J S S

ISSN 1615-9306 · JSSCCJ 38 (8) 1263–1440 (2015) · Vol. 38 · No. 8 · April 2015 · D 10609

JOURNAL OF

SEPARATION SCIENCE

Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment

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Meiyun Shi1 ∗∗ Yan Yang1 ∗∗ Xiaotong Zhou1 Lanlan Cai1 Chunxue Fang1 Can Wang1 Heping Sun1 Yantong Sun2 ∗ Yin Gao3 Jingkai Gu4,5 J. Paul Fawcett6 1 College

of Life Science, Jilin University, Changchun, P. R. China 2 School of Pharmaceutical Sciences, Jilin University, Changchun, P. R. China 3 Department of Medicine, Division of Rheumatology, Queen’s University, Kingston, Ontario, Canada 4 Research Center for Drug Metabolism, Jilin University, Changchun, P. R. China 5 Clinical Pharmacology Center, Research Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, P. R. China 6 School of Pharmacy, University of Otago, Dunedin, New Zealand Received October 27, 2014 Revised January 11, 2015 Accepted January 20, 2015

Research Article

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study The pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr, RKDVY) corresponds to amino acids 32–36 of the 49 amino acid immunomodulatory polypeptide, thymopoietin, whose biological activity is partially reproduced. Thymopentin is widely used in the clinic and represents a promising target for drug design but bioanalytical and pharmacokinetic data are limited due to its enzymatic instability. This paper reports a rapid and sensitive method based on liquid chromatography with tandem mass spectrometry for the determination of thymopentin in beagle dog blood. To inactivate peptidases and stabilize thymopentin, acetonitrile was added to blood samples immediately after collection followed by addition of stable isotope-labeled thymopentin as internal standard and washing with dichloromethane. Chromatography was carried out on an Ascentis Express Peptide ES-C18 column using gradient elution with methanol and aqueous 0.1% formic acid at a flow rate of 0.6 mL/min. Positive electrospray ionization mass spectrometry with selected reaction monitoring achieved linearity in the range of 1.5–800 ng/mL with good accuracy/precision and minimal matrix effects. The method was successfully applied to a pharmacokinetic study in beagle dogs after intravenous administration of 0.2 mg/kg thymopentin. Keywords: Dog blood / Liquid chromatography with tandem mass spectrometry / Peptides / Pharmacokinetics / Thymopentin DOI 10.1002/jssc.201401198

1 Introduction A specific immunomodulation by peptides or proteins is the immunization with recombinant protein and synthetic peptide vaccines. Peptides play a critical role in the immune system and some show promising immunomodulatory properties [1, 2]. A good example is thymopentin (TP-5), a pentapeptide (H-Arg-Lys-Asp-Val-Tyr-OH, RKDVY) that corresponds to amino acids (AAs) 32–36 of the 49-AA thymichormone, thymopoietin. Like thymopoetin, TP-5 actively affects the function of T cells and monocytes through enhancing cGMP level and triggering cellular signal transduction, respectively [3–6]. A number of clinical studies have demonstrated that TP-5 treatment alleviates an existing imbalance in the cell-cytokine network of the immune system without observable side effects even at very high doses [1, 5]. As a result, TP-5 injection Correspondence: Dr. Jingkai Gu, College of Life Science, Jilin University, 2699 Qianjin Street, Changchun 130012, P. R. China E-mail: [email protected] Fax: +86-431-85155380

Abbreviations: AA, amino acid; AUC, area under curve; ES, extra stability; IS, internal standard; LLOQ, lower limit of quantitation; TP-5, thymopentin  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

has been widely used for the treatment of hepatitis B, rheumatoid arthritis, AIDS, severe acute respiratory syndrome, cutaneous T-cell lymphoma/cancer immunodeficiency, and other primary immunodeficiencies [5–8]. However, its extremely short half-life means repeated injections are necessary to maintain effective plasma concentrations [9–11] unless it is administered as a biodegradable implant [12–14]. Although TP-5 has been applied in clinical treatment, to date there are very few reports on its pharmacokinetics study. It is a huge challenge to evaluate TP-5 in bio-matrices especially in blood samples at therapeutic doses and to clarify its pharmacokinetic properties in vivo, since serine protease and aminopeptidase M-like enzymes rapidly inactivate the peptide after administration [15]. The enzymic degradation of TP-5 also poses a significant problem in pharmacokinetic studies since it continues during blood collection and sample preparation. Strategies to overcome this problem and investigate TP-5 pharmacokinetics albeit indirectly have employed modified derivatives of TP-5 including fluorescein isothiocyanate labeled TP-5 [16] and a novel cyclic hexapeptide, cTP6 [17]. Although these ∗ Additional corresponding author: Dr. Yantong Sun E-mail: [email protected] ∗∗ These authors contributed equally to this work.

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M. Shi et al.

compounds provide useful data, with chemical construction changes, they perforce have pharmacokinetic properties different from those of TP-5. Accordingly, developing an effective way to stabilize TP-5 in blood samples remains an important issue. The determination of TP-5 is also challenged by the appropriate analytical tools. A sensitive and high throughput method for the quantitation of unmodified TP-5 especially in biological samples is essential. Although immunoassay remains a popular option for the determination of peptides [18], LC–MS/MS offers several advantages including improved precision, accuracy, and higher throughput, and has become the preferred analytical tool for their qualitative and quantitative analysis [18–21]. Here, we report a novel LC–MS/MS method for quantitation of TP-5 in dog blood using acetonitrile to stabilize the peptide in blood at the time of sampling. The method was successfully applied to a pharmacokinetic study in beagle dogs after single intravenous injection of 0.2 mg/kg TP-5.

2 Materials and methods

infusion of analyte and IS were as follows: nebulizer gas, 40 psi; curtain gas, 40 psi; collision gas, 10 psi; ionspray voltage, 5500 V; source temperature, 550⬚C; declustering potentials: 105 V for TP-5, 120 V for IS; collision energies: 45 eV for TP-5, 48 eV for IS. Data acquisition and calculation were performed by Analyst 1.4.2 software. 2.3 Stability in blood Stability of TP-5 in blood was assessed with different agents such as the anticoagulant, enzyme inhibitors, and organic solvent. Heparinized fresh blood samples (10 mL) were spiked with an aqueous solution of TP-5 at a fixed concentration (450 ng/mL) containing either EDTA (10 mM) or aprotinin (5 TIU/mL). Samples were then incubated at 37⬚C and aliquots (300 ␮L) removed after 0, 0.17, 0.33, 0.5, 0.75, 1, 2, 3, 5, 7.5, 10, 15, 20, and 30 min and put into 900 ␮L ice-cold acetonitrile. Blood samples spiked with TP-5 at 450 ng/mL were also added immediately into threefold volumes of acetonitrile and 1200 ␮L aliquots removed at the same time points. Aliquots were processed as described in Section 2.4. Each stability assessment was conducted in triplicate.

2.1 Chemicals and reagents 2.4 Sample preparation Thymopentin (purity> 98.0 %) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, P. R. China). Stable isotope labeled TP-5, R*(13 C6 ,15 N4 )-KDVY (purity> 99.8 %) for use as internal standard (IS) was synthesized by SciLight Biotechnology (Beijing, P.R. China). Formic acid and the serine protease inhibitor, aprotinin, were obtained from Sigma–Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and methanol were purchased from Fisher (Fair Lawn, NJ, USA). All other chemicals were analytical grade and used without further purification. High-purity water (18.2 M⍀) was obtained bypassing house-distilled water through a Milli-Q system (Millipore, Bedford, USA).

Immediately after collection, dog blood samples (300 ␮L) were accurately aliquoted into prechilled heparinized Eppendorf tubes containing 900 ␮L acetonitrile and 50 ␮L IS working solution (300 ng/mL). Tubes were vortexed for 30 s followed by centrifugation at 15 000 rpm for 10 min at 4⬚C. Supernatants were then transferred into new Eppendorf tubes containing 1200 ␮L dichloromethane, vortex-mixed for 30 s and centrifuged at 15000 rpm at 4⬚C for 5 min. Finally, 20 ␮L of clear supernatant was injected into the LC–MS/MS system for analysis. 2.5 Preparation of calibration standards and QC samples

2.2 LC–MS/MS conditions LC–MS/MS was carried out using an Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to an API 4000 mass spectrometer (AB SCIEX, Toronto, Canada) equipped with an ESI source operating in the positive ion (ESI+ ) mode. Chromatography was performed on an Ascentis Express Peptide ES C18 analytical column (2.7 ␮m, 150 × 4.6 mm id, Supelco, USA) maintained at 40⬚C protected by a Security Guard C18 guard column (4 × 3.0 mm id, Phenomenex, USA). Gradient elution employed 0.1% formic acid in water (solvent A) and methanol (solvent B) delivered at 0.6 mL/min according to the following linear gradient: 0–1.0 min, 10–80% B; 1.0–4.0 min, 80% B; 4.0– 4.5 min, 80–10% B; 4.5–8.0 min, 10% B. Detection involved multiple reaction monitoring using the transitions of the protonated molecular ions at m/z 680.7→400.3 for TP-5 and m/z 690.5→410.3 for IS. MS parameters optimized by direct  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Stock solutions (1 mg/mL) of TP-5 in water were diluted with water to produce series of standard and QC solutions. A similar stock solution of IS was diluted to give a 300 ng/mL IS working solution. TP-5 calibration standards were produced by diluting standard solutions with drug-free beagle dog blood to give concentrations of 1.50, 4.50, 15.0, 45.0, 150, 450, and 800 ng/mL. QC samples (4.50, 45.0, 640 ng/mL) were generated from QC solutions in the same way. All calibration standards and QC samples were treated immediately as described in Section 2.4. Processed samples were stored at 4⬚C until required.

2.6 Assay validation Assay validation was performed according to the US Food and Drug Administration Guidelines [22]. The validation www.jss-journal.com

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parameters included specificity, linearity, intra- and interassay precision and accuracy, extraction recovery, matrix effect and stability. Specificity was assessed by analyzing drug-free and drug-spiked blood from six beagle dogs to investigate any potential endogenous interference at the peak regions for TP5 and IS. Linearity in the range 1.50–800 ng/mL was evaluated by linear least-squares regression with a 1/x2 weighting of calibration curves based on peak area ratios prepared in triplicate on three separate batches. Intra- and interday precision (as percentage RSD) and accuracy (as relative error, RE) were estimated by analysis of six replicate QC samples on three separate days. The lower limit of quantification (LLOQ) was defined as the lowest concentration that could be determined with accuracy ±20% and precision

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

The pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr, RKDVY) corresponds to amino acids 32-36 of the 49 amino acid immunomodulatory polypeptide, thymopoi...
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