Differential mobility spectrometry tandem mass spectrometry with multiple ion monitoring for the bioanalysis of liraglutide Xiangjun Meng, Haitong Xu, Zhi Zhang, John Paul Fawcett, Junru Li, Yan Yang & Jingkai Gu Analytical and Bioanalytical Chemistry ISSN 1618-2642 Volume 409 Number 20 Anal Bioanal Chem (2017) 409:4885-4891 DOI 10.1007/s00216-017-0431-6
1 23
Your article is protected by copyright and all rights are held exclusively by SpringerVerlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.
1 23
Author's personal copy Anal Bioanal Chem (2017) 409:4885–4891 DOI 10.1007/s00216-017-0431-6
RESEARCH PAPER
Differential mobility spectrometry tandem mass spectrometry with multiple ion monitoring for the bioanalysis of liraglutide Xiangjun Meng 1 & Haitong Xu 1 & Zhi Zhang 1 & John Paul Fawcett 2 & Junru Li 1 & Yan Yang 1 & Jingkai Gu 1,3
Received: 17 March 2017 / Revised: 14 May 2017 / Accepted: 24 May 2017 / Published online: 28 June 2017 # Springer-Verlag Berlin Heidelberg 2017
Abstract Liraglutide is a glucagon-like peptide-1 analog for the treatment of type 2 diabetes. Major interference in plasma of human and animals and low fragment signal in tandem mass spectrometry are the main difficulties encountered in the bioanalysis of liraglutide. In this study, by combining differential mobility spectrometry (DMS) with multiple ion monitoring detection (MIM), a liquid chromatography differential mobility spectrometry tandem mass spectrometry with multiple ion monitoring detection (LC-DMS-MIM) method was developed for the quantitation of liraglutide in dog plasma. Mixed anion-exchange solid-phase extraction was used for sample preparation. The parameters of DMS were meticulously optimized to increase the signal-to-noise ratio of the analyte. The assay was linear in the range 1–100 ng/mL with good accuracy and precision. The lower limit of quantitation (LLOQ, the lowest standard on the calibration curve) of this method was 1 ng/mL. The research reveals that DMS is an effective tool for the elimination of interference in bioanalysis and that LC-DMS-MIM has better specificity and higher Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0431-6) contains supplementary material, which is available to authorized users. * Yan Yang
[email protected] * Jingkai Gu
[email protected] 1
School of Life Sciences, Jilin University, 2699 Qianjin Road, Changchun, Jilin 130012, China
2
School of Pharmacy, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
3
Clinical Pharmacology Center, Research Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin 130061, China
signal-to-noise ratio than classical liquid chromatographytandem mass spectrometry (LC-MS/MS) for the bioanalysis of liraglutide. Keywords Differential mobility spectrometry . Multiple ion monitoring . Liraglutide . Mixed anion-exchange
Introduction Glucagon-like peptide-1 (GLP-1) analogs have emerged as promising therapeutic agents for the treatment of type 2 diabetes (T2DM). Native human GLP-1 has an extremely short half-life of approximately 2 min due to its degradation by dipeptidyl peptidase (DPP)-4 [1]. There are currently several GLP-1 analogs approved viz. exenatide, liraglutide, lixisenatide, albiglutide, and dulaglutide [2, 3]. Liraglutide is a relatively long-acting agent requiring once-daily injection compared with twice-daily exenatide [4–6]. This is because it has greater reversible binding to serum albumin and is less susceptible to degradation by DPP-4 [7]. Besides its use in treating T2DM, high-dose liraglutide (marketed as Saxenda®) is an effective medication for the treatment of obesity [8]. To support the development of longer-acting and orally active GLP-1 analogs, a highly specific and sensitive bioanalytical method is required. Traditionally, enzymelinked immunosorbent assays (ELISA) have been used for quantitation of protein therapeutics, and liraglutide is no exception [9]. However, ELISA has several intrinsic shortcomings such as cross-reactivity and the trend in recent years has been to turn to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for protein and peptide analyses. Multiplereaction monitoring (MRM) is the most commonly used mode for quantitation in LC-MS/MS but, when analyzing peptides,
Author's personal copy 4886
it often suffers from low signal intensity of MS/MS fragments as found with liraglutide. An approach to increase the analytical sensitivity of peptides is to apply MIM, also called parent-to-parent pseudo-MRM. In this approach, the precursor and product ions selected in Q1 and Q3 respectively of a tandem mass spectrometer share the same m/z value [10, 11]. However, the method has significant drawbacks such as matrix interference and elevated background noise due to isobaric interference. For liraglutide, which shares 97% sequence identity with endogenous GLP-1, there is a lot of interference in plasma when using MIM. Therefore, action is required to decrease background noise and interference. DMS is an effective technology that can be used for background noise elimination [12, 13]. It separates ions in the gas phase prior to analysis by MS on the basis of their different mobilities [14, 15]. In a DMS device, an asymmetric separation voltage (SV) waveform that varies between high- and low-field regimes is applied between two planar electrodes perpendicular to the ion transport flow. Each ion exhibits a different mobility in the pulsed high- and low-electric fields and can be selected to pass out of the cell by applying a compound-specific compensation voltage (CoV) [12, 14, 16]. In this way, chemical background noise is significantly reduced or eliminated. By combining DMS and MIM, a high signal-to-noise ratio can be obtained. In this study, liquid chromatography differential mobility spectrometry tandem mass spectrometry with multiple ion monitoring detection (LC-DMS-MIM) was applied to the determination of liraglutide in dog plasma. On the basis of the results, it was concluded that the technique is superior to LC-MS/MS with MRM in reducing interference and improving sensitivity.
Meng X. et al.
(2.1 × 50 mm, 5 μm, Agela) column maintained at 40 °C and injection volume of 10 μL. Mobile phases A and B for gradient elution were 0.1% formic acid in water:acetonitrile (95:5, v/v) and acetonitrile respectively delivered at a flow rate of 0.4 mL/ min. The gradient was as follows: 0–0.5 min, 20% B; 0.5– 3.2 min, 20–70% B; 3.2–3.5 min, 70–99% B; 3.5–4.5 min, 99% B; 4.5–4.7 min, 99–20% B; and 4.7–5.5 min, 20% B. MS conditions A SCIEX QTRAP 6500 system equipped with a SelexION DMS system (SCIEX, Concord, ON, Canada) was used. The DMS device was mounted between the ion source and sampling orifice. The QTRAP system was operated in the positive electrospray ionization (ESI) mode using a TurboIonSpray® probe consists of a stainless steel tubing (0.012 in., o.d.). Optimized MS and DMS parameters are shown in Table 1. Preparation of calibration standards and quality control samples Two separate liraglutide standard solutions (1 mg/mL) were prepared in 7% aqueous acetic acid. The standard solutions were aliquoted into sets of 2.0 mL Eppendorf tubes and stored at −80 °C prior to use. Calibration standards were prepared by diluting one set of standard solutions with blank dog plasma to concentrations of 1, 2, 5, 10, 20, 50, 75, and 100 ng/mL. LLOQ and quality control (QC) samples at concentrations of 1, 4.5 (low), 45 (medium), and 80 ng/mL (high) were prepared in a similar way by diluting the other set of standard solutions. Sample preparation
Materials and methods Chemicals and reagents Liraglutide (purity 94.95%) was provided by Harbin Jixianglong Biotech Co Ltd. (Harbin, China). Exenatide (purity 91%) for use as internal standard (IS) was provided by Changchun Beyel Pharmaceutical Co Ltd. (Changchun, China). HPLC-grade acetonitrile was purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid, isopropanol, and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Acetic acid and ammonium hydroxide were purchased from Beijing Chemical Plant (Beijing, China). Deionized water was produced via a Millipore system. LC conditions Chromatography was performed using an Acquity® UPLC system (Waters, Milford, MA, USA) with a Venusil ASB C8
Samples (or calibration standards or QC samples) (400 μL) were added to 2.0 mL Eppendorf tubes followed by 50 μL IS Table 1 Optimum parameters for the determination of liraglutide in beagle dog plasma using LC-DMS-MIM Parameter
Liraglutide
IS
Curtain gas (N2, psi) Ionspray voltage (V) Turboheater temperature (°C) Nebulizer gas (N2, psi) Heater gas (N2, psi) m/z transition Declustering potential (V) Collision energy (eV) DMS temperature (°C) DMS resolution enhancement DMS separation voltage (V) DMS compensation voltage (V)
25 5500 500 40 45 939.0 → 939.0 55 14 150 Medium 3500 13
25 5500 500 40 45 1047.7 → 1047.7 60 10 150 Medium 3500 10.5
Author's personal copy Differential mobility spectrometry tandem mass spectrometry
4887
solution (5 ng/mL) and 1.2 mL methanol and the mixtures vortexed for 60 s. Mixtures were centrifuged at 13,300 rpm for 5 min and the supernatants transferred to 10 mL tubes. Then 5.5 mL 10% ammonium hydroxide was added, the mixtures vortexed for 60 s, and subjected to solid-phase extraction (SPE) using Oasis-mixed anion-exchange (MAX) cartridges (1cm3, 30 mg, Waters) pre-conditioned with 1.0 mL each of methanol and 10% ammonium hydroxide. After loading, cartridges were consecutively washed with 1.0 mL aliquots of 10% ammonium hydroxide, acetonitrile:water (10:90, v/v) and 0.1% formic acid before eluting with acetonitrile:isopropanol:water (75:20:5, v/v/v) containing 5% formic acid. The eluent was diluted 1:1 with water and 10 μL injected into the LC-MS system.
Method validation The procedure for method validation is detailed in the Electronic supplementary material (ESM).
Results and discussion Method validation Accuracy (as relative error (R.E.)) and precision (as relative standard deviation (R.S.D.)) values are given in Table 2. The values of
