A method for detecting luperamide, descarbo-xyl-desloratadine and 3-hydroxydesloratadine in human plasma by HPLC-MS / MS

Abstract

The present application relates to a kind of HPLC-MS / MS detection method of detecting in human plasma, desloratadine and 3-hydroxydesloratadine in human plasma, it includes the following steps: (1) human plasma sample pretreatment;(2) liquid chromatography-mass spectrometry detection, using mobile phase A and mobile phase B as mixed mobile phase is carried out gradient elution, wherein: mobile phase A is methanol-acetonitrile mixed solution, in mixed solution methanol and acetonitrile volume ratio is 4-6:6-4;Mobile phase B is 2-20mM ammonium formate aqueous solution;(3) the determination of the concentration of human plasma in lopatadine, desloratadine and 3-hydroxydesloratadine.The present application uses lopatadine-d4, desloratadine-d5, 3-hydroxydesloratadine-d4 as deuterium internal standard, using Agilent, ZORBAX Eclipse XDB-Phenyl is carried out gradient elution, deuterium internal standard and the measured substance has the same retention time, chemical property and matrix effect, the reproducibility, accuracy of determining the concentration of lopatadine, desloratadine and 3-hydroxydesloratadine in plasma are good.The method of the present application can be used to evaluate the bioequivalence of lopatadine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological analysis, and particularly relates to a method for detecting luperamide, desloratadine and 3-hydroxydesloratadine in human plasma by HPLC-MS / MS. BACKGROUND

[0002] Lupatadine fumarate belongs to N-alkyl pyridine derivatives, is a new second-generation H1 antihistamine, can selectively and long-acting antagonize peripheral H1 receptors, and has the effect of antagonizing platelet activation factor. It can be used for allergic rhinitis and urticaria in adults and adolescents (over 12 years old). Lupatadine fumarate tablets are marketed in many European countries, and were initially approved for marketing in Spain in March 2003, then in most European Union countries including the United Kingdom in September 2009, and in China in August 2020.

[0003] At present, domestic literature has reported a method for determining the content of luperamide and its metabolites by liquid chromatography-mass spectrometry. For example, the literature "Simultaneous determination of luperamide concentration in human plasma by solid phase extraction-liquid chromatography-tandem mass spectrometry" (Cao Jing, Zeng Xin, Zhou Xiaolin (Hunan Children's Hospital, Changsha 410007, Hunan), disclosed a method for detecting luperamide in plasma, but did not detect its metabolites desloratadine and 3-hydroxydesloratadine, and the pretreatment method before detection was solid phase extraction, which was complicated in operation, and the minimum quantitative concentration of luperamide was 0.2 ng / mL.

[0004] The literature "LC-MS / MS method for simultaneous determination of luperamide and its active metabolite desloratadine in human plasma" (Zhang Jingjing 1 , Tian Yuan 2 , Zhang Zhenjian 2,1.Jiangsu Center for Disease Control and Prevention, Nanjing 210009, China; 2.Analytical Testing Center of China Pharmaceutical University, Nanjing 210038, China) also discloses a method for detecting lu- patid and its active metabolite desloratadine in plasma, but the method uses non-deuterated internal standard estazolam as internal standard, which cannot control the same matrix effect of analyte and internal standard, and may cause the difference between sample detection result and true value. The plasma dosage is 1000 μL, which is not suitable for large batch of analysis samples, and the metabolite 3-hydroxydesloratadine is not detected. The minimum quantification concentration of desloratadine in the detection method is 0.05 ng / mL, while the minimum quantification concentration of desloratadine in the invention is 0.01 ng / mL. The extraction recovery rates of lu- patid at low, medium and high concentration levels in the detection method are 80.22%, 82.10% and 87.40% respectively, and the extraction recovery rates of desloratadine at low, medium and high concentration levels are 80.81%, 79.91% and 82.41% respectively. The extraction recovery rates of lu- patid at low, medium and high concentration levels in the invention are 101.18%, 100.77% and 100.56% respectively, and the extraction recovery rates of desloratadine at low, medium and high concentration levels are 95.66%, 93.45% and 94.28% respectively.

[0005] Determination of Desloratadine and Its Metabolite 3-Hydroxydesloratadine in Human Plasma by LC-MS / MS (Liu Linlin 1 Xu Jianan 2 Chu Nannan 2 Chen Weili 2 Xu Hongrong 2 Li Xuening 2,1. Jiangxi Province Nanchang City First People's Hospital Pharmacy, Nanchang 330046; 2. Fudan University Affiliated Zhongshan Hospital Clinical Hospital Pharmacology Room, Shanghai 200032) also disclosed the detection method of desloratadine and its metabolite 3-hydroxydesloratadine in plasma, but the plasma dosage in the detection method is 1 mL, the plasma dosage is relatively large and is not suitable for large batch of analysis samples, and the pretreatment is liquid-liquid extraction, which needs to add 0.15 mL of sodium hydroxide solution and 6 mL of diethyl ether, then take the supernatant, and nitrogen blowing is carried out in a 40℃ water bath, which is relatively complex and the reagent dosage is relatively large; the plasma dosage of the present application is only 0.1 mL, if the plasma dosage of the detection method in the literature is changed to 0.1 mL, the final injection solution is only 20 μL, but the injection volume is 10 μL, so the method is not suitable; the minimum quantitative concentration of desloratadine and its metabolite 3-hydroxydesloratadine in the detection method in the literature is 0.05 ng / mL, while the minimum quantitative concentration of desloratadine and 3-hydroxydesloratadine in the present application is 0.01 ng / mL; the extraction recoveries of desloratadine at low, medium and high concentration levels in the detection method in the literature are 48.6%, 37.1% and 36.1% respectively, the extraction recoveries of 3-hydroxydesloratadine at low, medium and high concentration levels are 38.6%, 25.2% and 24.8% respectively, the extraction recoveries of desloratadine at low, medium and high concentration levels in the present application are 95.66%, 93.45% and 94.28% respectively, and the extraction recoveries of 3-hydroxydesloratadine at low, medium and high concentration levels are 94.08%, 93.66% and 94.07% respectively; the chromatographic peak of 3-hydroxydesloratadine in the detection method in the literature is tailing, which affects the calculation of peak area.

[0006] In order to meet the needs of clinical large batch of sample analysis and evaluation of drug bioequivalence, a simpler, more reliable, high-throughput sample pretreatment method and a method for detecting the concentrations of lumaritine, desloratadine and 3-hydroxydesloratadine in human plasma are needed. SUMMARY

[0007] The purpose of the present application is to provide a method for detecting the concentrations of lumaritine, desloratadine and 3-hydroxydesloratadine in human plasma, which has good reproducibility, high sensitivity, fast analysis speed, small matrix effect and high extraction recovery on the basis of the prior art.

[0008] The technical solution of the present application is as follows:

[0009] A method for detecting lumaritine, desloratadine and 3-hydroxydesloratadine in human plasma by HPLC-MS / MS, which comprises the following steps:

[0010] (1) Pretreatment of human plasma samples: Add internal standard working solution and precipitant to human plasma samples, and then perform centrifugation and dilution.

[0011] (2) Detection by liquid chromatography-mass spectrometry, using mobile phase A and mobile phase B as mixed mobile phases for gradient elution, wherein mobile phase A is a methanol-acetonitrile mixed solution, and the volume ratio of methanol to acetonitrile in the mixed solution is 4-6:6-4; mobile phase B is a 2-20 mM ammonium formate aqueous solution;

[0012] (3) Determination of the concentrations of rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma; gradient elution process as follows:

[0013] Within 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B is 50:50;

[0014] Within 0.5-1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 50:50 to 70:30 at a constant rate.

[0015] Within 1.0–2.6 minutes, the volume ratio of mobile phase A to mobile phase B was 70:30;

[0016] Within 2.6–3.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually and uniformly changes from 70:30 to 95:5.

[0017] Within 3.0–4.5 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5;

[0018] Within 4.5-5.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 95:5 to 50:50 at a constant rate.

[0019] Within 5.0–5.5 minutes, the volume ratio of mobile phase A to mobile phase B is 50:50.

[0020] The specific gradient elution process is shown in Table 1:

[0021] Table 1: Liquid Chromatographic Gradients of Rupatadine, Desloratadine, and 3-Hydroxydesloratadine

[0022]

[0023] In this invention, when detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS, the weak wash solution is methanol, and the strong wash solution is methanol:acetonitrile:isopropanol (1:1:1, v / v / v) (0.1% formic acid). Preferably, the volume ratio of methanol, acetonitrile, isopropanol, and formic acid in the strong wash solution is 1000:1000:1000:3.

[0024] In this invention, when detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS, in a preferred embodiment, the volume ratio of methanol to acetonitrile in mobile phase A is 1:1. Further, mobile phase B is a 10 mM ammonium formate aqueous solution. To improve chromatographic selectivity, the polarity of the mobile phase can be adjusted. Based on 100% of the total volume of the ammonium formate aqueous solution, mobile phase B contains 0.05–0.20% formic acid by volume. Without affecting the effectiveness of this invention, in a preferred embodiment, based on 100% of the total volume of the ammonium formate aqueous solution, mobile phase B contains 0.1% formic acid by volume, i.e., mobile phase B is a 0.1% formic acid-10 mM ammonium formate aqueous solution.

[0025] In this invention, when HPLC-MS / MS is used to detect rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma, an Agilent ZORBAX Eclipse XDB-Phenyl column is used. Specifically, the column has a length of 75 mm, a diameter of 4.6 mm, and a packing particle size of 3.5 μm, namely Agilent ZORBAX Eclipse XDB-Phenyl (4.6 × 75 mm, 3.5 μm).

[0026] In chromatography, the selection of the chromatographic column is crucial, requiring high column efficiency, good selectivity, and fast analysis speed. This invention employs the aforementioned mobile phase for gradient elution, using an Agilent ZORBAX Eclipse XDB-Phenyl column. Under suitable conditions, endogenous substances do not interfere with sample determination, and the method exhibits good reproducibility, high sensitivity, fast analysis speed, and minimal matrix effect.

[0027] When using the internal standard method, the selection of the internal standard is crucial. An ideal internal standard should be able to be added to the sample in an accurate and known amount, and should have essentially the same or as consistent as possible with the physicochemical properties, chromatographic behavior, and response characteristics as the analyte. Under chromatographic conditions, the internal standard must be able to be fully separated from the components in the sample. In this invention, when using HPLC-MS / MS to detect rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma, rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 were used as internal standards, respectively, with deuterated internal standards. The deuterated internal standard and the analyte have the same retention time, chemical properties, and matrix effect, resulting in good reproducibility and accuracy in determining the concentrations of rupatadine, desloratadine, and 3-hydroxydesloratadine in plasma.

[0028] In step (1), preferably, the present invention uses protein precipitation to pretreat human plasma samples, with methanol as the precipitant. Using protein precipitation to pretreat human plasma samples avoids the cumbersome and time-consuming liquid-liquid extraction process, while achieving surprisingly high recovery rates. In the present invention, protein precipitation is used to pretreat human plasma samples using an Agilent ZORBAX Eclipse XDB-Phenyl column. Under other conditions, the overall extraction recoveries of rupatadine, desloratadine, and 3-hydroxydesloratadine are 107.25%, 99.55%, and 97.74%, respectively.

[0029] In one embodiment, step (1) of the human plasma sample pretreatment includes: adding an internal standard working solution and a precipitant to the human plasma sample, vortexing and centrifuging, taking the supernatant, and mixing it with a diluent to obtain the sample to be tested; wherein, the internal standard working solution contains rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 as internal standards; and the precipitant is methanol. The diluent is a mixed solution of methanol and water, preferably, the volume ratio of methanol to water in the diluent is 40-60:6-40; more preferably, the volume ratio of methanol to water in the diluent is 50:50.

[0030] The detection method of the present invention further includes the preparation of internal standard working solutions: weigh rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 reference standards, dissolve them in methanol to obtain internal standard stock solutions with a concentration of 1.00 mg / mL, and then dilute them with a methanol-water mixture with a volume ratio of 50:50 to obtain internal standard working solutions with a concentration of 10.0 ng / mL for each of rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4.

[0031] In a more preferred embodiment, in step (1), the pretreatment of human plasma sample includes: taking 100 μL of human plasma sample, adding 50.0 μL of internal standard working solution and 250 μL of methanol, vortexing and centrifuging, taking 150 μL of supernatant, and then mixing it with 50.0 μL of diluent to obtain the sample to be tested; the volume ratio of methanol to water in the diluent is 50:50.

[0032] The present invention uses protein precipitation to pretreat human plasma samples, wherein the vortexing and centrifugation conditions are as follows: vortexing for 5 min, and centrifuging at 4000 rpm / min for 10 min at 4℃.

[0033] In this invention, the sample to be tested is placed in an autosampler for LC-MS / MS analysis during chromatographic detection. The injection volume is 10 μL and the autosampler temperature is 4 °C.

[0034] The detection method of the present invention, step (2) of using liquid chromatography-mass spectrometry for detection includes the following detailed chromatographic conditions: using an Agilent ZORBAX Eclipse XDB-Phenyl (4.6×75mm, 3.5μm) column, gradient elution is performed according to the elution process mentioned above, the column temperature is 30~45℃, preferably 40℃; the flow rate is 0.5~0.9mL / min, preferably 0.7mL / min.

[0035] The mass spectrometry conditions of this invention include: electrospray ionization source, positive ion multiple reaction monitoring (MRM) scanning, spray voltage of 3500 V, and ion source temperature of 550 °C; rupatadine, [M+H] + m / z 416.1→309.2, DP value 100V, CE value 26V; rupatadine-d4, [M+H] + m / z 420.4→284.2, DP value 100V, CE value 47V; desloratadine, [M+H] + m / z 311.1→259.1, DP value 100V, CE value 31V; desloratadine-d5, [M+H] + m / z 316.3→264.2, DP value 100V, CE value 47V; 3-hydroxydesloratadine, [M+H] + m / z 327.2→275.2, DP value 100V, CE value 31V; 3-hydroxydesloratadine-d4, [M+H] + m / z 331.4→279.2, DP value 100V, CE value 47V.

[0036] The detection method of the present invention, step (3) of determining the concentrations of rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma includes: preparing the plasma to be tested according to the sample pretreatment method in step (1), detecting it according to the liquid chromatography-mass spectrometry in step (2), recording the peak areas corresponding to rupatadine, desloratadine, and 3-hydroxydesloratadine respectively, and weighting the peak area ratios of rupatadine, desloratadine, 3-hydroxydesloratadine, and their corresponding internal standards using a weighting coefficient w = 1 / x. 2 Linear regression was performed, with the equation y = ax + b, to calculate the concentrations of rupatadine, desloratadine, and 3-hydroxydesloratadine in the plasma to be tested.

[0037] The detection method of this invention can be used for clinical pharmacokinetic sample monitoring. The steps for calculating clinical drug metabolism kinetic parameters include: calculating pharmacokinetic parameters using WinNonlin 8.0, including: C max T max t 1 / 2 AUC0-t At the same time, the mean and standard deviation of each parameter are calculated.

[0038] The advantages of using the technical solution of this invention are as follows:

[0039] (1) The present invention can simultaneously determine the concentrations of rupatadine, desloratadine and 3-hydroxydesloratadine in human plasma.

[0040] (2) The detection method of the present invention uses deuterium as an internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties and matrix effect. The reproducibility and accuracy of determining the concentrations of rupatadine, desloratadine and 3-hydroxydesloratadine in plasma are good.

[0041] (3) The detection method of the present invention uses a methanol-acetonitrile-isopropanol mixed solution as the washing solution (e.g., volume ratio of 1:11:1) during the gradient elution process, which can effectively remove residues and make the detection results accurate and reliable.

[0042] (4) The sample volume used in this detection method is only 100 μL, which is relatively small and suitable for large-scale plasma sample detection.

[0043] (5) In the detection method of the present invention, the linear range of rupatadine is 50.0 pg / mL to 40,000 pg / mL, the linear range of desloratadine is 10.0 pg / mL to 8,000 pg / mL, and the linear range of 3-hydroxydesloratadine is 10.0 pg / mL to 8,000 pg / mL. This linear range is relatively wide, the limit of quantitation is low, and the application range is relatively wide.

[0044] (6) The extraction and recovery rates of rupatadine, desloratadine and 3-hydroxydesloratadine in the detection method of the present invention are high, all around 100%.

[0045] (7) In the detection method of the present invention, the retention times of rupatadine, desloratadine and 3-hydroxydesloratadine are appropriate and the peak shapes are good.

[0046] (8) The detection method of the present invention selects a specific mobile phase and optimizes the elution time and the ratio of the mobile phase during gradient elution. It has the advantages of good reproducibility, high sensitivity, fast analysis speed, small matrix effect and high recovery rate. It has also conducted full method validation including specificity, accuracy, precision, matrix effect, extraction recovery rate and stability. It can be reliably used to evaluate the bioequivalence of rupatadine in humans. Attached Figure Description

[0047] Figure 1-1 This is a scan of the Lupata stator ion.

[0048] Figure 1-2This is a scan of desloratadine stator ions;

[0049] Figure 1-3 This is a scan of the 3-hydroxydechloratadine ion;

[0050] Figure 2-1 This is a scan of the lumatadin-d4 ion;

[0051] Figure 2-2 This is a scan of the desloratadine-d5 ion;

[0052] Figure 2-3 This is a scan of the 3-hydroxydesloratadine-d4 ion;

[0053] Figure 3 is a specific chromatogram for the determination of rupatadine / desloratadine / 3-hydroxydesloratadine-d4 in plasma by LC-MS / MS.

[0054] (3-1~3-18) Chromatograms of blank plasma from 6 batches of different individuals;

[0055] in, Figures 3-1 to 3-6 In each figure, the chromatogram on the left is rupatadine, and the chromatogram on the right is rupatadine-d4;

[0056] in, Figures 3-7 to 3-12 In each figure, the chromatogram on the left is desloratadine, and the chromatogram on the right is desloratadine-d5;

[0057] in, Figures 3-13 to 3-18 In each figure, the chromatogram on the left is 3-hydroxydesloratadine, and the chromatogram on the right is 3-hydroxydesloratadine-d4;

[0058] Figure 4 is a chromatogram of mixed blank plasma;

[0059] in, Figure 4-1 The chromatogram on the left is rupatadine, and the chromatogram on the right is rupatadine-d4;

[0060] in, Figure 4-2 The chromatogram on the left is desloratadine, and the chromatogram on the right is desloratadine-d5;

[0061] in, Figure 4-3 The chromatogram on the left is 3-hydroxydesloratadine, and the chromatogram on the right is 3-hydroxydesloratadine-d4;

[0062] Figure 5 is the chromatogram of a sample with the lower limit of quantitation.

[0063] in, Figure 5-1 The chromatogram on the left is rupatadine, and the chromatogram on the right is rupatadine-d4;

[0064] in, Figure 5-2The chromatogram on the left is desloratadine, and the chromatogram on the right is desloratadine-d5;

[0065] in, Figure 5-3 The chromatogram on the left is 3-hydroxydesloratadine, and the chromatogram on the right is 3-hydroxydesloratadine-d4;

[0066] Figure 6 This document describes the simultaneous determination of the concentrations of rupatadine and its active metabolite dilatadine in human plasma using LC-MS / MS (Zhang Jingjing). 1 Tian Yuan 2 Zhang Zunjian 2 Chromatograms from (1. Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, Jiangsu 210009; 2. Analytical and Testing Center of China Pharmaceutical University, Nanjing, Jiangsu 210038), where 1 is desloratadine, 2 is 3-hydroxydesloratadine, 3 is internal standard desloratadine-D4, and 4 is internal standard 3-hydroxydesloratadine-D4. Detailed Implementation

[0067] The detection method of the present invention will be further described through the following embodiments and in conjunction with the accompanying drawings, but these embodiments do not constitute any limitation on the present invention.

[0068] Materials and Methods

[0069] 1. Instruments and reagents

[0070] High-performance liquid chromatography (Shimadzu LC-30AD series); mass spectrometry (API 5500, Applied Biosystems / Sciex); pure water system (Milli-Q, Merck Millipore); microbalance (XP6, METTLER TOLEDO); centrifuge (Heraeus Muitifuge X1R, Thermo Fisher); micro-orifice shaker (MIX-1500, Hangzhou Mio Instrument Co., Ltd.).

[0071] Methanol (Merck, HPLC grade), acetonitrile (Merck, HPLC grade), water (ultrapure water, lab-made), formic acid (Aladdin, HPLC grade), ammonium formate (Aladdin, HPLC grade), isopropanol (Sinopharm Chemical Reagent Co., Ltd., HPLC grade). Blank plasma was obtained from healthy subjects. Rupatadine fumarate (TRC, batch number: 3-AZP-170-1), desloratadine (TRC, batch number: 20-GHZ-17-1), 3-hydroxydesloratadine (TLC, batch number: 3794-043A1), rupatadine fumarate-d4 (TRC, batch number: 7-DRR-162-1), desloratadine-d5 (TLC, batch number: 2246-083A5), 3-hydroxydesloratadine-d4 (TLC, batch number: 2211-086A5).

[0072] 2. Liquid chromatography-mass spectrometry (LC-MS) conditions

[0073] Liquid chromatography conditions: Column: Agilent, ZORBAX Eclipse XDB-Phenyl (4.6 × 75 mm, 3.5 μm); Column temperature: 40℃; Injector temperature: 4℃; Mobile phase A: methanol-acetonitrile (1:1, v / v); Mobile phase B: 0.1% formic acid-10 mM ammonium formate aqueous solution (based on a total volume of 10 mM ammonium formate aqueous solution of 100%, containing a volume ratio of 0.1% formic acid); Gradient elution process as follows: Within 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B is 50:50; Within 0.5-1.0 minutes, the volume ratio of mobile phase A to mobile phase B changes from 50:50 to 50:50. The volume ratio of mobile phase A to mobile phase B was gradually changed from 70:30 to 70:50 over 1.0–2.6 minutes; from 2.6–3.0 minutes, it gradually changed to 95:5; from 3.0–4.5 minutes, it remained at 95:5; from 4.5–5.0 minutes, it gradually changed to 50:50; and from 5.0–5.5 minutes, it remained at 50:50. The flow rate was 0.7 mL / min; the wash method was Rinse Pump Then Port; the weak wash buffer was methanol; the wash volume was 1000 μL; and the strong wash buffer was methanol:acetonitrile:isopropanol (1:1:1, v / v / v) (0.1% formic acid).

[0074] Mass spectrometry conditions: Ion detection mode: Multiple reaction monitoring (MRM); Ionization mode: Pneumatic-assisted electrospray ionization (ESI); Ion polarity: Positive; Detector: Rupatadine, [M+H] +m / z 416.1→309.2, DP value 100V, CE value 26V; rupatadine-d4, [M+H] + m / z 420.4→284.2, DP value 100V, CE value 47V; desloratadine, [M+H] + m / z 311.1→259.1, DP value 100V, CE value 31V; desloratadine-d5, [M+H] + m / z 316.3→264.2, DP value 100V, CE value 47V; 3-hydroxydesloratadine, [M+H] + m / z 327.2→275.2, DP value 100V, CE value 31V; 3-hydroxydesloratadine-d4, [M+H] + m / z 331.4→279.2, DP value 100V, CE value 47V. Mass spectrometry parameters: IonSpray Voltage: 3500V; TEM: 550℃. The specific ion scans for rupatadine, desloratadine, 3-hydroxydesloratadine, and rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 are shown below. Figures 1-1 to 1-3 and Figures 2-1 to 2-3 As shown.

[0075] 3. Preparation of standard solutions

[0076] Preparation of analyte working solutions: Accurately weigh two portions each of rupatadine, desloratadine, and 3-hydroxydesloratadine reference standards, correct them with mass correction factors, and dissolve them in methanol to obtain two stock solutions of each analyte with a concentration of 1.00 mg / mL. The stock solutions are stored in a -20°C freezer. After the stock solutions passed inspection, rupatadine, desloratadine, and 3-hydroxydesloratadine stock solutions were accurately measured and diluted with methanol:water (50:50, v / v) to prepare a series of rupatadine / desloratadine / 3-hydroxydesloratadine concentrations of 1,000 / 200 / 200 pg / mL (LLOQ), 2,000 / 400 / 400 pg / mL, 5,000 / 1,000 / 1,000 pg / mL, 50,000 / 10,000 / 10,000 pg / mL, 150,000 / 30,000 / 30,000 pg / mL, 400,000 / 80,000 / 80,000 pg / mL, 640,000 / 128,000 / 128,000 / 128,000 pg / mL, and 3-hydroxydesloratadine / 3-hydroxydesloratadine concentrations. Standard curve sample working solutions of 800,000 / 160,000 / 160,000 pg / mL and 800,000 / 160,000 / 160,000 pg / mL were prepared. Another portion of rupatadine, desloratadine and 3-hydroxydesloratadine stock solutions were accurately measured and diluted with methanol:water (50:50, v / v) to prepare quality control working solutions with rupatadine concentration / desloratadine concentration / 3-hydroxydesloratadine concentration of 2,500 / 500 / 500 pg / mL (LQC), 30,000 / 6,000 / 6,000 pg / mL (GMQC), 350,000 / 70,000 / 70,000 pg / mL (MQC), and 600,000 / 120,000 / 120,000 pg / mL (HQC).

[0077] Preparation of internal standard working solutions: Accurately weigh rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 reference standards, correct for mass, and dissolve in methanol to obtain internal standard stock solutions with a concentration of 1.00 mg / mL. Store the stock solutions at -20°C. Accurately measure a certain amount of the internal standard stock solution and dilute with methanol:water (50:50, v / v) to prepare internal standard working solutions with a concentration of 10.0 ng / mL for each of rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4.

[0078] 4. Preparation of standard curve samples and quality control samples

[0079] For the standard curve samples and quality control samples at each concentration level, the preparation process is as follows: Add 20.0 μL of the corresponding working solution to 380 μL of blank plasma, mix well, and adjust the volume as needed. Prepare samples with rupatadine / deloratadine / 3-hydroxydeloratadine concentrations of 50.0 / 10.0 / 10.0 pg / mL (LLOQ), 100 / 20.0 / 20.0 pg / mL, and 250 / 50 pg / mL, respectively. Standard curve samples and concentrations of 0.0 / 50.0 pg / mL, 2,500 / 500 / 500 pg / mL, 7,500 / 1,500 / 1,500 pg / mL, 20,000 / 4,000 / 4,000 pg / mL, 32,000 / 6,400 / 6,400 pg / mL and 40,000 / 8,000 / 8,000 pg / mL were prepared, along with LLOQ values ​​of 50.0 / 10.0 / 10.0 pg / mL. Quality control samples with values ​​of 125 / 25.0 / 25.0 pg / mL (LQC), 1,500 / 300 / 300 pg / mL (GMQC), 17,500 / 3,500 / 3,500 pg / mL (MQC), and 30,000 / 6,000 / 6,000 pg / mL (HQC).

[0080] 5. Sample pretreatment

[0081] Add 100 μL of sample (biological sample to be tested, standard curve sample, quality control sample) to a 96-well plate; for double blank samples and blank samples, add 100 μL of blank matrix. Add 50.0 μL of solvent methanol:water (1:1, v / v) to the double blank samples. Except for the double blank samples, add 50.0 μL of internal standard working solution (rupatadine concentration-d4 / deloratadine concentration-d5 / 3-hydroxydeloratadine concentration-d4: 10.0 / 10.0 / 10.0 ng / mL) to all wells, then add 250 μL of precipitant methanol. Vortex the 96-well plate at 1000 rpm / min for 5 min, and then centrifuge the 96-well plate at 4000 rpm / min for 10 min at 4 °C. Add 150 μL of supernatant to a clean 96-well plate, then add 50.0 μL of diluent methanol:water (1:1, v / v), mix well, and place in the injection chamber or a refrigerator at the same temperature for testing.

[0082] 6. Methodological Examination Content

[0083] The detection method was validated according to the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in the 2020 edition of the Chinese Pharmacopoeia to ensure the accuracy, repeatability, and stability of the assay. Validation included the following: specificity, standard curve, precision and accuracy, matrix effect, extraction recovery, and stability.

[0084] Results and Discussion

[0085] 1. Exclusivity

[0086] Under the chromatographic conditions used in this experiment, the retention time of rupatadine was approximately 2.58 min. Figure 5-1 The retention time of desloratadine is approximately 2.25 minutes. Figure 5-2 The retention time of 3-hydroxydesloratadine is approximately 1.97 min. Figure 5-3 The retention time of the internal standard rupatadine-d4 is approximately 2.55 minutes. Figure 4-1 The retention time of the internal standard desloratadine-d5 is approximately 2.23 min. Figure 4-2 The retention time of the internal standard 3-hydroxydesloratadine-d4 is approximately 1.96 min. Figure 4-3 Take 100 μL of blank plasma from each of six different sources. Except for the absence of an internal standard, perform sample pretreatment procedures to obtain the chromatograms of the blank plasma samples, as shown below. Figures 3-1 to 3-18 The chromatogram of the sample with the lower limit of quantitation is shown in [reference needed]. Figures 5-1 to 5-3 The results indicate that endogenous substances in plasma do not affect the detection of rupatadine, desloratadine, and 3-hydroxydesloratadine. Furthermore, internal standards also do not affect the detection of rupatadine, desloratadine, and 3-hydroxydesloratadine.

[0087] 2. Accuracy and precision tests

[0088] Prepare solutions containing 50.0 / 10.0 / 10.0 pg / mL of rupatadine / deloratadine / 3-hydroxydeloratadine. Quality control samples were prepared at concentrations of 125 / 25.0 / 25.0 pg / mL (LQC), 1,500 / 300 / 300 pg / mL (GMQC), 17,500 / 3,500 / 3,500 pg / mL (MQC), and 30,000 / 6,000 / 6,000 pg / mL (HQC). Six samples were prepared for each concentration, and two standard curves were prepared (obtained by regression analysis of two sets of standard curve samples). The ratio f of the peak area As of rupatadine, desloratadine, and 3-hydroxydesloratadine to the corresponding internal standard peak area Ai was calculated and denoted as f. Substituting f into the standard curve of the day, the measured concentration, the average measured concentration, and the accuracy were obtained. Intra-batch precision and accuracy were calculated, and the results are shown in Table 2. The results showed that, except for the limit of quantitation (LLOQ), the RSD of the intra-batch quality control samples for rupatadine, desloratadine, and 3-hydroxydesloratadine was less than 15%, the RE was at least 67% not exceeding ±15%, and at least 50% of the quality control samples at each concentration level deviated from their theoretical values ​​by no more than ±15%. For the LLOQ samples, the RSD of the intra-batch quality control samples for rupatadine, desloratadine, and 3-hydroxydesloratadine was less than 20%, the RE was at least 67% not exceeding ±20%, and at least 50% of the quality control samples at each concentration level deviated from their theoretical values ​​by no more than ±20%. In summary, both precision and accuracy met the requirements.

[0089] Table 2 Precision and accuracy of intra-batch and inter-batch sample testing

[0090] Analyte: Lupatadine

[0091]

[0092]

[0093] Analyte: desloratadine

[0094]

[0095]

[0096] Analyte: 3-hydroxydesloratadine

[0097]

[0098] 3. Investigation of matrix effect

[0099] Matrix sample preparation: Six batches of blank plasma from different donors were used, and nine replicate double blank samples were prepared from each batch of blank plasma. The blank matrix extract was obtained by following the sample pretreatment procedure. After extraction, a certain amount of analyte and internal standard were added to make the final concentration consistent with the injection concentration of the low, medium and high concentration quality control samples (three replicates for each concentration level).

[0100] Sample preparation: Pure water was used instead of blank plasma for pretreatment. The working solution was then diluted to the appropriate concentration so that the concentration after adding 100 μL of blank matrix extract or 100 μL of pure water extract was consistent with the injection concentration of the low, medium, and high concentration quality control samples after pretreatment. Three replicate samples were prepared for each concentration.

[0101] The results showed that the total matrix effect factor (calculated as peak area ratio) for rupatadine was 99.7–102.6, with a precision of less than 1.3%; the total matrix effect factor (calculated as peak area ratio) for desloratadine was 98.5–102.0, with a precision of less than 2.2%; and the total matrix effect factor (calculated as peak area ratio) for 3-hydroxydesloratadine was 98.9–103.2, with a precision of less than 2.0%. The plasma matrix did not affect the accurate quantification of rupatadine, desloratadine, and 3-hydroxydesloratadine. The matrix effect data are shown in Table 3.

[0102] Table 3 Matrix Effects

[0103] Analyte: Lupatadine

[0104]

[0105] Analyte: desloratadine

[0106]

[0107]

[0108] Analyte: 3-hydroxydesloratadine

[0109]

[0110] 4. Analysis of extraction recovery rate

[0111] Matrix sample preparation: Plasma was prepared by mixing blank plasma from 6 batches of different donors to form 9 replicate double blank samples. The blank plasma extract was obtained by following the sample pretreatment procedure. After extraction, a certain amount of analyte and internal standard were added to make the final concentration consistent with the injection concentration of the low, medium and high concentration quality control samples (3 replicates for each concentration level).

[0112] Quality control sample preparation: Take three concentrations of quality control samples (low, medium, and high) and process them according to the sample processing method. Prepare 6 samples for each concentration level.

[0113] Recovery is evaluated by comparing the response values ​​of the analyte or internal standard in a single quality control sample with the mean response values ​​of a double blank sample after extraction with the addition of the analyte and internal standard.

[0114] The acceptance criteria for recovery were: the precision of the recovery at each concentration level and across all concentration levels should be within 15.0%. The recovery rate of rupatadine (calculated as peak area ratio) was 100.84%, with recoveries of 101.18%, 100.77%, and 100.56% at low, medium, and high concentrations, respectively. The recovery rate of desloratadine (calculated as peak area ratio) was 94.46%, with recoveries of 95.66%, 93.45%, and 94.28% at low, medium, and high concentrations, respectively. The recovery rate of 3-hydroxydesloratadine (calculated as peak area ratio) was 93.94%, with recoveries of 94.08%, 93.66%, and 94.07% at low, medium, and high concentrations, respectively. The results are shown in Table 4.

[0115] Table 4 Extraction Recovery Rate

[0116] Analyte: Lupatadine

[0117]

[0118] Analyte: desloratadine

[0119]

[0120]

[0121] Analyte: 3-hydroxydesloratadine

[0122]

[0123] 5. Stability Study

[0124] Stability of processed samples: After the first injection analysis of the analytical batch to examine precision and accuracy, the samples were placed in the autosampler (4℃) for 171 h. Freshly prepared standard curve samples and previously analyzed samples were then injected and analyzed, and the chromatograms were recorded. The results are shown in Table 5. The injection solutions of rupatadine, desloratadine and 3-hydroxydesloratadine plasma samples showed good stability after being placed in the autosampler for 171 h, which meets the requirements for biological sample analysis.

[0125] Room temperature stability: The prepared low and high concentration levels of quality control samples, containing rupatadine / deloratadine / 3-hydroxydeloratadine concentrations of 125 / 25.0 / 25.0 pg / mL and 30,000 / 6,000 / 6,000 pg / mL respectively, were mixed thoroughly and left at room temperature for 26 h before LC-MS / MS analysis. The chromatograms were recorded, and the results are shown in Table 6. The plasma samples showed good stability after being left at room temperature for 26 h.

[0126] Freeze-thaw stability: Freshly prepared samples containing 125 / 25.0 / 25.0 pg / mL of rupatadine / deloratadine / 3-hydroxydeloratadine and 30,000 / 6,000 / 6,000 pg / mL were subjected to four freeze-thaw cycles at -80°C. Acceptance criteria were: the %RE (mean reactivity ratio) of the average measured value of the stable sample should not exceed ±15.0% from its theoretical value, and the %RSD (mean relative standard deviation) of the measured values ​​of the stable samples at each concentration level should be ≤15.0%. The results are shown in Table 7. The samples exhibited good stability after four freeze-thaw cycles at -80°C.

[0127] Long-term stability: Freshly prepared samples containing 125 / 25.0 / 25.0 pg / mL of rupatadine / desloratadine / 3-hydroxydesloratadine and 30,000 / 6,000 / 6,000 pg / mL were stored at -80°C for 106 days before testing. Acceptance criteria were: the %RE (mean reactivity ratio) of the average measured value of the stable sample should not exceed ±15.0%, and the %RSD (mean relative standard deviation) of the measured values ​​of the stable samples at each concentration level should be ≤15.0%. The results are shown in Table 8. The samples showed good stability after being stored at -80°C for 106 days.

[0128] Table 5. Stability of treated samples

[0129] Analyte: Lupatadine

[0130]

[0131] Analyte: desloratadine

[0132]

[0133] Analyte: 3-hydroxydesloratadine

[0134]

[0135] Table 6. Stability during biological sample pretreatment (room temperature stability)

[0136] Analyte: Lupatadine

[0137]

[0138]

[0139] Analyte: desloratadine

[0140]

[0141] Analyte: 3-hydroxydesloratadine

[0142]

[0143]

[0144] Table 7 Freeze-thaw stability

[0145] Analyte: Lupatadine

[0146]

[0147] Analyte: desloratadine

[0148]

[0149] Analyte: 3-hydroxydesloratadine

[0150]

[0151] Table 8 Long-term stability

[0152] Analyte: Lupatadine

[0153]

[0154] Analyte: desloratadine

[0155]

[0156]

[0157] Analyte: 3-hydroxydesloratadine

[0158]

[0159] This invention establishes an HPLC-MS / MS method for the determination of rupatadine, desloratadine, and 3-hydroxydesloratadine in plasma. This method exhibits good specificity, and endogenous substances in plasma do not interfere with the determination of samples. The linear range of the rupatadine standard curve is 50.0 pg / mL to 40,000 pg / mL, the linear range of the desloratadine standard curve is 10.0 pg / mL to 8,000 pg / mL, and the linear range of the 3-hydroxydesloratadine standard curve is 10.0 pg / mL to 8,000 pg / mL, all showing good linearity: rupatadine concentration / desloratadine concentration / The intra-assay and inter-assay precision of the quality control samples at three concentration levels of 3-hydroxydesloratadine—high (30,000 / 6,000 / 6,000 pg / mL), medium (17,500 / 3,500 / 3,500 pg / mL), mid-medium (1,500 / 300 / 300 pg / mL), and low (125 / 25.0 / 25.0 pg / mL)—were all less than 15.0%. The intra-assay and inter-assay precision of the quality control samples at the limit of quantitation (50.0 / 10.0 / 10.0 pg / mL) were both less than 20.0%.

[0160] The total matrix effect factor (MIF) of rupatadine ranged from 99.7 to 102.6, with a precision of less than 1.3%. The plasma matrix did not affect the accurate quantification of rupatadine. The MIF of desloratadine ranged from 98.5 to 102.0, with a precision of less than 2.2%. The plasma matrix did not affect the accurate quantification of desloratadine. The MIF of 3-hydroxydesloratadine ranged from 98.9 to 103.2, with a precision of less than 2.0%. The plasma matrix did not affect the accurate quantification of 3-hydroxydesloratadine. The extraction recoveries of rupatadine, desloratadine, and 3-hydroxydesloratadine were 100.84%, 94.46%, and 93.94%, respectively. The plasma samples of rupatadine / desloratadine / 3-hydroxydesloratadine showed good stability after being placed at room temperature for 24 hours; good stability after 4 cycles of freezing / thawing; good stability after being placed in an autosampler at 4°C for 171 hours; and good stability after being placed at -80°C for 106 days, meeting the requirements for biological sample analysis.

[0161] In summary, the HPLC-MS / MS method established by this invention for determining the concentrations of rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma meets the relevant requirements of the 2020 edition of the Pharmacopoeia "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples" and can be used for plasma sample analysis and detection in clinical trials.

[0162] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS, characterized in that, It includes the following steps: (1) Pretreatment of human plasma samples includes: adding internal standard working solution and precipitant to human plasma samples, vortexing and centrifuging, taking the supernatant, and mixing it with diluent to obtain the sample to be tested; the internal standard working solution contains rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 as internal standards; the precipitant is methanol; the diluent is a mixed solution of methanol and water. (2) Detection by liquid chromatography-mass spectrometry, using mobile phase A and mobile phase B as mixed mobile phases for gradient elution. Mobile phase A is a methanol-acetonitrile mixed solution with a volume ratio of 1:1; mobile phase B is a 10 mM ammonium formate aqueous solution, containing 0.05~0.20% formic acid with a volume ratio of 100% based on the total volume of the ammonium formate aqueous solution; the chromatographic column in the liquid chromatography is an Agilent ZORBAX Eclipse XDB-Phenyl; (3) Determination of the concentrations of rupatadine, desloratadine and 3-hydroxydesloratadine in human plasma; the gradient elution process is as follows: Within 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B is 50:50; Within 0.5-1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 50:50 to 70:30 at a constant rate. Within 1.0–2.6 minutes, the volume ratio of mobile phase A to mobile phase B was 70:30; Within 2.6–3.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually and uniformly changes from 70:30 to 95:

5. Within 3.0–4.5 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; Within 4.5-5.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 95:5 to 50:50 at a constant rate. Within 5.0–5.5 minutes, the volume ratio of mobile phase A to mobile phase B is 50:50; During gradient elution, the weak washing solution is methanol; the strong washing solution is a mixed solution of methanol-acetonitrile-isopropanol-formic acid. Mass spectrometry conditions included: electrospray ionization source, positive ion multiple reaction monitoring (MRM), spray voltage 3500 V, ion source temperature 550 °C; rupatadine, [M+H] + m / z 416.1→309.2, DP value 100 V, CE value 26 V; rupatadine-d4, [M+H] + m / z 420.4→284.2, DP value 100 V, CE value 47 V; desloratadine, [M+H] + m / z 311.1→259.1, DP value 100 V, CE value 31 V; desloratadine-d5, [M+H] + m / z 316.3→264.2, DP value 100 V, CE value 47 V; 3-hydroxydesloratadine, [M+H] + m / z 327.2→275.2, DP value 100 V, CE value 31 V; 3-hydroxydesloratadine-d4, [M+H] + m / z 331.4→279.2, DP value 100 V, CE value 47 V.

2. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 1, characterized in that, The volume ratio of methanol, acetonitrile, isopropanol and formic acid in the strong washing solution is 1000:1000:1000:

3.

3. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 1, characterized in that, In mobile phase B, based on a total volume of 100% ammonium formate aqueous solution, there is a volume ratio of 0.1% formic acid.

4. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 1, characterized in that, The liquid chromatography conditions included: a column length of 75 mm, a diameter of 4.6 mm, a packing particle size of 3.5 µm, a column temperature of 30–45 °C, and a flow rate of 0.5–0.9 mL / min.

5. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 4, characterized in that, The liquid chromatography conditions included: column temperature of 40℃ and flow rate of 0.7 mL / min.

6. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 1, characterized in that, The volume ratio of methanol to water in the diluent is 40~60:6~40.

7. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 6, characterized in that, The volume ratio of methanol to water in the diluent is 50:

50.

8. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 1, characterized in that, In step (1), the internal standard working solution is prepared as follows: Weigh rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4 reference standards, dissolve them in methanol to obtain internal standard stock solutions with a concentration of 1.00 mg / mL, and then dilute them with a methanol-water mixture with a volume ratio of 50:50 to obtain internal standard working solutions with a concentration of 10.0 ng / mL for each of rupatadine-d4, desloratadine-d5, and 3-hydroxydesloratadine-d4.

9. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 8, characterized in that, In step (1), the pretreatment of human plasma samples includes: taking 100 μL of human plasma sample, adding 50.0 μL of internal standard working solution and 250 µL of methanol, vortexing and centrifuging, taking 150 µL of supernatant, and then mixing it with 50.0 μL of diluent to obtain the sample to be tested; the volume ratio of methanol to water in the diluent is 50:

50.

10. The method for detecting rupatadine, desloratadine, and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS according to claim 9, characterized in that, The conditions for vortexing and centrifugation are as follows: vortex for 5 min, centrifuge at 4000 rpm / min for 10 min at 4℃; place the sample to be tested in an autosampler for LC-MS / MS analysis, with an injection volume of 10 μL and an autosampler temperature of 4℃.

11. The method for detecting rupatadine, desloratadine and 3-hydroxydesloratadine in human plasma using HPLC-MS / MS as described in claim 1 is used for sample monitoring of clinical pharmacokinetics.

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