Qualitative and quantitative determination of anaprazole and its major metabolites in human plasma

Journal Pre-proof Qualitative and quantitative determination of anaprazole and its major metabolites in human plasma Chongzhuang Tang, Liang Li, Xifeng Ma, Jin Wang, Bo Chen, Xiaojian Dai, Yifan Zhang, Xiaoyan Chen

PII:

S0731-7085(19)32465-3

DOI:

https://doi.org/10.1016/j.jpba.2020.113146

Reference:

PBA 113146

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

9 October 2019

Revised Date:

30 December 2019

Accepted Date:

4 February 2020

Please cite this article as: Tang C, Li L, Ma X, Wang J, Chen B, Dai X, Zhang Y, Chen X, Qualitative and quantitative determination of anaprazole and its major metabolites in human plasma, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113146

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Qualitative and quantitative determination of anaprazole and its major metabolites in human plasma. Chongzhuang Tanga,b, Liang Lia, Xifeng Mac, Jin Wangc, Bo Chenc, Xiaojian Daia, Yifan Zhanga, Xiaoyan Chena,b a

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road,

Shanghai 201203, P.R. China University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,

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b

China c

XuanZhu Pharma, 2518 Tianchen Street, Jinan, Shandong, China

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Corresponding author:

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Xiaoyan Chen, Ph. D.

Shanghai 201203, China.

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Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike road,

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Highlighta

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E-mail: [email protected]

A total of 14 metabolites from anaprazole, a novel proton pump inhibitor, in

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human plasma were first investigated using UPLC-UV/Q-TOF MS, where the UV chromatograms provided a reliable relative amount of the metabolites.

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A characteristic loss of SH was applied to characterize thioether-related metabolites.

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Anaprazole and its four major metabolites were simultaneously determined by LC-MS/MS in human plasma.

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Metabolites with exact mass difference of 0.04 Da were chromatographically 1

separated to avoid mutual crosstalk in MS/MS analysis.

Abstract Anaprazole is a novel proton pump inhibitor under development for the treatment of gastric and duodenal ulcers. In the present study, an ultra-performance liquid chromatography-ultraviolet detector/quadrupole time-of-flight mass spectrometry method was developed for the metabolic profiling of human plasma after an oral

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administration of 40 mg anaprazole. The principal metabolic pathways were identified as sulfoxide reduction to thioether (M8-1), dehydrogenation (M21-1), sulfoxide

oxidation to sulfone (M16-3), and sulfoxide reduction with O-demethylation to form

carboxylic acid (M7-1). Anaprazole, M8-1, M16-3, M21-1, and M7-1 were selected

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and further quantified in human plasma by using a rapid and sensitive liquid

chromatography–tandem mass spectrometry method. Anaprazole and its four

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metabolites were extracted from 50 of μL plasma by acetonitrile protein precipitation. Chromatographic retention and separation were achieved on an Kinetex XB-C18 column

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(50 mm × 4.6 mm i.d., 5 μm) under gradient elution using 5 mM ammonium acetate with 0.005% ammonium hydroxide and methanol with 0.005% ammonium hydroxide

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as the mobile phase. Positive electrospray ionization was performed using multiple reaction monitoring with transitions of m/z 402.2→242.2, 386.2→226.2, 400.2→242.2, 418.2→282.2, and 386.2→161.2 for anaprazole, M8-1, M21-1, M16-3, and M7-1,

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respectively. This method was linear in the range of 5.00–3000 ng/mL for anaprazole and 1.00–600 ng/mL for the four metabolites. The lower limit of quantitation was

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established at 5.00 ng/mL for anaprazole and 1.00 ng/mL for the metabolites. The quantitative method was used to evaluate the pharmacokinetics of anaprazole in phase I clinical trials.

Keywords: Metabolite identification; LC-MS/MS; UPLC-UV/Q-TOF; Anaprazole

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1. Introduction Proton pump inhibitors (PPIs), a series of weak basic benzimidazole drugs, can be enriched in the acidic compartment of the parietal cell and be protonated to convert into active electrophilic intermediates. These active intermediates of PPIs covalently bind to gastric hydrogen/potassium adenosine triphosphatase enzyme (H+/K+ ATPase) and directly inhibit H+ secretion into the gastric lumen, resulting in strong and long-lasting reduction of gastric acid production[1]. Therefore, PPIs are developed for the treatment of patients with gastric acid-related diseases[2]. In recent years, researchers have also

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found that PPIs have inhibitory effects on tumor cell growth, metastasis, chemoresistance, and autophagy[3]. Given the safety, tolerability, and potential

selectivity for targeting the acidic microenvironment of tumors, PPIs are likely to serve as antitumor drugs in the future.

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PPIs are mainly eliminated by metabolism in the body[4]. According to the difference in metabolic properties, the marketed PPIs are divided into two categories:

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those mainly eliminated by oxidative metabolism and those mainly eliminated by reductive metabolism. The first type of PPIs, including omeprazole, lansoprazole, and

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pantoprazole, are oxidized mainly by cytochrome P450s, especially CYP2C19 and CYP3A4, in the liver to produce corresponding hydroxylated and sulfone metabolites.

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Therefore, the pharmacokinetic and pharmacodynamic properties of these PPIs and their metabolites are susceptible to individual CYP2C19 gene polymorphism[5]. There is also a class represented by rabeprazole and ilaprazole, which mainly undergo

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reductive metabolism in vivo, and their pK properties and acid suppression abilities are less affected by the polymorphism of CYP2C19[6].

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The drug metabolites in the systemic circulation are often associated with efficacy

and/or toxicity. The thioether metabolites of PPIs have certain pharmacological activities. Rabeprazole thioether was reported to markedly inhibit the migration of Helicobacter pylori[7], which is associated with various upper gastrointestinal diseases, including chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer[8]. Rabeprazole thioether also has a stronger 3

inhibitory effect on several CYP enzymes compared with other PPIs, such as omeprazole, pantoprazole, lansoprazole, and its parent drug[9]. Lansoprazole kills Mycobacterium tuberculosis by targeting its cytochrome bc1 complex through the reduction of intracellular sulfoxide to lansoprazole thioether[10]. In a previous study, we found that thioether metabolites are the intermediates of the in vivo chiral inversion of PPIs[11]. Anaprazole is a structural analogue of rabeprazole. It is currently in phase II clinical trials and is administered orally in the form of enteric-coated tablets. To date, no literature has reported the metabolism and pharmacokinetic properties of anaprazole

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in humans. The present study aims to identify the major metabolites of anaprazole in human plasma

by

using

an

ultra-performance

liquid

chromatography–ultraviolet

detector/quadrupole time-of-flight mass spectrometry (UPLC-UV/Q-TOF MS) system

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and to establish a rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to determine anaprazole and its main

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metabolites in humans. The validated method was used to study the pharmacokinetic properties of anaprazole and its major metabolites after a single oral administration of

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anaprazole enteric-coated tablet in healthy Chinese adult subjects in phase I clinical

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trials.

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2. Materials and Methods 2.1. Materials (R)-anaprazole sodium (M0, 98.8% purity), M8-1 (99.8% purity), M21-1 (97.3% purity), M16-3 (98.7% purity), M7-1 (98.6% purity), and stable isotope-labeled internal standards (IS; 13

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C,d3-anaprazole (94.1% purity),

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C,d3-M8-1 (98.0% purity), and

C,d3-M16-3 (97.4% purity)) were kindly provided by Xuanzhu Pharmaceutical Co.,

Ltd. (Shandong, China). HPLC-grade methanol, acetonitrile, ammonium acetate, and ammonium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO, USA).

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Deionized water was generated using the Millipore Milli-Q Gradient Water Purification System (Molsheim, France). Blank human plasma with EDTA-K2 anticoagulant was purchased from BioIVT (New York, USA). 2.2. Study protocol and sample collection

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Human blood samples were collected from five healthy male subjects during the course of a phase I clinical trial of anaprazole performed at Peking University Third

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Hospital (Beijing, China). The study was conducted in accordance with the Declaration of Helsinki and was approved by the Peking University Third Hospital Medical Science

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Research Ethics Committee. All subjects gave their voluntary informed consent and received a single dose of 40 mg of anaprazole. Blood samples were collected at 0 (pre-

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dose), 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 15, and 24 h after administration and were centrifuged immediately to isolate the plasma fraction. The plasma samples were stored frozen at −70 °C until analysis.

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2.3. Metabolite profiling and identification by UPLC-UV/Q-TOF MS Plasma samples that were collected at pre-dosage, 4.5 h (approximately at the time

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to reach peak concentration), and 12 h (at the elimination phase) were segregated by sample collection time. Equal volumes of plasma at each collection time from all individuals were pooled for analysis. Acetonitrile (300 μL) was added to a 150-μL aliquot of the pooled samples. This mixture was vortexed and centrifuged at 14,000 × g for 5 min. The supernatant was transferred into a plastic tube, evaporated to dryness under a stream of nitrogen at 40 °C, and reconstituted in 150 μL of methanol. A 7.0-μL 5

aliquot of the reconstituted solution was injected into the UPLC-UV/Q-TOF MS system for analysis. Anaprazole and its metabolites were separated on a Capcell PAK MG C18 column (100 mm × 4.6 mm, 5 μm) equilibrated at 45 °C. The mobile phase consisted of 5 mM ammonium acetate in water (A) as the aqueous phase and 5 mM ammonium acetate in methanol (B) as the organic phase. The gradient elution was maintained at 20% B for 1 min, increased linearly to 80% over 14 min and to 99% over the next 1 min, returned to 20% B for 1 min, and held for 4 min at an eluent flow rate of 0.5 mL/min. The UV

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detection wavelength used was set at 315 nm. MS detection was achieved using a triple TOF 5600+ MS/MS system (AB Sciex, Concord, Ontario, Canada) in the positive electrospray ionization (ESI) mode. The

mass range was set at m/z 80–1000. The following parameter settings were used: ion

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spray voltage, 5500 V; declustering potential, 60 V; ion source heater, 550 °C; curtain gas, 40 psi; ion source gas 1, 55 psi; and ion source gas 2, 50 psi. For the TOF MS scans,

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the collision energy was 10 eV. For the product ion scans, the collision energy was 35 eV, with a spread of 25 eV in the MS/MS experiment. Information-dependent

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acquisition (IDA) was used to trigger the acquisition of MS/MS spectra for ions matching the IDA criteria. A real-time multiple mass defect filter was used for the IDA

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criteria.

2.4. Simultaneous determination of anaprazole and its four major metabolites in human plasma by LC-MS/MS

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The LC-20AD LC system (Shimadzu, Kyoto, Japan) was employed in this experiment. Chromatographic separation was performed on a Kinetex 5u XB-C18

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column (50 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) with a C18 guard column (4.0 mm × 3.0 mm, 5 μm; Phenomenex, Torrance, CA, USA). The mobile phase consisted of (A) 5 mM ammonium acetate with 0.005% ammonium hydroxide and (B) methanol with 0.005% ammonium hydroxide. The column temperature and flow rate were set at 40 °C and 0.6 mL/min, respectively. The gradient elution was started from 20% B and maintained for 1.2 min, increased linearly to 60% B in the next 1.3 min, and 6

held for 1 min. Afterward, the gradient was rapidly increased to 95% B for 0.1 min, maintained at 95% B for 1 min and reduced to 20% B for 0.1 min, and finally maintained at 20% B for 1.8 min to equilibrate the column. MS detection was conducted on an AB Sciex Triple Quad 5500 triple quadrupole mass spectrometer (Concord, ON, Canada) equipped with a Turbo Ion ESI source in positive multiple reaction monitoring (MRM) mode. The spray voltage and source temperature were set to 4500 V and 550 °C, respectively. The curtain gas, nebulizer gas (GS1), and heating gas (GS2) were optimized at 30, 50, and 50 psi, respectively. The

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dwell time for each transition was set at 200 ms. The MRM transitions and optimized MS parameters for each analyte are summarized in Table 1. Analyst 1.6.2 (AB Sciex) was used for data acquisition and processing.

2.5. Preparation of standards and quality control (QC) samples.

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Stock solutions of anaprazole, M8-1, M16-3, M21-1, and M7-1 at concentrations of approximately 1.00 mg/mL were prepared in methanol. The solutions were serially

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diluted with methanol to produce working solutions of 60.0, 30.0, 15.0, 5.00, 1.50, 0.500, 0.200 and 0.100 µg/mL for anaprazole and of 12.0, 6.00, 3.00, 1.00, 0.300, 0.100,

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0.0400 and 0.0200 µg/mL for the respective metabolites. Calibration standard samples were prepared by spiking working solutions into drug-free plasma to obtain final

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concentrations of 3000, 1500, 750, 250, 75.0, 25.0, 10.0, and 5.00 ng/mL for anaprazole and 600, 300, 150, 50.0, 15.0, 5.00, 2.00, and 1.00 ng/mL for other metabolites. QC solutions were prepared by separately weighing the standard references. QC samples

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were independently prepared in blank plasma at four concentrations: 5.00 (lower limit of quantitation, LLOQ), 15.0 (low QC, LQC), 500 (middle QC, MQC), and 2400 ng/mL

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(high QC, HQC) for anaprazole, and 1.00 (LLOQ), 3.00 (LQC), 100 (MQC), and 480 (HQC) for other metabolites. The mixed working solutions of ISs (13C,d3-M0, 13C,d3M8-1, and

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C,d3-M16-3) at concentration of 250/100/100 ng/mL were diluted in

methanol from stock solutions. All working and stock solutions were kept at −20 °C, whereas the QC samples and calibration standards were kept at −70 °C until analysis. 2.6. Sample preparation for LC-MS/MS analysis. 7

Working solution of 25 μL of the ISs and 200 μL of acetonitrile were added to a 50-μL aliquot of the plasma sample. The mixture was vortex-mixed for 1 min and centrifuged at 14,000 × g for 5 min. A 4-μL aliquot of supernatant was injected into the LC-MS/MS system for quantitative analysis. 2.7. Method validation The validation of LC-MS/MS assay was fulflled according to the guidelines issued by the U.S. Food and Drug Administration (FDA)[12]. Selectivity was evaluated by analyzing the drug-free plasma samples and the

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corresponding LLOQs from six different sources. The peak areas of coeluting interfering peaks or background noise should be ≤20% and ≤5% those of analytes and isotopic ISs, respectively, at the LLOQs.

The calibration curve was established by plotting the peak area ratio of analytes to

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ISs versus the nominal concentrations. A linear least-squares and 1/x2-weighted (x denotes the nominal level of each analyte) regression was used for constructing the

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curves. Calibration standards were prepared in duplicate for three independent days for

required to be >0.99.

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the evaluation of linearity. The value of r2 (correlation coefficient of determination) was

The accuracy and precision were assessed by calculating the relative error (RE)

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and the relative standard deviation (RSD) of QCs at four concentrations (LLOQ, LQC, MQC, and HQC), which were assayed in six replicates for three consecutive days. The accuracy was considered acceptable if RE was within ±20% of the theoretical value for

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LLOQs and ±15% for the other concentrations. The intra- and inter-day precision was considered acceptable if the RSD was <20% for the LLOQs and <15% for the

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remaining concentrations.

The matrix effect was evaluated by analyzing two sets of samples at LQC and

HQC levels: (A) blank extracts from six different lots that was spiked with five analytes and ISs, and (B) solutions containing the same concentrations of five analytes and ISs. The ratios of the peak area of analytes versus ISs in the presence of matrix to those in absence of matrix were defined as the IS-normalized matrix factor (IMF). The inter8

subject (n = 6) variability of IMF should be <15%. Furthermore, some special type of matrices, such as hemolysis and hyperlipemia plasma, may have an effect on the MS response. Thus, these matrix effects were assessed similarly as described above. The recoveries of each analyte were measured at LQC, MQC, and HQC by comparing their mean peak area in six QC samples with that in three post-extraction samples spiked with an equivalent concentration of QC working solutions. The recoveries of ISs were calculated in a similar way. Stability was evaluated by measuring the replicates (n = 6) of LQC and HQC

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samples that were exposed to different conditions. The conditions included room temperature (6 h at 22 °C), long-term storage (114 days at −70 °C), post-preparation in autosampler (24 h at 4 °C), and three freeze/thaw cycles (from −70 °C to room temperature). The analytes were considered stable when the calculated concentration

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was within ±15% of the theoretical value. 3. Results and discussion

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3.1. UPLC-UV/Q-TOF MS Analysis of Anaprazole

Understanding the chromatographic and mass fragmentation patterns of the

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reference is the basis for the subsequent identification of metabolites using the UPLC/Q-TOF MS approach. Under the chromatographic conditions employed,

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anaprazole was eluted at 13.8 min (Fig. 1A). In the ESI (+) mode, anaprazole gave a protonated molecule at m/z 402.149 (Fig. 1B). The main characteristic product ions of m/z 402.149 were observed at m/z 242.085, 212.128, 195.126, 191.028, 170.023,

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161.071, 150.091, and 136.076 (Fig. 1C), and the tentative fragmentation patterns are shown in Fig. 1D. The fragment ion at m/z 161.071 was formed via the cleavage of the

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C–S bond between the benzimidazole and sulfur atom, leading to a substituted benzimidazole ion. Meanwhile, the fragment ion at m/z 242.085 was an ion corresponding to the O=S–CH2 fraction containing pyridine derivative, which underwent further cleavage to form the fragment ions at m/z 212.128, 195.126, 170.023, 150.091, and 136.076. These diagnostic fragmentations were used to facilitate the spectral interpretation of unknown metabolites from anaprazole. 9

3.2. Anaprazole Metabolite Profiling and Identification Anaprazole and its metabolites in human plasma were identified by UPLC-UV/QTOF MS. In addition to anaprazole, 14 metabolites were observed in the pooled human plasma (Fig. 2). The MS response alone cannot represent the relative amount of the metabolites, because the metabolic modification of the parent drug changes the polarity and their tendency to form ions in the ESI source, resulting in different ionization efficiencies and diverse mass responses. However, common phase I metabolites, such as hydroxylated and dealkylated metabolites, cause little changes to the chromophoric

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groups of the parent drug and UV properties. Thus, the UV absorbance, rather than MS response, can provide a more reliable relative amount of the metabolites. Figs. 2D and E show the corresponding UPLC-UV chromatograms of the plasma samples.

Comparing the UV chromatogram of plasma with that of drug-free plasma (Fig. 2C),

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the parent drug was the most abundant circulating drug-related component in human plasma at 4.5 h post-dose (Fig. 2D). Other predominant circulating metabolites were

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eluted at 13.5 (M16-3), 14.8 (M21-1), 15.9 (M8-1), and 8.7 (M11-2) min, and two end metabolites were detected at 10.78 (M7-1) and 12.05 (M6) min in plasma at 12 h post-

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dose (Fig. 2E). The proposed metabolic pathways of anaprazole in humans are shown in Fig. 3.

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Table 1 lists the detailed information about these metabolites, including the proposed structures, protonated molecules, retention times, and characteristic fragment ions. The metabolites were named in accordance with their protonated molecular

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weights, and metabolites with the same molecular weight were named in the sequential order of their retention times. The metabolites were identified as follows:

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Parent Drug M0. A chromatographic peak was detected at 14.0 min and gave a protonated molecule at m/z 402.147. Its tentative elemental composition was C20H23N3O4S. The retention time and mass spectral fragmentation patterns were identical to the parent drug, anaprazole. These results indicated that this component was unchanged anaprazole, designated as M0. M8-1. Metabolite M8-1, which had a protonated molecular mass of 386.153, was eluted 10

at 15.9 min. The elemental composition of M8-1 was C20H23N3O3S, indicative of deoxidation compared with anaprazole. The fragment ions of M8-1 at m/z 195.126, 161.072, and 150.092 were identical to those of anaprazole. The fragment ions at m/z 226.091 and 154.033 were 16 Da lower than those of the parent drug at m/z 242.085 and 170.027, indicating that the sulfoxide moiety was reduced to a thioether. In addition, a unique fragmentation was observed at m/z 353.175, which was derived from the loss of SH (−33 Da). The chromatographic retention time and mass spectral fragmentation patterns of M8-1 were identical to those of the reference standard. Therefore, M8-1 was

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accordingly confirmed as the reduced metabolite of anaprazole. In addition to M8-1, several metabolites that displayed [M+H]+ ions at m/z 314.096 (M1), 372.137 (M5), 386.117 (M7-1), 384.101 (M6), 412.112 (M11-2),

400.096 (M10), 367.153 (M21-2), 404.163 (M14-1), and 418.143 (M16-1 and M16-2)

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had the characteristic fragment ions resulting from the loss of SH (−33 Da), suggesting that they were thioether-related metabolites derived from M8-1.

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M1. M1 was eluted at 10.7 min. The fragment ions of M1 at m/z 161.070 and 154.031 were identical to those of the parent drug and M8-1, respectively. The elemental

metabolite of M8-1.

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composition of M1 was C16H15N3O2S, indicating that it was the O-dealkylated

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M5. M5 was eluted at 13.9 min, and the chemical formula of M5 was C19H21N3O3S. The major fragment ions at m/z 339.156 and 212.064 were proposed to be the result of the demethylation from the fragment ions of M8-1 at m/z 353.173 and 226.090,

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respectively. M5 was accordingly identified as the O-demethylated metabolite of M8-

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M7-1. M7-1 was the most predominant metabolite in urine (unpublished data), which was also detected in circulation. M7-1 was eluted at 10.8 min, and its chemical formula was C19H19N3O4S. The nominal mass of fragment ions of M7-1 at m/z 353.133, 226.048, and 195.082 was identical to those of M8-1, indicating O-demethylation followed by carboxylation of the corresponding fragment ions of M8-1. The fragment ions at m/z 161.066 and 154.029 were identical to those of M8-1. The chromatographic retention 11

time and mass spectral fragmentation pattern of M7-1 were identical to those of the reference standard. Therefore, M7-1 was accordingly identified as the carboxylic acid metabolite of M8-1, which was formed by O-demethylation. M6. M6 was eluted at 12.1 min, and its elemental composition was C19H17N3O4S. The fragment ions of M6 at m/z 351.120 and 159.051 were 2 Da lower than those of M7-1, indicating that dehydrogenation occurred on the dihydrofuran ring, whereas fragment ions at m/z 226.048 and 154.027 were identical to those of M7-1. Thus, M6 was proposed as the dehydrogenated metabolite of M7-1.

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M11-2. M11-2 was eluted at 8.7 min, and its elemental composition was C19H19N3O5S. The fragment ions of M11-2 at m/z 369.129 and 177.067 were 16 Da larger than those of M7-1, indicating that oxidation occurred on the benzimidazole ring, whereas

fragment ions at m/z 226.048 and 154.026 were identical to those of M7-1. Thus, M11-

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2 was proposed as the mono-oxidized metabolite of M7-1.

M10. M10 was eluted at 10.4 min, and its elemental composition was C19H17N3O5S.

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The fragment ions at m/z 367.113 and 175.064 were 2 Da lower than those of M11-2, whereas the fragment ions at m/z 226.052 and 154.032 were identical to those of M11-

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2, indicating that dehydrogenation occurred on the dihydrofuran ring. M10 was proposed as the dehydrogenated metabolite of M11-2.

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M21. M21-1 and M21-2 were eluted at 14.8 and 15.6 min, respectively, exhibiting the same [M+H]+ ions at m/z 400.133. Their elemental composition was C20H21N3O4S. The fragment ions of M21-1 at m/z 242.077, 195.119, 170.022, 152.013, and

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150.082 were identical to those of anaprazole, suggesting that M21-1 was a M0-related metabolite, instead of M8-1. The chromatographic retention time and mass spectral

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fragmentation pattern of M21-1 were identical to those of the reference standard. Therefore, M21-1 was accordingly confirmed as the dehydrogenated metabolite of M0, and the reaction site was the dihydrofuran ring. The fragment ion of M21-2 at m/z 367.153 was 14 Da larger than that of M8-1 at m/z 353.173, and the fragment ion at m/z 226.083 was identical to that of M8-1. Thus, M21-2 was proposed as the oxidized and dehydrogenated metabolite of M8-1. 12

M14-1. M14-1 was eluted at 13.5 min, and its elemental composition was C20H25N3O4S. The mass spectrum of m/z 404.163 gave major fragment ions at m/z 371.186, 226.089, 179.082, and 154.035. The fragment ions at m/z 371.186 and 179.082 were 18 Da larger than those of M8-1, indicating that oxidation and hydrogenation occurred on the dihydrofuran ring part, whereas the fragment ions at m/z 226.089 and 154.035 were identical to those of M8-1. M14-1 was proposed as the oxidized and hydrogenated metabolite of M8-1. M16. M16-1, M16-2, and M16-3 were eluted at 11.6, 12.0, and 13.6 min, respectively,

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and exhibited [M+H]+ ion at m/z 418.143. They had an elemental composition of C20H23N3O5S.

The major fragment ions of M16-1 and M16-2 were at m/z 385.163, 226.088, 193.061, and 154.032. The fragment ions at m/z 385.163 and 193.061 were 32 Da larger

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than those of M8-1, whereas the fragment ions at m/z 226.088 and 154.032 were identical to those of M8-1, indicating that dioxidation occurred on the benzimidazole

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part. M16-1 and M16-2 were proposed as the dioxidized metabolites of M8-1. The mass spectrum of M16-3 gave major fragment ions at m/z 354.179, 282.121,

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and 258.077. The fragment ion at m/z 354.179 was formed via the neutral loss of SO2 and underwent further C–O bond cleavage to form the fragment ion at m/z 282.121.

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The fragment ion at m/z 258.080 was 16 Da larger than that of M0, indicating that oxidation metabolism occurred in the sulfoxide. The chromatographic retention time and mass spectral fragmentation pattern of M16-3 were identical to those of the

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reference standard. Therefore, M16-3 was accordingly identified as the sulfone metabolite of anaprazole.

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M9-2. M9-2 was eluted at 11.9 min and exhibited [M+H]+ ion at m/z 388.133. Its chemical formula was C19H21N3O4S. No indicative fragment ion of M9-2 was observed due to its trace level in samples. However, based on the exact mass information, M9-2 was readily identified as the O-demethylated metabolite of anaprazole. 3.3. Optimization of LC-MS/MS conditions to determine anaprazole and its major metabolites in plasma. 13

A LC–tandem triple quadrupole mass spectrometer was used for the absolute quantification of anaprazole and its major metabolites (M8-1, M16-3, M21-1, and M71) in human plasma due to the limited detection sensitivity of the Q-TOF instruments. However, establishing an LC-MS/MS method for the simultaneous determination of anaprazole and its major metabolites is challenging, which is described below. In the Q1 full scan mode, [M+H]+ ions of five analytes and isotopic ISs were readily observed. The product ion spectra and fragmentation profiles of the analytes and ISs are presented in Fig. 4. Generally, under the MRM mode of triple quadrupole

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MS, coeluted components can be distinguished and quantitated separately due to different precursor ions and/or product ions. However, the exact mass difference

between the precursor ions of M7-1 (385.110) and M8-1 (385.146) was too small to be differentiated by triple quadrupole MS analysis. The major product ion of M8-1 at m/z

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226.090 was also very similar to that of M7-1 at m/z 226.053, and they shared the same fragment ion at m/z 161.071. Therefore, adequate chromatographic separation was

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required to prevent mutual interference between the two metabolites. The product ion of M7-1 at m/z 161.071 was selected for MRM transitions for lower ion suppression at

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this retention time compared with the product ion at m/z 226.053. Dehydrogenation (M21-1) and reduction (M8-1) to thioethers led to an increase in

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lipophilicity compared with the parent drug. However, reduction to thioether with Odemethylation to carboxylic acid (M7-1) increased the polarity. LogD values at pH 7.4 were calculated as 2.09, 3.49, 2.9, 2.26, and −0.52 for anaprazole, M8-1, M16-3, M21-

ur

1, and M7-1, respectively (predicted by ACD/Percepta, Advanced Chemistry Development). Five analytes exhibited different chromatographic behaviors due to

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diff erent chemical polarities. Thus, gradient elution was chosen to obtain sufficient resolution in a shorter analysis time. PPIs are unstable in neutral and acidic solutions as reported[13, 14]. Therefore, the

alkaline mobile phase was selected to ensure the stability of analytes and increase the chromatographic retention. The buffer mobile phase made the peak shape symmetrical and improved the reproducibility. Finally, 5 mM ammonium acetate containing 0.005% 14

ammonium hydroxide and methanol containing 0.005% ammonium hydroxide were used as aqueous and organic phases, respectively. 3.4. Sample preparation The sample preparation methods reported to determine PPIs and their metabolites in human plasma included solid phase extraction[15] and liquid–liquid extraction[1620]. As described before, the physical properties of different metabolites varied widely. Therefore, protein precipitation was chosen for sample preparation because of its

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excellent recovery rates and simplicity. Stable isotope-labeled ISs were employed to avoid the impact of ion enhancement or suppression in MS detection caused by the coeluting endogenous substances on the quantification.

13

C,d3-anaprazole and

13

C,d3-

M16-3 were used as the ISs for M0 and M16-3, respectively, whereas 13C,d3-M8-1 was

-p

a common IS for other analytes. Stable isotope-labeled compounds can compensate for

the variations during sample preparation and matrix effect due to the almost identical

re

physicochemical properties to those of the unlabeled analytes. Given the instability of PPIs in aqueous solution, the extracted supernatant was injected to LC-MS/MS directly

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without further dilution with the mobile phase. Given the good MS response and high concentrations of M21-1, the optimal CE value of M21-1 was set at 17 eV rather than

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the best MS response CE value at 40 eV to avoid MS detector saturation. 3.5. Method validation

3.5.1. Assay selectivity and carryover

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Fig. 5 shows the MRM chromatograms of blank human plasma samples (Fig. 5A), blank human plasma spiked only with ISs (Fig. 5B), LLOQ (Fig. 5C), and the plasma

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samples collected 6 h after the administration of 40 mg of anaprazole (Fig. 5D). The chromatographic peaks of M8-1 and M7-1 did not interfere with each other due to sufficient chromatographic separation. The peak areas of blank samples for each analyte were <20% compared with those observed at LLOQs. The peak areas of blank samples injected immediately after ULOQ were <20% compared with those of each analyte at LLOQs, suggesting that little carryover existed in the present conditions. 15

3.5.2. Linearity and LLOQ The linear regression curves were fitted to the concentration ranges of 5.00–3000 ng/mL for anaprazole and 1.00–600 ng/mL for M8-1, M21-1, M16-3, and M7-1. Excellent linearities (r2 > 0.99) were observed for the five analytes. The regression equations for the calibration curves were as follows. M0: y = 0.00184 x + 0.000276, r2 = 0.9940 M8-1: y = 0.0288 x + 0.00294, r2 = 0.9974 M21-1: y = 0.0332 x + 0.000487, r2 = 0.9940

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M16-3: y = 0.0260 x + 0.00224, r2 = 0.9944 M7-1: y = 0.0169 x + 0.000569, r2 = 0.9924

where y represents the peak area ratio of each analyte/ISs and x represents the nominal plasma concentrations of each analyte. LLOQs of 5.00 and 1.00 ng/mL were achieved

-p

with acceptable accuracy and precision. 3.5.3. Precision and accuracy

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The results for precision and accuracy are listed in Table 2. The intra- and interbatch precision values were <12.2% and <12.6%, respectively, and the accuracy ranged

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from −7.6% to 7.6%, which were in the acceptable ranges. 3.5.4. Matrix effect and extraction recovery

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The IMFs of five analytes determined at the LQC and HQC levels for normal, hemolysis, and hyperlipemia plasma ranged from 90.0% to 106%. The intersubject variability of the IMFs, as measured by their RSD, was lower than 7%. These results

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indicated that the matrix effect in the current method could be ignored. The mean extraction recoveries obtained from three concentrations of QC plasma

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samples were 98.5%, 93.9%, and 92.3% for anaprazole; 98.5%, 93.9%, and 92.3% for M8-1; 107%, 96.1%, and 93.8% for M16-3; 108%, 101%, and 95.9% for M21-1; and 99.1%, 87.1%, and 93.0% for M7-1. The mean extraction recovery rates of ISs were between 100% and 105%. These results indicated sufficient extraction efficiency. 3.5.5. Stability The results of the stability test are presented in Table 3. All analytes were stable 16

under various tested conditions, such as ambient temperature short-term storage, −70 °C long-term storage, auto-sampler storage after sample preparation, and three freeze–thaw cycles. 3.6. Pharmacokinetic study The validated analytical method was used to determine the plasma concentrations of anaprazole and its four metabolites in five healthy volunteers after a single oral administration of 40 mg of anaprazole. The mean plasma concentration–time profiles are shown in Fig. 6. Accurate quantitative results show that the thioether metabolites

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M8-1 and dehydrogenated metabolites M21-1 are the most important metabolites in the systemic circulation, and the clearance rate of metabolites in the body is slower than

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that of the parent drug.

17

4. Conclusion In this study, 14 metabolites in human plasma were characterized by UPLC/QTOF after the administration of a single dose of 40 mg of anaprazole in healthy subjects. The predominant metabolic pathways of anaprazole, including sulfoxide reduction to thioether (M8-1), dehydrogenation (M21-1), sulfoxide oxidation to sulfone (M16-3), and sulfoxide reduction with O-demethylation to form carboxylic acid (M7-1), were identified by the UPLC-UV system. A simple and accurate LC-MS/MS method was established for the simultaneous determination of parent drug and its major metabolites

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(M8-1, M21-1, M16-3, and M7-1). After method optimization, M8-1 and M7-1, whose exact mass difference was below 0.04 Da, were chromatographically separated and avoided mutual crosstalk in MS/MS analysis. Analytes with different polarities were

simultaneously determined within the 6.5-min chromatographic time. The quantitative

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method has been successfully applied in pharmacokinetic experiments of anaprazole, providing effective and stable support for clinical trials, including metabolite

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experiments, safety assessments, and PK/PD correlation studies.

Funding information This research was financially supported by the National Natural

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Science Foundation of China [81573500; 81573351].

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Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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Author Statement

Chongzhuang Tang: Writing - Original Draft; Visualization; Methodology; Formal

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analysis

Liang Li: Methodology; Data Curation Xifeng Ma: Project administration; Resources Jin Wang: Project administration; Resources Bo Chen: Project administration; Resources Xiaojian Dai: Methodology; Data Curation 18

Yifan Zhang: Project administration; Formal analysis Xiaoyan Chen: Writing - Review & Editing ; Supervision; Project administration; Resources; Formal analysis

Acknowledgments We greatly appreciate Xuanzhu Pharmaceutical Co.,Ltd. (Shandong, China) for

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na

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providing the reference standards of anaprazole and its metabolites.

19

Table 1 Mass spectrometric conditions for analytes and internal standards. Collision (eV)

M0

402.2 → 242.2

38

M8-1

386.2 → 226.2

30

M21-1

400.2 → 242.2

17

M16-3

418.2 → 282.2

40

M7-1

386.2 → 161.2

40

13

C,d3-M0

406.2 → 246.2

38

13

C,d3-M8-1

390.2 → 230.2

30

C,d3-M16-3

422.2 → 282.2

40

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na

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-p

13

20

energy

ro of

MRM transitions

Analyte

Table 2 Precision and accuracy data for the analysis of anaprazole and its four metabolites in human plasma (three days with six replicates per day).

M21-1

Intra-day

Inter-day

5.00

4.76

7.5

6.6

-4.9

15.0

15.3

3.1

5.4

2.2

500

504

4.4

4.4

0.9

2400

2258

4.6

6.3

-5.9

1.00

0.986

6.6

8.9

-1.4

3.00

3.12

6.9

6.4

4.1

100

101

6.4

5.6

0.7

480

451

4.9

4.9

-6.1

1.00

0.953

12.2

12.6

-4.8

3.00

3.22

7.0

8.4

7.2

100

103

8.3

7.1

2.8

480

452

6.9

7.3

-5.8

1.00

0.983

8

5.9

1.7

3.00

3.23

7.9

7.2

7.6

100

103

3.1

4.1

3.3

ro of

Mean calculated

480

445

4.6

5.2

-7.2

1.00

1.01

5.9

9.6

0.6

3.00

3.19

5.8

7.5

6.4

100

104

9.0

9.3

3.9

480

444

6.7

8.7

-7.6

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M7-1

RE (%)

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M16-3

Nominal

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M8-1

RSD (%)

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M0

Concentration (ng/mL)

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Analyte

21

f oo

Table 3 Summary of stability of anaprazole and its four metabolites in human plasma under various storage conditions (n = 6). Storage conditions

M8-1

M16-3

Added

Found RE

Added Found RE

Added

Found RE

Short-term

15.0

14.9

-0.3

3.00

2.98

3.00

2.85

-4.9 3.00

3.07

2.2

3.00

3.05

1.7

(6 h at 22 °C)

2400

2465

2.8

480

494

3.0

480

478

-0.5 480

484

0.8

480

503

4.8

Autosampler

15.0

15.9

6.1

3.00

3.04

1.3

3.00

2.83

-5.8 3.00

3.01

0.3

3.00

2.99

-0.3

(24 h at 22°C)

2400

2440

1.7

480

474

-1.2

480

495

3.1

480

488

1.7

480

484

0.8

Three freeze/thaw 15.0

15.4

2.2

3.00

2.98

-0.6

3.00

3.42

14

3.00

2.83

-5.8

3.00

2.87

-4.3

cycles

2390

-0.5

480

501

4.3

480

483

0.7

480

516

7.6

480

488

1.6

Long-term

(114 15.0 2400

Blood

15.0

(2 h at 22 °C)

2400

Pr

e-

-0.7

M21-1

M7-1

Added Found RE

Added Found RE

14.2

-5.8

3.00

3.02

0.5

3.00

3.18

6.1

3.00

2.87

-4.5

3.00

3.03

0.9

2475

3.1

480

500

4.2

480

496

3.4

480

514

7

480

493

2.8

16.0

6.4

3.00

2.87

-4.2

3.00

3.18

6.0

3.00

3.11

3.7

3.00

2.99

-0.3

480

505

5.2

480

508

5.9

480

496

3.4

480

482

0.4

Jo ur

days at −70 °C)

na l

2400

pr

anaprazole

2410

0.4

22

ro of -p re lP na ur Jo

Fig. 1 Chromatograms and mass spectra of anaprazole obtained by UPLC-UV/Q-TOF MS analysis. (A) Chromatograms, (B) mass spectrum, (C) MS2 spectrum, and (D) tentative MS/MS fragmentation pattern of anaprazole.

23

ro of -p re lP na

ur

Fig. 2 Metabolic profiles of anaprazole in human plasma detected by UPLC-UV/QTOF MS: pooled plasma samples at 4.5 (A) and 12 h (B), UPLC-UV chromatograms

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of pooled human plasma and drug-free plasma (C), pooled plasma samples at 4.5 (D) and 12 h (E).

24

f oo pr ePr na l Jo ur

Fig. 3 Proposed metabolic pathways of anaprazole in human plasma.

25

ro of -p re lP na

Fig. 4 Product ion mass spectra of anaprazole (A), 13C,d3-anaprazole (B), M16-3 (C), C,d3-M16-3 (D), M8-1 (E),

13

C,d3-M16-3 (F), M21-1 (G), and M7-1 (H) and their

ur

13

Jo

proposed fragmentation patterns acquired by LC-MS/MS.

26

27

na l

Jo ur

oo

pr

e-

Pr

f

f

oo

Fig. 5 Typical MRM chromatograms of anaprazole (I), 13C,d3-anaprazole (II), M8-1 (III), 13C,d3-M8-1 (IV), M16-3 (V), 13C,d3-M16-3 (VI), M211 (VII), and M7-1 (VIII). (A) Blank plasma sample; (B) blank plasma sample spiked with ISs (250/100/100 ng/mL 13C,d3-anaprazole/13C,d3-M8-

pr

1/13C,d3-M16-3 in plasma); (C) blank plasma sample spiked with anaprazole, M8-1, M16-3, M21-1, and M7-1 at the LLOQ (5.00/1.00/1.00/1.00/1.00 ng/mL for anaprazole/M8-1/M16-3/M21-1/M7-1) and ISs; and (D) plasma sample collected 6 h after the administration

e-

of 40 mg of anaprazole.

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na l

Pr

*: Crosstalk peak between M7-1 and M8-1 due to slight mass difference of MRM transitions as described in Section 3.3.

28

ro of -p re lP

na

Fig. 6 Mean plasma concentration–time profiles of anaprazole (A), M8-1 (B), M16-3

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(C), M21-1 (D), and M7-1 (E) after a single oral administration of 40 mg of anaprazole.

29

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