Journal of Pharmaceutical and Biomedical Analysis 83 (2013) 75–81
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Highly sensitive liquid chromatography-tandem mass spectrometry method for quantification of TAK-448 in human plasma夽 Yoji Kuze ∗ , Fumihiro Jinno, Takahiro Kondo, Satoru Asahi Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
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Article history: Received 16 January 2013 Received in revised form 8 April 2013 Accepted 10 April 2013 Available online 29 April 2013 Keywords: TAK-448 Human plasma Bioanalysis LC–MS/MS Validation
a b s t r a c t TAK-448 is a nonapeptide analogue and a novel metastin receptor agonist. The aim of this study was to develop a bioanalytical method for TAK-448F (the free base of TAK-448) in human plasma with LC/MS/MS that is sensitive and applicable for the clinical PK studies, and to evaluate the reliability and robustness of the developed method through a validation study in accordance with the regulatory guidance/guideline. The bioanalytical method developed in this study can be outlined as follows. The structural analogue, TAK-683, was used as the internal standard (IS). TAK-448F and the IS were extracted from human plasma using solid phase extraction (SPE) with a polymer-based weak cationic exchanger. After evaporating, the residue was reconstituted and injected into a LC–MS/MS system with ESI probe and analyzed by the selected reaction monitoring (SRM) in the positive ion mode. Separation was performed through an UPLC BEH Phenyl column with the mobile phase of water/methanol/formic acid mixture at a flow rate of 0.2 mL/min. The total run time was 10 minutes. The LLOQ was achieved to be 5 pg/mL with 0.5 mL of human plasma sample. All the validation results met the acceptance criteria in accordance with the regulatory guidance/guideline proving its reliability and robustness. As a result of the clinical study, the human PK profiles of TAK-448F were successfully obtained with this method. © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
1. Introduction Peptide drugs often have potent activity and are well known as challenging drugs for the bioanalytical works due to the low plasma concentrations at therapeutic dose levels [1,2]. Traditionally, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been employed for the determination of peptides in biological samples [3]. These assays generally have high sensitivity and can provide high sample throughput although there are some inherent issues such as a risk of cross-reactivity with structurally similar peptides including metabolites and endogenous components, and requirement of the specific antibody to an analyte. Other bioanalytical platforms such as liquid chromatography (LC) with ultraviolet, fluorescence and electrochemical detections have been reported for the quantitative determination of peptides [4–6]. These methods have good
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author at: 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan. Tel.: +81 466 32 4157; fax: +81 466 29 4435. E-mail address:
[email protected] (Y. Kuze).
linearity with wide calibration ranges and good repeatability but have limited sensitivity and selectivity on the detection. Recently, the bioanalysis of liquid chromatography tandem mass spectrometry (LC/MS/MS) has become common not only for small molecules but also for proteins and peptides [1,7]. Since LC/MS/MS-based assays have certain advantages that it does not require any specific materials such as antibodies, and most equipment can be shared with the bioanalysis of small molecules, it would be preferably chosen if there is no issue on the sensitivity. Thus, it should be critical if the sensitivity can be improved on the LC/MS/MS-based assays of proteins and peptides. In most cases, the sensitivity is largely influenced by the processes of electrospray ionization (ESI) and collision induced dissociation (CID) in addition to sample treatment procedures [2]. On the selected reaction monitoring (SRM) analysis of proteins and peptides, a number of ions with different charged states at ESI and various fragmentations at CID lead to reduction of the intensity. To overcome these challenges, optimization of LC/MS/MS conditions should be more carefully done compared to small molecules. The sensitive SRM analyses for proteins and peptides have been reported by several studies. Lovgren et al. and Zhang et al. reported highly sensitive SRM analyses of peptides [8,9]. They improved the sensitivity by optimizing all the conditions carefully and achieved the low LLOQs which are almost equivalent to ELISA assays. Simon et al. demonstrated that peptide signals on the SRM analysis were improved by
0731-7085/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.04.023
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modifying the charged status of peptides [10]. These reports have encouraged the use of LC/MS/MS for the bioanalysis of peptides. TAK-448, 2-(N-Acetyl-d-tyrosyl-trans-4-hydroxy-l-prolyll-asparaginyl-l-threonyl-l-phenylalanyl)hydrazinocarbonyl-lmonoacetate, leucyl-Nω -methyl-l-arginyl-l-tryptophanamide is a nonapeptide analogue and a new oral metastin receptor agonist discovered by Takeda and currently under development for potential use in the treatment of prostate cancer [11]. TAK-448 has potent pharmacological activity with presumed effective concentrations at low levels in the pg/mL range in human plasma. This suggests the need for a highly sensitive bioanalytical method to monitor the drug concentrations in the clinical studies, and also the reliability and robustness as a regulated bioanalysis is imperative. In the previous study, the sandwich ELISA method for the determination of TAK-448 was established and validated [12]. This method is highly sensitive with 3 pg/mL of the LLOQ in rat plasma, although as inherent issues for ELISA methods, specific monoclonal antibodies are required and the calibration range is relatively narrow (3–300 pg/mL) compared to general chromatographic assay methods resulting in the necessity of frequent dilution for samples with higher drug concentration. The aim of this study was to develop a bioanalytical method for TAK-448 in human plasma based on an LC/MS/MS technology that should be highly sensitive with a sufficient calibration range for the use in the clinical studies, and to evaluate the reliability and robustness of the developed method through a validation study in accordance with the regulatory guidance/guideline [13,14]. Additionally, human PK profiles in the phase I study were obtained by using the validated method.
2. Material and methods 2.1. Chemicals and reagents The reference standard of TAK-448 and the internal standard (IS) of TAK-683, 2-(N-Acetyl-d-tyrosyl-d-tryptophyl-l-asparaginyl-lthreonyl-l-phenylalanyl)hydrazinocarbonyl-l-leucyl-Nω -methyll-arginyl-l-tryptophanamide monoacetate were prepared by Takeda Pharmaceutical Company Limited. The chemical purity for these standards was more than 90%. Blank human plasma containing EDTA-2K as an anticoagulant was purchased from Nippon Bio-supp. Center. All the other chemicals and reagents were obtained from commercial sources.
2.2. Instrumentation and conditions for LC/MS/MS analysis The LC–MS/MS system consisted of an Acquity UPLC system (Waters Corp., MA, USA) and an API5000 triple quadruple mass spectrometer (AB Sciex, CA, USA) with a turbo ion spray interface. An Acquity UPLC BEH Phenyl (2.1 mm I.D., 50 mm, particle size 1.7 m, Waters Corp.) was used for the HPLC column at 40 ◦ C. The following gradient was used: 0–0.5 min, B 5%; 0.5–1.2 min, B 5–50%; 1.2–8.5 min, B 50%; 8.5–8.6 min, B 50–100%; 8.6–9.0 min, B 100%; 9.0–9.1 min, B 100–5%; 9.1–10.0 min, where the mobile phase A and B were water/methanol/formic acid (900:100:1, v/v/v) and (100:900:1, v/v/v), respectively. The flow rate of the mobile phase was 0.2 mL/min from 0 to 8.6 min and 0.4 mL/min from 8.6 to 10.0 min. The SRM with the positive ion mode was used and the monitoring ions (precursor ion → product ion) were set at m/z 614 → 511 for TAK-448F and m/z 650 → 545 for the IS. Instrument control and data analysis were performed with the Analyst software (version 1.4.1, AB Sciex).
2.3. Preparation of the stock solutions and standard/IS solutions The stock solutions for TAK-448 and the IS were prepared in water/methanol/formic acid (100:400:1, v/v/v) at a concentration of 100 g/mL. The stock solutions were serially diluted with water/methanol/formic acid (100:400:1, v/v/v) to prepare the working solutions at appropriate concentrations and the IS solution at 200 ng/mL. 2.4. Preparation of the calibration standards and quality control (QC) samples The calibration standards were fleshly prepared for each batch by adding the working solutions to the blank human plasma. The concentrations in plasma for the calibration standards were 5, 20, 50, 200, 500, 2000, 5000 and 10,000 pg/mL. The QC samples were prepared on the first day in bulk by adding the working solutions to the blank human plasma, and stored at −20 ◦ C. The concentrations in plasma for the QC samples were 5, 15, 400 and 8000 pg/mL. 2.5. Sample extraction procedure A 500 L aliquot of each plasma sample was mixed with 500 L of a 500 mmol/L ammonium acetate–ammonia buffer (pH 7.0) and 10 L of the IS solution. The sample was loaded into a solid phase extraction cartridge, OASIS WCX (30 mg/1 cc, 30 m, Waters Corp.), preconditioned with 1 mL of methanol and 1 mL of distilled water. After the cartridge was washed with 1 mL of a 500 mmol/L ammonium acetate–ammonia buffer (pH 7.0) and 1 mL of methanol, TAK-448F and the IS were eluted with 1 mL of a mixture of methanol/formic acid (98:2, v/v). The eluted solutions were evaporated to dryness under a stream of nitrogen gas at room temperature. The residues were reconstituted with 250 L of a methanol/distilled water/formic acid (500:500:1, v/v/v) and 50 L of the reconstituted solutions were injected into the LC–MS/MS system. 2.6. Method validation The developed method was validated for the items of selectivity, matrix effects, linearity of calibration curves, precision and accuracy, recovery, stability in accordance with the regulatory guidance/guideline [13,14]. 2.6.1. Selectivity Six lots of blank human plasma were analyzed without TAK448F and the IS, and the potential for interference peaks on the SRM chromatograms was visually evaluated. When an interfering peak is detected at the eluting position of the analyte or the IS, the response of the interfering peak should not exceed 20% of the response of the analyte at the LLOQ or it should not exceed 5% of the response of the IS. 2.6.2. Matrix effect The matrix effect for TAK-448F was evaluated using 6 lots of the blank human plasma. Six lots of the blank human plasma were spiked with TAK-448F at the LQC level (15 pg/mL) and analyzed. The accuracy of each sample calculated as the relative error to the nominal concentrations (%RE) and the precision calculated as the coefficient of variation (%CV) were evaluated. The acceptance criteria were set at within ±15% of the %RE and at
