 days. The results are shown in Table 2:

[0071] Table 2 Stability results

[0072]

[0073] The results showed that the accuracy of abaparatide plasma sample testing at various concentration levels was between 91.1% and 112%, and the precision RSD was no more than 15%, indicating that it was stable under all storage conditions.

[0074] The above test results show that the method established by the present invention has high sensitivity, strong specificity, and good precision and accuracy.

[0075] Example 3: Preclinical plasma sample testing

[0076] Abaparatide pharmacokinetic study: Six female SD rats were fasted for 12 h before the experiment and allowed to drink water freely. 24 μg·kg -1 Before and after administration of abalaparatide, 0.4 mL of blood was collected from the eye sockets at different time points, placed in a pre-cooled heparinized centrifuge tube, and immediately placed in an ice bath at 4°C and 12000 r·min -1 The mixture was centrifuged at 400 rpm for 5 min, and the supernatant plasma was collected. The concentration of abaparatide in the plasma was determined by the LC-MS / MS method established in the present invention. WinNonlin was used to calculate the main pharmacokinetic parameters using a non-compartmental model, including the elimination half-life t 1 / 2 , area under the plasma concentration-time curve AUC 0-t and AUC 0-∞ , peak concentration C max and peak time T maxwait.

[0077] Among them, the blood drug concentration-time curve is shown in Figure 6 The main pharmacokinetic parameters are shown in Table 3:

[0078] Table 3 Main pharmacokinetic parameters

[0079]

[0080] Depend on Figure 6 As shown in Table 3, rats were injected subcutaneously with 24 μg kg -1 After taking abaparatide, the peak concentration can be reached in 10 minutes, and the peak concentration is C max It is 4.16±0.528 ng / mL, which is eliminated quickly in the body, with a half-life of t 1 / 2 The in vivo exposure AUC was 20.4±2.46min. 0-t It is 99.9±12.5 min·ng / mL.

[0081] In summary, the method of the present invention can achieve rapid and sensitive detection of abaparatide in plasma, and can be applied to the pharmacokinetics and bioequivalence studies of abaparatide.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims

1. A LC-MS / MS detection method for determining abaparatide in plasma, characterized in that: The following steps are involved: (1) Pretreatment of plasma samples: Take the plasma sample containing abaparatide to be tested, thaw it at 4-8°C, and place it in an ice bath. Add the internal standard teriparatide and a methanol solution containing 0.1% (v / v) formic acid, vortex and precipitate, centrifuge, take the supernatant and put it in another centrifuge tube, blow dry the supernatant with a nitrogen blower, re-dissolve the residue with 20% (v / v) methanol, and take the supernatant after centrifugation as the sample solution to be tested; (2) Detection by liquid chromatography-mass spectrometry: Liquid chromatography conditions: chromatographic column: C 18 Column; mobile phase A: 0.3% (v / v) formic acid in water; mobile phase B: acetonitrile; gradient elution; autosampler temperature was 10°C; analysis time was 7.5 min; The mass spectrometry conditions were set as follows: the ion source used an electrospray ionization source ESI, positive ion detection, ionization voltage 3500 V, ion source temperature 350°C; the scanning mode was multiple reaction monitoring scanning MRM; In step (2), the conditions of the gradient elution are as follows: The liquid chromatography conditions are as follows: column temperature 40-50°C; injection volume 30-50 μL; flow rate 0.2-0.4 mL / min; The mass spectrometry conditions are as follows: sheath gas N2 is 35Arb, auxiliary gas N2 is 15Arb; The parameters of the multiple reaction monitoring scan MRM are as follows: 。 2. The LC-MS / MS detection method for determining abaparatide in plasma according to claim 1, characterized in that: In step (1), the volume ratio of the methanol solution containing the internal standard teriparatide and 0.1% (v / v) formic acid to the plasma sample to be tested is 3-5:

1.

3. The LC-MS / MS detection method for determining abaparatide in plasma according to claim 1, characterized in that: In step (1), the concentration of the internal standard teriparatide is 1-2 ng / mL.

4. The LC-MS / MS detection method for determining abaparatide in plasma according to claim 1, characterized in that: In step (1), the rotation speed of the vortex is 3000 rpm and the time is 1 min; the centrifugal conditions are a rotation speed of 12000 rpm, a temperature of 4° C., and a time of 5 min.

5. The LC-MS / MS detection method for determining abaparatide in plasma according to claim 1, characterized in that: In step (1), the setting temperature of the nitrogen blowdown apparatus is 40-50°C.

6. The LC-MS / MS detection method for determining abaparatide in plasma according to claim 1, characterized in that: In step (2), the liquid chromatography conditions are: column temperature 45°C; injection volume 30 μL; flow rate 0.3 mL / min.

Images