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Determination of liraglutide in rat plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetics study
Accepted Manuscript Determination of liraglutide in rat plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetics study
Shiqi Dong, Yuan Gu, Guangli Wei, Duanyun Si, Changxiao Liu PII: DOI: Reference:
S1570-0232(18)30235-6 doi:10.1016/j.jchromb.2018.05.020 CHROMB 21184
To appear in: Received date: Revised date: Accepted date:
6 February 2018 9 May 2018 15 May 2018
Please cite this article as: Shiqi Dong, Yuan Gu, Guangli Wei, Duanyun Si, Changxiao Liu , Determination of liraglutide in rat plasma by a selective liquid chromatographytandem mass spectrometry method: Application to a pharmacokinetics study. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Chromb(2017), doi:10.1016/j.jchromb.2018.05.020
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ACCEPTED MANUSCRIPT Determination of liraglutide in rat plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetics study
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
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a
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Shiqi Donga,b, Yuan Gub, Guangli Weib, Duanyun Sib※, Changxiao Liu a,b※
China
State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin
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b
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D
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Institute of Pharmaceutical Research, Tianjin 300193, China
ACCEPTED MANUSCRIPT Abstract A simple, sensitive and selective LC-MS/MS method was developed for the quantitative analysis of liraglutide and validated in rat plasma. Human insulin was used as the internal standard. After a simple protein precipitation step, liraglutide was
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chromatographically separated using an InertSustain Bio C18 column with mobile
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phases comprising acetonitrile with 0.1% formic acid (A) and water with 0.1% formic
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acid (B). Detection was achieved using positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the
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concentration range 0.5~250 ng/mL (r2＞0.99). The intra- and inter-day precision
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values (expressed as relative standard deviation, RSD) of liraglutide ranged from 1.97~7.63% and 5.25~11.9, respectively. The accuracy (expressed as relative error,
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RE) ranged from -8.79~11.4%. Both the recovery and matrix effect were within
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acceptable limits. This method was successfully applied for the pharmacokinetics study of liraglutide in rats after subcutaneous administration.
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Keywords: liraglutide; LC-MS/MS; rat plasma; selective; pharmacokinetics.
ACCEPTED MANUSCRIPT 1. Introduction Accounting for approximately ninety percent of all diabetes cases, type 2 diabetes mellitus is a major disease severely affecting human health and a leading cause of mortality worldwide. According to the International Diabetes Federation
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(IDF), more than 425 million people worldwide suffered from diabetes in 2017, and
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this rate is expected to reach 629 million by 2045. Furthermore, more than 5 million
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people die of diabetes and its complications every year [1]. Although insulin and its analogs have played a huge role in the treatment of diabetes, their side effects,
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including hypoglycemia, obesity and allergies, cannot be ignored [2]. Hence, new
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treatment drugs to reduce blood glucose levels and prevent side effects are urgently needed. After years of research, the discovery of glucagon-like peptide-1 (GLP-1)
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receptor agonists has substantially helped diabetic patients, as these agents not only
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effectively reduce blood glucose levels and maintain normal glucose levels but also significantly reduce the risk of side effects. In recent years, GLP-1 receptor agonists
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diabetes.
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have become powerful competitors of insulin drugs in the treatment of type 2
Liraglutide is a recombinant human GLP-1 analog with an amino acid sequence 97% homologous to native GLP-1, which has been approved for use in adult type 2 diabetic patients [3]. Liraglutide consists of 31 amino acids with molecular weight of 3751 Da. Since its launch in 2009, liraglutide has been widely recognized by clinical doctors and patients because of its superior performance in stimulating glucose-dependent insulin secretion and inhibiting glucagon release [4]. In addition,
ACCEPTED MANUSCRIPT liraglutide exerts an obvious weight loss effect by decreasing energy intake [5] and is thus popular with obese patidents. In view of this, many pharmaceutical companies have recently began focusing on the development of liraglutide analogs or new GLP-1 receptor agonists, some of which are undergoing clinical trials. For example, Eli Lilly
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and Company launched Trulicity in 2014, and Novo Nordisk is actively promoting the
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development of Semaglutide. In the next several years, GLP-1 receptor agonists will
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usher in a new wave of international research and development. Therefore, developing a highly efficient analytical platform that is simple, robust and
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high-throughput is necessary to better facilitate liraglutide applications and accelerate
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the development of other novel GLP-1 receptor agonists. In 2002, Agerso utilized an enzyme-linked immunosorbent assay (ELISA)
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method to quantify liraglutide and applied it to a pharmacokinetics (PK) study for the
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first time. His findings were published in the journal Diabetologis [6]. Since then, this ELISA method has been widely recognized and has become the standard method for
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liraglutide determination in PK studies [7-11]. Ligand binding assays (LBA)
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including ELISA and RIA are the classical method for the quantitative analysis of peptides and proteins. The sensitivity of the LBA method are usually very high, and this method is applicable for almost all peptides and proteins. However, the shortcomings of ligand binding assays (LBAs) have become more fully elucidated with the recent advancement of liquid chromatography-mass spectrometry (LC-MS) and can be summarized as follows: LBA method development processes are time consuming and costly [12, 13], critical reagents are generally variable [14], LBA
ACCEPTED MANUSCRIPT methods that are developed for specific matrices/species cannot be readily transferred to others [15], and quantifying multiple compounds simultaneously is unrealistic [16]. Although shortcomings of the LBA method compared to the LC-MS/MS method have gradually been recognized, only a few LC-MS/MS methods have been developed to
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explore the PK profiles of peptides and proteins.
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Traditionally, LC-MS/MS methods have been used to provide robust
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bioanalytical support for drugs exposure studies [17-20], while peptides and proteins are exception because of the poor ionization, serious endogenous interference and low
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concentrations [21]. In recent years, LC-MS/MS methods have become a promising
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alternative to LBA methods as a platform for support of peptides and proteins studies, as enzyme digestion and immune-purification are applied to biological sample
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preprocessing [22-25]. Peptides and proteins can be hydrolyzed into multiple peptides
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with smaller molecular weights firstly, and then specific peptide is selected from them. The concentrations of peptides and proteins can be indirectly measured by the
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quantification of the specific peptide. This method can not only achieve the same
avoid
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sensitivity as the LBA method, but also has a stronger specificity and can effectively cross-reactions.
While
generally
effective,
enzyme
digestion
and
immune-purification increases the complexity of already-complex matrices. Therefore, many studies used for academic research are available, but it is difficult to achieve high-throughput application. In this study, we tried to develop a simple and direct LC-MS/MS method to quantify liraglutide in rat plasma. Fortunately, we successfully developed and fully validated an LC-MS/MS analytical method to quantify liraglutide
ACCEPTED MANUSCRIPT in rat plasma and applied this method to a preclinical PK study after subcutaneous administration to rats. 2. Experimental Methods 2.1. Chemicals and reagents
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Liraglutide and human insulin were manufactured by Novo Nordisk
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(Copenhagen, Denmark), and human insulin was used as the internal standard (IS).
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HPLC-grade methanol and acetonitrile (ACN) were purchased from Concord Co., Inc. (Tianjin, China), and formic acid (FA), used for HPLC, was purchased from Tianjin
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Guangfu Fine Chemical Research Institute (Tianjin, China). Ammonium acetate was
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purchased from Tianjin Guangfu Technology Development Co., LTD (Tianjin, China). Water was prepared in-house with the BM-40 water purification system from
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Zhongsheng Maoyuan Technology Development Co. Ltd. (Beijing, China). All other
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chemicals were of analytical grade. Drug-free heparinized rat plasma was freshly collected from Sprague Dawley (SD) rats in our laboratory and stored at -20°C before
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use.
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2.2. LC-MS/MS conditions Analysis was performed on an LC-MS/MS system consisted of a binary LC-30AD delivery pump, a DGU-20A5R vacuum degasser, a CTO-20A column oven, a SIL-30AC auto-sampler, a CBM-20A system controller (Shimadzu, Japan) and an API LCMS-8060 mass spectrometer (Shimadzu, Japan). The LC system was coupled to a mass spectrometer through an electro-spray ionization (ESI) source. Chromatographic separation was performed on an InertSustain Bio C18 column (GL
ACCEPTED MANUSCRIPT Sciences, 100 × 2.1 mm, 1.9 µm particle size) at a flow rate of 0.3 mL/min for 6.5 min at a column temperature of 40°C. The gradient elution solvents comprised 0.1% FA in ACN (mobile phase A) and 0.1% FA in water (mobile phase B). The gradient was initially employed at 55% B and then held for 0.5 min. The analytes were
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separated using a linear gradient changing from 55% B to 40% B within 3.5 min.
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Mobile phase B was then ramped from 40% to 5% over 0.1 min and held for 0.9 min
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to clean the column. The gradient ended at the initial conditions for 1.4 min to equilibrate the column. The auto-sampler temperature was maintained at 6°C, and the
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injection volume was set to 3 μL. Quantification was performed in the positive ion
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multiple reaction monitoring (MRM) mode of the transitions m/z 938.4→1128.3 for liraglutide and m/z 1162.2→143.2 for the IS. Full scan and product ion spectra of
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liraglutide are shown in Figure 1. The mass spectrometry parameters, including
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nebulizing gas flow, heating gas flow, interface temperature, desolventizer (DL) temperature, heat block temperature and drying gas flow, and the compound
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parameters containing collision energy, Q1 and Q3 voltages were optimized to obtain
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the highest sensitivity for the monitored transitions, and the final optimized parameters are summarized in Table 1. 2.3 Preparation of calibration curve and quality control (QC) samples A stock solution of liraglutide was prepared in solvent consisting of 70/30 ACN/ 5mM ammonium acetate at a concentration of 1.00 mg/mL. Working solutions at the concentrations of 10, 20, 40, 100, 500, 2000 and 5000 ng/mL and QC solutions at 25, 500 and 4000 ng/mL were prepared from the stock solution with the solvent. The
ACCEPTED MANUSCRIPT working solution for the IS was prepared in the above solvent at a concentration of 100 ng/mL. All solutions were stored at 4°C and placed 10 min at room temperature before use. Calibration curve and QC samples were prepared by spiking 10 µL of either standard or QC working solutions into 190 µL of blank rat plasma in
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polypropylene tubes. The final concentrations of liraglutide in calibration curves were
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0.5, 1, 2, 5, 25, 100 and 250 ng/mL, and those in QC samples were 1.25, 25, and 200
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ng/mL. All calibration and QC samples were prepared daily to avoid potential degradation or adsorption issues.
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2.4 Sample preparation
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Samples were thawed at room temperature for approximately 20 min and then the plasma was vortexed for 10 s. A 50 μL plasma sample and 10 μL of the IS
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working solution were transferred into 1.5 mL tubes, and then a 100 μL precipitant
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consisting of 70/30 ACN/ methanol (v/v) was added. The mixture was vortexed for 2 min and centrifuged at 12,000 rpm for 10 min; then, 3 μL of supernatant fluid were
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injected into the LC–MS/MS system. Serial standard and quality control samples (QC
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samples) were prepared following the method described above. 2.5 Method validation 2.5.1 Sensitivity and specificity The specificity was defined as non-interference at retention times of liraglutide and IS from the endogenous plasma components and no cross-interference between liraglutide and IS using the developed LC-MS/MS method. To investigate the specificity of the method, blank rat plasma samples collected from six different
ACCEPTED MANUSCRIPT batches were analyzed. The signal-to-noise ratio at the lower limit of quantification (LLOQ) was calculated to evaluate the sensitivity of this method. 2.5.2 Carryover A blank sample was analyzed following the injection of an upper limit of
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quantification (ULOQ) sample in order to evaluate carryover. The peak area of
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liraglutide obtained from the blank plasma sample should be less than 20% of that
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obtained from the LLOQ sample. 2.5.3 Cross-interference
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A control sample containing only IS and a ULOQ containing only liraglutide
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sample were analyzed in order to evaluate cross-interference between the liraglutide and IS. The peak area of liraglutide obtain from control sample should be less than 20%
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of that obtained from the LLOQ sample. The peak area of IS obtain from ULOQ
2.5.4 Linearity
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sample should be less than 5% of that obtained from the control sample.
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Linear calibration curves in rat plasma were generated by plotting the peak area
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ratio of liraglutide-IS versus the nominal concentration of calibration standards over the range 0.5-250 ng/mL. The calibration curves were fitted using a least square linear regression model y=ax + b, weighted by 1/x2 using Labsolution software. 2.5.5 Accuracy and precision The intra- and inter-day precision and accuracy were evaluated by parallel analytical runs performed on three consecutive days. Six replicates of liraglutide at four QC levels (0.5, 1.25, 25 and 200 ng/mL) were analyzed. Intra-day precision was
ACCEPTED MANUSCRIPT calculated using replicate (n=6) determinations for each concentration of the spiked plasma samples during a single analytical run. Inter-day precision and accuracy were calculated using replicate (n=18) determinations of each concentration made on three separate days. The precision was determined as the relative standard deviation (%
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RSD) and the accuracy was expressed as a percentage of the nominal concentration
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(relative error, % RE).
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2.5.6 Extraction recovery and matrix effect
The extraction recovery and matrix effect of the method were determined by
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comparing the peak areas of liraglutide at two concentrations (1.25, 25 and 200
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ng/mL) and those of IS at 10 ng/mL with six replicates, including standards, standards spiked before extraction and standards spiked after extraction. The extraction
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recoveries of liraglutide and IS were evaluated by comparing the peak areas of
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standards spiked before extraction with those of standards spiked after extraction. The matrix effects of liraglutide and IS were assessed by comparing the peak areas of
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standards with those of standards spiked after extraction.
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2.5.7 Stability.
The stability of QC samples for liraglutide were studied under different possible conditions, which included stability of three cycles of freeze/thaw (-80°C/ 22°C), stability of the analyte in the auto-sampler at 6°C for 24 h, stability of plasma samples at room temperature for 2.5 h. The stability of liraglutide was demonstrated by evaluating the relative error (% RE) and the relative standard deviation (% RSD) of the mean measured QC concentrations from their nominal concentrations.
ACCEPTED MANUSCRIPT 2.6. Pharmacokinetics study SD rats (weighted 200±20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The protocols for animal studies were carried out by the guidelines of the Animal Care and Use Committee of
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Tianjin Institute of Pharmaceutical Research, which was approved by Tianjin
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Municipal Science and Technology Commission (Tianjin, China). All animals were
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allowed free access to food and water during the experiments. Four rats (2 males and 2 females) were subcutaneously administered a single dose of 100 μg/kg liraglutide.
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200 μL of blood were collected from each rat at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 48
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and 72 h after subcutaneous administration. Heparin was used as the anticoagulant to obtain the rat plasma. All samples were immediately centrifuged at 3000 ×g for 5 min
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3. Result and discussion
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at 4°C immediately and separated plasma samples were stored at -80°C until analysis.
3.1 LC-MS/MS conditions
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Initially, liraglutide was dissolved in solvent consisting of 30/70 ACN/ water.
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However, poor linearity was observed in the concentration range 0.5~250 ng/mL when analyzing the working solutions, and we suspect that this was due to strong peptides adsorption. Then we enhanced the elution strength of the solvent and replaced the initial solvent with 70/30 ACN/ 5mM ammonium acetate. The results showed that the linearity of the working solutions in the range 0.5~250 ng/mL was very good. Therefore, peptides adsorption is directly related to the elution strength of the solvent, and the presence of ammonium ions is beneficial for the stabilization of
ACCEPTED MANUSCRIPT the peptides and proteins. In this study, the Shimadzu 8060 mass spectrometer was selected to quantify liraglutide in rat plasma. Since the mass-to-charge ratio range of the Shimadzu 8060 was less than 2000, the multiply charged precursor of liraglutide (the 4+ precursor at
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m/z 938.4) was selected (Fig. 1A). Many ion fragments with similar intensities were
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produced under a certain collision energy (Fig. 1B). Due to the similar structure of the
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peptide bond in liraglutide, multiple cleavages of the peptide bond occur at a certain collision energy, which makes it difficult to confirm the specific ion fragments with a
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high intensity for quantification. In addition, the multiply charged form of precursors
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and product ions of liraglutide also increase the difficulty to confirm the specific ion fragments. Initially a number of possible ion fragments ( m/z 1064, m/z 1128, m/z
and
pre-analysis
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optimization
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1185, m/z 1353 and m/z 523) were selected for investigation. After careful MRM of
biological
samples,
the
transitions
m/z
938.4→1128.3, which was high intensity with minimum interference, was finally
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selected to quantify liraglutide in rat plasma. After careful analysis we speculate that
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m/z 1128 is the b11 ion fragment. The transitions from precursor ion to product ion are m/z 1162.2→143.2 for human insulin (IS), and m/z 143.2 is the a2 ion fragment. Optimization of the chromatographic conditions was relatively simple, and focused mainly on evaluating the compositions of the mobile phase and the type of analytical column [26]. Based on our previous research experience, we found that ACN is more suitable than methanol as the organic phase, and the presence of FA was beneficial for the sensitivity of liraglutide. So the final mobile phases comprised 0.1%
ACCEPTED MANUSCRIPT FA in ACN (mobile phase A) and 0.1% FA in water (mobile phase B). The Diamonsil C18 column (Dikma Technologies Inc. 100 × 4.6 mm, 5 µm, pore diameter: 10 nm), the ZORBAX Eclipse Plus C8 column (Agilent, 100 × 4.6 mm, 5 µm, pore diameter: 10 nm) and the InertSustain Bio C18 column (GL Sciences, 100 × 2.1 mm, 1.9 µm,
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pore diameter: 20 nm) were tested to achieve the minimum matrix interference and
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symmetric peak shapes. Both the Diamonsil C18 column and the ZORBAX Eclipse
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Plus C8 column showed a poor peak shape and a serious peak tailing. In contrast, the InertSustain Bio C18 column provided minimum matrix interference and a good
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chromatographic resolution with sharp peaks. This difference was caused by the
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difference in the particle size of the analytical columns. The smaller the particle size, the higher the efficiency.
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As we all know, solid-phase extraction (SPE) is the preferred way for the
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biological sample preprocessing of peptides and proteins when using LC-MS/MS. In our study, the WondaSep MCX and Monospin C18 SPE cartridges were initially
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tested to separate and enrich liraglutide; however, only 15% of liraglutide was
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recovered because of a high plasma protein binding ratio. Then, protein precipitation was tested to both clean the sample and disrupt protein binding. To find the best precipitant, methanol, acetonitrile and the mixed solution of them were tested. When acetonitrile was used as a precipitant, coprecipitation of partial liraglutide with plasma proteins resulted in lower recovery. 55% recovery was achieved using an acetonitrile: plasma ratio of 1:1. When the above ratio became 2:1 and 3:1, the recoveries were 35% and 30%, respectively. Although methanol did not cause precipitation of the analyte,
ACCEPTED MANUSCRIPT it was not as effective as acetonitrile in removing interference. Fortunately, the ACN and methanol combination (70/30 ACN/methanol) provided the best sensitivity and recoveries (80~90%) for the liraglutide from rat plasma after continuous testing. 3.2 Method validation
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3.2.1 Sensitivity and specificity
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Representative chromatograms of liraglutide and IS are shown in Figure 2,
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including a blank plasma sample (Fig. 2A), a blank plasma sample spiked with liraglutide at 0.5 ng/mL (Fig. 2B) and a rat plasma sample obtained 6 h after
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subcutaneous administration (Fig. 2C). No interfering peaks were observed at the
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retention times of liraglutide and IS in the blank sample. The response intensity of liraglutide was sufficient for quantification at the concentration of 0.5 ng/ mL
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3.2.2 Carryover
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(LLOQ).
A blank sample was analyzed following the ULOQ to evaluate carryover. No
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peak was found at the retention time of liraglutide in the blank sample, indicating that
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the carryover of the method was acceptable. 3.2.3 Cross-interference No peak was found at the retention time of liraglutide in the control sample, and no peak was found at the retention time of IS in the ULOQ sample. The result showed that there was no cross-interference between the liraglutide and IS. 3.2.4 Linearity The plasma calibration curve was constructed by seven calibrators over the
ACCEPTED MANUSCRIPT concentration range of 0.5~250 ng/mL. A typical standard curve for liraglutide was y=0.0467x-0.00145 (r2=0.9978) with a weighting factor of 1/x2. The ratio of liraglutide to IS was calculated, and x represented the corresponding plasma concentrations of liraglutide.
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3.2.5 Accuracy and precision
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The accuracy and precision of liraglutide at four concentrations (0.5, 1.25, 25
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and 200 ng/mL) were calculated (Table 2). The intra- and inter-day precision (expressed as RSD) of liraglutide were in the range of 1.97~7.63% and 5.25~11.9,
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respectively. The accuracy (expressed as RE) ranged from -8.79~11.4%. The results
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indicate that the LC/MS/MS method had good accuracy and excellent precision. 3.2.6 Extraction recovery and matrix effect
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The matrix effect can be regarded as the ion suppression or enhancement of the
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analyte [27]. The extraction procedure and chromatographic conditions were optimized to decrease or even eliminate the unfavorable influence of the matrix effect
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[28]. In this study, the matrix values were 109±3.40%, 112±2.10% and 107±3.28% for
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liraglutide and 102±3.12% for IS, indicating that no matrix effect for liraglutide and IS existed in the method. Under these operating conditions, the detailed values of recoveries in rat plasma were 82.3±1.98%, 85.1±2.46% and 84.2±2.07% for liraglutide and 65.6±3.42% for IS.
ACCEPTED MANUSCRIPT 3.2.7 Stability The stability results, which are listed in Table 3, demonstrate that the analyte displayed excellent stability under all experimental conditions. The RSD ranged from 0.541% to 3.12%, and the RE ranged from -10.4~10.0%.
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3.2.8 Incurred sample reanalysis
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In this study, about 20% of the study sample size were selected for reanalysis.
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The result showed that 90% of the repeated sample results were within 20%. The detail results were shown in Table 4.
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3.3 Comparison of methods
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In recent years, a few LC-MS/MS methods [29-31], which are summarized in the Table 5, have been developed to explore the PK profiles of liraglutide as the
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shortcomings of LBA methods are gradually recognized. Compared with the
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published LC-MS/MS methods, we believe that three aspects of this manuscript will make it interesting to general readers. Firstly, the key technique of our LC-MS/MS
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method compared to the reported methods was the simplification and improvement of
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the sample preprocessing. In the published reports, the researchers failed to directly measure liraglutide in the biological samples after precipitating because the precipitant they used was not suitable. In Meng’s study, methanol was selected to disrupt the binding of liraglutide to albumin and to inactivate neutral endopeptidases. But methanol was not enough to remove all interference in plasma. Therefore, after the protein precipitation procedure, SPE was utilized for the sample preprocessing of liraglutide to enhance the sample cleanup. In Shah’s study, initially ice cold (-20ºC)
ACCEPTED MANUSCRIPT acetonitrile was selected as a plasma protein denaturing agent, but a strong interference and low recovery were observed at analyte RT in lower concentration. Therefore, protein precipitation was eventually replaced by SPE for plasma sample preprocessing. In this study, the new precipitant we formulated provided the best
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sensitivity and recoveries (80~90%) for the liraglutide from rat plasma, and the
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biological sample could be analyzed directly by the mass spectrometer after a simple
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step of precipitation. In addition, the sensitivity and linearity of our method are superior to Meng’s study. In Shah’s study and Kellie’s study, the linearity is quite
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unreasonable because the exposure level of liraglutide in vivo range from 1~200
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ng/mL [7,8,10,32]. Moreover, the plasma volume used for analysis is smaller in our study, which is a huge benefit to experimental animals or people.
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3.4 Pharmacokinetics study
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Our method was successfully applied to the PK study after the subcutaneous administration of 100 μg/kg liraglutide to rats. The mean plasma concentration-time
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profile of liraglutide with a single subcutaneous administration to four rats is
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illustrated in Fig. 3. The mean PK parameters, including Cmax, Tmax, t1/2, AUC0-12h, AUC0-∞, Vd, CL and MRT, obtained after the subcutaneous administration of liraglutide are summarized in Table 6. Liraglutide could balance blood glucose for a long time, and the elimination of the drug was slow in rats. The level of liraglutide was lower than the LLOQ in rat plasma 48 h after dosing. The successful application of the LC–MS/MS method indicated that this analytical method was suitable and sufficient for the PK study of liraglutide.
ACCEPTED MANUSCRIPT 4. Conclusion In this manuscript, a simple, specific and high-throughput LC-MS/MS analytical method was successfully developed and fully validated to quantify liraglutide in rat plasma. This method was successfully applied to the pharmacokinetics study of
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liraglutide after subcutaneous administration to rats. In addition, a simple precipitation
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method was developed for the biological sample pretreatment, which was simpler,
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more efficient and more inexpensive than previously reported methods. The biological samples could be analyzed directly by the mass spectrometer after a simple protein
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precipitation step. Moreover, the sensitivity was reasonable, and the LLOQ of this
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method was 0.5 ng/ mL. The plasma volume used for analysis is smaller in our study, which is a huge benefit to experimental animals or people. I believe our method will
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dramatically enhance the analytical efficiency of liraglutide and greatly reduce the
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costs caused by time-consuming method development, sample analysis and critical reagents, such as antibodies. We hope that this study will serve as a valuable tool to
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accelerate the development of liraglutide and its analogues.
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Acknowledgments
The work was supported by “National Natural Science Foundation of China” [grant number 81503154].
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[10] D.J. Klein, T. Battelino, D.J. Chatterjee, L.V. Jacobsen, P.M. Hale, et al,
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Liraglutide’s safety, tolerability, pharmacokinetics, and pharmacodynamics in
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pediatric type 2 diabetes: a randomized, double-blind, placebo-controlled trial, Diabetes Technology & Therapeutics 16 (2014) 679-687.
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[11] A. Plum, L.B. Jensen, J.B. Kristensen, In vitro protein binding of liraglutide in
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human plasma determined by reiterated stepwise equilibrium dialysis, Journal of Pharmaceutical Sciences 102 (2013) 2882-2888.
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[12] N. Savoie, F. Garofolo, A.P. van, S. Bansal, C. Beaver, P. Bedford, B.P. Booth, C.
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Evans, M. Jemal, M. Lefebvre, et al, 2010 white paper on recent issues in regulated bioanalysis & global harmonization of bioanalytical guidance, Bioanalysis 2 (2010) 1945-1960. [13] W.L. Nowatzke, K. Rogers, E. Wells, R.R. Bowsher, C. Ray, and S. Unger, Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs, Bioanalysis 3 (2011) 509-521. [14] D.M. O’Hara, V. Theobald, A.C. Egan, J. Usansky, M. Krishna, J. TerWee, M.
ACCEPTED MANUSCRIPT Maia, F.P. Spriggs, J. Kenney, A. Safavi, et al, Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents, AAPS J 14 (2012) 316-328. [15] A.N. Hoofnagle, M.H. Wener, The fundamental flaws of immunoassays and
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potential solutions using tandem mass spectrometry, J Immunol Methods 347
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(2009) 3-11.
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[16] Y. Xiao, L. Guo, Y. Wang, A targeted quantitative proteomics strategy for global kinome profiling of cancer cells and tissues, Mol Cell Proteomics 13 (2014)
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1065-1075.
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[17] R. Gajula, N.R. Pilli, V.B. Ravi, R. Maddela, J.K. Inamadugu, S.R. Polagani, S. Busa, Simultaneous determination of atorvastatin and aspirin in human plasma
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(2011) 439-449.
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by LC-MS/MS: its pharmacokinetic application, Biomedical Chromatography 26
[18] J. Déglon, A. Thomas, Y. Daali, E. Lauer, C. Samer, J. Desmeules, P. Dayer, P.
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Mangin, C. Staub, Automated system for on-line desorption of dried blood spots
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applied to LC-MS/MS pharmacokinetic study of flubiprofen and its metabolite, Journal of Pharmaceutical & Biomedical Analysis 54 (2011) 359-367. [19] X. Feng, Y. Liu, X. Wang, X. Di, A rapid and sensitive LC−MS/MS method for the determination of linarin in small-volume rat plasma and tissue samples and its application to pharmacokinetic and tissue distribution study, Biomedical Chromatography 30 (2016) 618-624. [20] C.P. Vieira, D.V. Neves, E.J. Cesarino, A. Rocha, S. Poirier, V.L. Lanchote, An
ACCEPTED MANUSCRIPT indirect stereoselective analysis of nebivolol glucuronides in plasma by LC-MS/MS: application to clinical pharmacokinetics, Journal of Pharmaceutical & Biomedical Analysis 144 (2017) 25-30. [21] J.D. Russell, M. Scalf, A.J. Book, D.T. Ladror, R.D. Vierstra, L.M. Smith, J.J.
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Coon, Characterization and quantification of intact 26S proteasome proteins by
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real-time measurement of intrinsic fluorescence prior to top-down mass
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spectrometry, Plos One 8 (2013) 1-8.
[22] P. Bults, R. Bischoff, H. Bakker, J.A. Gietema, N.C. Van de Merbel,
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LC-MS/MS-based monitoring of in vivo protein biotransformation: quantitative
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determination of trastuzumab and its deamidation products in human plasma, Analytical Chemistry 88 (2016) 1871-1877.
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[23] K.J. Bronsema, R. Bischoff, N.C.V.D. Merbel, High-sensitivity LC-MS/MS
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quantification of peptides and proteins in complex biological samples: the impact of enzymatic digestion and internal standard selection on method performance,
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Analytical Chemistry 85 (2013) 9528-9535.
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[24] H.H. Wang, S.K. Drake, J.H. Youn, A.Z. Rosenberg, Y. Chen, M. Gucek, A.F. Suffredini, J.P. Dekker, Peptide markers for rapid detection of KPC carbapenemase by LC-MS/MS, Scientific Reports 7 (2017) 1-10. [25] E.N. Fung, P. Bryan, A. Kozhich, Techniques for quantitative LC-MS/MS analysis
of protein therapeutics:
advances in enzyme digestion and
immunocapture, Bioanalysis 8 (2016) 847-856. [26] Y. Yuan, X. Zhou, J. Li, S.F. Ye, X.W. Ji, L. Li, T.Y. Zhou, W. Lu, Development
ACCEPTED MANUSCRIPT and validation of a highly sensitive LC-MS/MS method for the determination of dexamethasone in nude mice plasma and its application to a pharmacokinetic study, Biomed Chromatogr. 29 (2015) 578-583. [27] F.T. Peters, D. Remane, Aspects of matrix effects in applications of liquid
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chromatography–mass spectrometry to forensic and clinical toxicology-a review, Analytical and Bioanalytical Chemistry 403 (2012) 2155-2172.
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[28] R. Earla, K. Cholkar, S. Gunda, R.L. Earla. A.K. Mitra, Bioanalytical method validation of rapamycin in ocular matrix by QTRAP LC-MS/MS: Application to
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rabbit anterior tissue distribution by topical administration of rapamycin nanomicellar formulation, Journal of Chromatography B: Analytical Technology
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in Biomedicine and Life Sciences 908 (2012) 76-86.
[29] J.F. Kellie, J.R. Kehler, M.E. Szapacs, Application of high-resolution MS for
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development of peptide and large-molecule drug candidates, Bioanalysis 8 (2016) 169-177.
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[30] X. Meng, H. Xu, Z. Zhang, J.P. Fawcett, J. Li, Y.Yang, J. Gu, Differential
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mobility spectrometry tandem mass spectrometry with multiple ion monitoring for the bioanalysis of liraglutide, Anal Bioanal Chem. 409 (2017) 4885-4891. [31] M.H. Shah, P.R.Y. Reddy, M. Bhawanishankar, M.J. Raja, M. Pillai,
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UHPLC-MS/MS determination of GLP-1 analogue, liraglutide a bioactive peptide in human plasma, European Journal of Biomedical and Pharmaceutical
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Sciences 4 (2017) 304-311. [32] E. Streckel, C. Braunreichhart, N. Herbach, M. Dahlhoff, B. Kessler, et al, Effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition, Journal of Translational Medicine 13 (2015) 1-13.
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Table 1 Mass Spectrometry Conditions for liraglutide and IS
Compound name
MRM
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Q1 pre bias
Interface
DL
Heat block
Drying
gas flow
gas flow
temperature
temperature
temperature
gas flow
(L/min)
(℃)
(℃)
(℃)
(L/min)
10
300
250
400
10
10
300
250
400
10
N A
(eV)
(V)
(V)
(L/min)
M
-29
-40
-32
IS
1162.2→143.2
-58
-44
D E
T P E
A
C C
C S U
Heating
transition
938.4→1128.3
-29
I R
Nebulizing Q3 pre bias
liraglutide
T P
3 3
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Table 2 Intra- and inter-day precision and accuracy of the method for the determination of liraglutide in rat plasma
Spiked concentration
Measured concentration
Intra-day precision (n=6)
(ng/mL)
(ng/mL)
(%, RSD)
0.5
0.510±0.0391
7.63
1.25
1.14±0.05
4.19
25
24.4±1.10
1.97
200
223±7.33
T P E
D E
A
C C
T P
I R
Inter-day precision (n=18)
C S U
Accuracy (n=18)
(%, RSD)
(%, RE)
7.91
2.02
5.25
-8.79
M
11.9
-2.30
2.33
7.16
11.4
N A
ACCEPTED MANUSCRIPT Table 3 Stability of liraglutide in rat plasma under different storage conditions (n=6)
Concentration levels (ng/mL)
Precision
Accuracy
Storage conditions Measured (mean ± SD)
RSD (%)
RE (%)
Stability for 2.5 h at room
1.25
1.28 ± 0.0398
3.12
2.21
temperature
200
220 ± 1.33
0.571
10.0
Stability for extracted samples in
1.25
1.12 ± 0.0237
2.31
-10.4
auto-sample for 24 h
200
189 ± 3.66
1.94
-5.55
Stability for three freeze-thaw
1.25
1.30 ± 0.007
0.541
3.60
211 ± 3.84
1.82
5.34
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CE
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D
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Cycles (-80oC /22oC）
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Spiked
200
ACCEPTED MANUSCRIPT Table 4 The result of incurred sample reanalysis
Difference Number
Sample name
Original
Repeat
Mena (%)
124
118
2
ISR-Dog-SC-1#-24h
19.5
20.2
3
ISR-Dog-SC-2#-10h
61.5
85
4
ISR-Dog-SC-2#-12h
61.8
5
ISR-Dog-SC-2#-24h
6.29
6
ISR-Dog-SC-3#-10h
101
7
ISR-Dog-SC-3#-24h
8
ISR-Dog-SC-4#-8h
9 10
121
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ISR-Dog-SC-1#-10h
19.9
-4.96 3.53 32.1
60.4
61.1
-2.29
6.44
6.365
2.36
103
102
1.96
22.1
20.8
21
-6.06
101
100.0
100.5
-1.00
ISR-Dog-SC-4#-10h
102
104
103
1.94
ISR-Dog-SC-4#-24h
9.45
10.2
9.8
7.63
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D
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ISR number
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Accept ISR number
Accept ISR percentage (%)
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73
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1
10 9 90
ACCEPTED MANUSCRIPT Table 5 The comparison of our LC-MS/MS method with other published LC-MS/MS methods for the bioanalysis of liraglutide
Plasma Publication date
Sample preparation
Sensitivity
Linearity
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volume ng/mL
ng/mL
µL
100
100~10000
25
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year 2015
Incubation + precipitation
Meng’s study
2017
Precipitation + SPE
1.0
1.0~100
400
Shah’s study
2017
SPE
0.0005
0.0005~0.1
1000
0.5
0.5~250
50
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Precipitation
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My manuscript
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Kellie’ study
ACCEPTED MANUSCRIPT Table 6 Pharmacokinetic parameters of liraglutide in rats after subcutaneous administration
subcutaneous Pharmacokinetic parameter
T1/2
h
4.01 ± 0.227
Cmax
ng/ml
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administration (100 μg/kg)
Tmax
h
AUC0-t
ng·h/ml
AUC0-∞
ng·h/ml
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10 ± 0.476
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Vd
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1361 ± 21.2 1385 ± 19.6
mL
100 ± 10.4
mL/h
84 ± 2.12
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CL MRT
103 ± 31.1
h
11 ± 0.289
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T1/2, Elimination half-life; Cmax, The peak plasma concentration; Tmax, Time to reach Cmax; AUC0-t,
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Area under the curve the concentration-time curve from 0 to 48h; AUC0-∞, Area under the curve from 0 to infinity; Vd, Volume of distribution; CL, Clearance; MRT, Mean residence time.
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Legend of figure Fig.1. Full scan (A) and product ion (B) spectra of liraglutide
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Fig.2. Representative chromatograms of liragultide (4.08 min) and IS (3.17 min). (A)
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Blank rat plasma sample. (B) Blank plasma sample spiked with 0.5 ng/mL of
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liraglutide (LLOQ) and 10 ng/mL of IS. (C) A plasma sample collected at 6 h after subcutaneous administration of 100 μg/kg liraglutide.
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Fig.3. The mean plasma concentration–time profile of liraglutide in rat plasma after
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CE
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subcutaneous administration of 100 μg/kg liraglutide (n=4).
ACCEPTED MANUSCRIPT Highlights (1) A simple, specific and high-throughput LC-MS/MS analytical method was successfully developed and validated to quantify liraglutide in rat plasma. (2) The sample preprocessing is simple and the biological samples can be analyzed
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directly by the mass spectrometer after a simple protein precipitation step.
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more reasonable than previously reported methods.
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(3) The calibration curve range and the sensitivity of our LC-MS/MS method are
(4) The plasma volume used for analysis is smaller in our study, which is a huge
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CE
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D
MA
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benefit to experimental animals or people.
Figure 1
Figure 2
Figure 3
Shiqi Dong, Yuan Gu, Guangli Wei, Duanyun Si, Changxiao Liu PII: DOI: Reference:
S1570-0232(18)30235-6 doi:10.1016/j.jchromb.2018.05.020 CHROMB 21184
To appear in: Received date: Revised date: Accepted date:
6 February 2018 9 May 2018 15 May 2018
Please cite this article as: Shiqi Dong, Yuan Gu, Guangli Wei, Duanyun Si, Changxiao Liu , Determination of liraglutide in rat plasma by a selective liquid chromatographytandem mass spectrometry method: Application to a pharmacokinetics study. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Chromb(2017), doi:10.1016/j.jchromb.2018.05.020
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ACCEPTED MANUSCRIPT Determination of liraglutide in rat plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetics study
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
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a
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Shiqi Donga,b, Yuan Gub, Guangli Weib, Duanyun Sib※, Changxiao Liu a,b※
China
State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin
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b
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CE
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D
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Institute of Pharmaceutical Research, Tianjin 300193, China
ACCEPTED MANUSCRIPT Abstract A simple, sensitive and selective LC-MS/MS method was developed for the quantitative analysis of liraglutide and validated in rat plasma. Human insulin was used as the internal standard. After a simple protein precipitation step, liraglutide was
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chromatographically separated using an InertSustain Bio C18 column with mobile
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phases comprising acetonitrile with 0.1% formic acid (A) and water with 0.1% formic
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acid (B). Detection was achieved using positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the
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concentration range 0.5~250 ng/mL (r2＞0.99). The intra- and inter-day precision
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values (expressed as relative standard deviation, RSD) of liraglutide ranged from 1.97~7.63% and 5.25~11.9, respectively. The accuracy (expressed as relative error,
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RE) ranged from -8.79~11.4%. Both the recovery and matrix effect were within
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acceptable limits. This method was successfully applied for the pharmacokinetics study of liraglutide in rats after subcutaneous administration.
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Keywords: liraglutide; LC-MS/MS; rat plasma; selective; pharmacokinetics.
ACCEPTED MANUSCRIPT 1. Introduction Accounting for approximately ninety percent of all diabetes cases, type 2 diabetes mellitus is a major disease severely affecting human health and a leading cause of mortality worldwide. According to the International Diabetes Federation
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(IDF), more than 425 million people worldwide suffered from diabetes in 2017, and
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this rate is expected to reach 629 million by 2045. Furthermore, more than 5 million
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people die of diabetes and its complications every year [1]. Although insulin and its analogs have played a huge role in the treatment of diabetes, their side effects,
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including hypoglycemia, obesity and allergies, cannot be ignored [2]. Hence, new
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treatment drugs to reduce blood glucose levels and prevent side effects are urgently needed. After years of research, the discovery of glucagon-like peptide-1 (GLP-1)
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receptor agonists has substantially helped diabetic patients, as these agents not only
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effectively reduce blood glucose levels and maintain normal glucose levels but also significantly reduce the risk of side effects. In recent years, GLP-1 receptor agonists
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diabetes.
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have become powerful competitors of insulin drugs in the treatment of type 2
Liraglutide is a recombinant human GLP-1 analog with an amino acid sequence 97% homologous to native GLP-1, which has been approved for use in adult type 2 diabetic patients [3]. Liraglutide consists of 31 amino acids with molecular weight of 3751 Da. Since its launch in 2009, liraglutide has been widely recognized by clinical doctors and patients because of its superior performance in stimulating glucose-dependent insulin secretion and inhibiting glucagon release [4]. In addition,
ACCEPTED MANUSCRIPT liraglutide exerts an obvious weight loss effect by decreasing energy intake [5] and is thus popular with obese patidents. In view of this, many pharmaceutical companies have recently began focusing on the development of liraglutide analogs or new GLP-1 receptor agonists, some of which are undergoing clinical trials. For example, Eli Lilly
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and Company launched Trulicity in 2014, and Novo Nordisk is actively promoting the
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development of Semaglutide. In the next several years, GLP-1 receptor agonists will
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usher in a new wave of international research and development. Therefore, developing a highly efficient analytical platform that is simple, robust and
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high-throughput is necessary to better facilitate liraglutide applications and accelerate
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the development of other novel GLP-1 receptor agonists. In 2002, Agerso utilized an enzyme-linked immunosorbent assay (ELISA)
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method to quantify liraglutide and applied it to a pharmacokinetics (PK) study for the
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first time. His findings were published in the journal Diabetologis [6]. Since then, this ELISA method has been widely recognized and has become the standard method for
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liraglutide determination in PK studies [7-11]. Ligand binding assays (LBA)
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including ELISA and RIA are the classical method for the quantitative analysis of peptides and proteins. The sensitivity of the LBA method are usually very high, and this method is applicable for almost all peptides and proteins. However, the shortcomings of ligand binding assays (LBAs) have become more fully elucidated with the recent advancement of liquid chromatography-mass spectrometry (LC-MS) and can be summarized as follows: LBA method development processes are time consuming and costly [12, 13], critical reagents are generally variable [14], LBA
ACCEPTED MANUSCRIPT methods that are developed for specific matrices/species cannot be readily transferred to others [15], and quantifying multiple compounds simultaneously is unrealistic [16]. Although shortcomings of the LBA method compared to the LC-MS/MS method have gradually been recognized, only a few LC-MS/MS methods have been developed to
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explore the PK profiles of peptides and proteins.
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Traditionally, LC-MS/MS methods have been used to provide robust
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bioanalytical support for drugs exposure studies [17-20], while peptides and proteins are exception because of the poor ionization, serious endogenous interference and low
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concentrations [21]. In recent years, LC-MS/MS methods have become a promising
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alternative to LBA methods as a platform for support of peptides and proteins studies, as enzyme digestion and immune-purification are applied to biological sample
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preprocessing [22-25]. Peptides and proteins can be hydrolyzed into multiple peptides
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with smaller molecular weights firstly, and then specific peptide is selected from them. The concentrations of peptides and proteins can be indirectly measured by the
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quantification of the specific peptide. This method can not only achieve the same
avoid
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sensitivity as the LBA method, but also has a stronger specificity and can effectively cross-reactions.
While
generally
effective,
enzyme
digestion
and
immune-purification increases the complexity of already-complex matrices. Therefore, many studies used for academic research are available, but it is difficult to achieve high-throughput application. In this study, we tried to develop a simple and direct LC-MS/MS method to quantify liraglutide in rat plasma. Fortunately, we successfully developed and fully validated an LC-MS/MS analytical method to quantify liraglutide
ACCEPTED MANUSCRIPT in rat plasma and applied this method to a preclinical PK study after subcutaneous administration to rats. 2. Experimental Methods 2.1. Chemicals and reagents
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Liraglutide and human insulin were manufactured by Novo Nordisk
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(Copenhagen, Denmark), and human insulin was used as the internal standard (IS).
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HPLC-grade methanol and acetonitrile (ACN) were purchased from Concord Co., Inc. (Tianjin, China), and formic acid (FA), used for HPLC, was purchased from Tianjin
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Guangfu Fine Chemical Research Institute (Tianjin, China). Ammonium acetate was
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purchased from Tianjin Guangfu Technology Development Co., LTD (Tianjin, China). Water was prepared in-house with the BM-40 water purification system from
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Zhongsheng Maoyuan Technology Development Co. Ltd. (Beijing, China). All other
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chemicals were of analytical grade. Drug-free heparinized rat plasma was freshly collected from Sprague Dawley (SD) rats in our laboratory and stored at -20°C before
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use.
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2.2. LC-MS/MS conditions Analysis was performed on an LC-MS/MS system consisted of a binary LC-30AD delivery pump, a DGU-20A5R vacuum degasser, a CTO-20A column oven, a SIL-30AC auto-sampler, a CBM-20A system controller (Shimadzu, Japan) and an API LCMS-8060 mass spectrometer (Shimadzu, Japan). The LC system was coupled to a mass spectrometer through an electro-spray ionization (ESI) source. Chromatographic separation was performed on an InertSustain Bio C18 column (GL
ACCEPTED MANUSCRIPT Sciences, 100 × 2.1 mm, 1.9 µm particle size) at a flow rate of 0.3 mL/min for 6.5 min at a column temperature of 40°C. The gradient elution solvents comprised 0.1% FA in ACN (mobile phase A) and 0.1% FA in water (mobile phase B). The gradient was initially employed at 55% B and then held for 0.5 min. The analytes were
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separated using a linear gradient changing from 55% B to 40% B within 3.5 min.
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Mobile phase B was then ramped from 40% to 5% over 0.1 min and held for 0.9 min
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to clean the column. The gradient ended at the initial conditions for 1.4 min to equilibrate the column. The auto-sampler temperature was maintained at 6°C, and the
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injection volume was set to 3 μL. Quantification was performed in the positive ion
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multiple reaction monitoring (MRM) mode of the transitions m/z 938.4→1128.3 for liraglutide and m/z 1162.2→143.2 for the IS. Full scan and product ion spectra of
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liraglutide are shown in Figure 1. The mass spectrometry parameters, including
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nebulizing gas flow, heating gas flow, interface temperature, desolventizer (DL) temperature, heat block temperature and drying gas flow, and the compound
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parameters containing collision energy, Q1 and Q3 voltages were optimized to obtain
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the highest sensitivity for the monitored transitions, and the final optimized parameters are summarized in Table 1. 2.3 Preparation of calibration curve and quality control (QC) samples A stock solution of liraglutide was prepared in solvent consisting of 70/30 ACN/ 5mM ammonium acetate at a concentration of 1.00 mg/mL. Working solutions at the concentrations of 10, 20, 40, 100, 500, 2000 and 5000 ng/mL and QC solutions at 25, 500 and 4000 ng/mL were prepared from the stock solution with the solvent. The
ACCEPTED MANUSCRIPT working solution for the IS was prepared in the above solvent at a concentration of 100 ng/mL. All solutions were stored at 4°C and placed 10 min at room temperature before use. Calibration curve and QC samples were prepared by spiking 10 µL of either standard or QC working solutions into 190 µL of blank rat plasma in
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polypropylene tubes. The final concentrations of liraglutide in calibration curves were
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0.5, 1, 2, 5, 25, 100 and 250 ng/mL, and those in QC samples were 1.25, 25, and 200
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ng/mL. All calibration and QC samples were prepared daily to avoid potential degradation or adsorption issues.
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2.4 Sample preparation
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Samples were thawed at room temperature for approximately 20 min and then the plasma was vortexed for 10 s. A 50 μL plasma sample and 10 μL of the IS
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working solution were transferred into 1.5 mL tubes, and then a 100 μL precipitant
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consisting of 70/30 ACN/ methanol (v/v) was added. The mixture was vortexed for 2 min and centrifuged at 12,000 rpm for 10 min; then, 3 μL of supernatant fluid were
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injected into the LC–MS/MS system. Serial standard and quality control samples (QC
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samples) were prepared following the method described above. 2.5 Method validation 2.5.1 Sensitivity and specificity The specificity was defined as non-interference at retention times of liraglutide and IS from the endogenous plasma components and no cross-interference between liraglutide and IS using the developed LC-MS/MS method. To investigate the specificity of the method, blank rat plasma samples collected from six different
ACCEPTED MANUSCRIPT batches were analyzed. The signal-to-noise ratio at the lower limit of quantification (LLOQ) was calculated to evaluate the sensitivity of this method. 2.5.2 Carryover A blank sample was analyzed following the injection of an upper limit of
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quantification (ULOQ) sample in order to evaluate carryover. The peak area of
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liraglutide obtained from the blank plasma sample should be less than 20% of that
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obtained from the LLOQ sample. 2.5.3 Cross-interference
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A control sample containing only IS and a ULOQ containing only liraglutide
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sample were analyzed in order to evaluate cross-interference between the liraglutide and IS. The peak area of liraglutide obtain from control sample should be less than 20%
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of that obtained from the LLOQ sample. The peak area of IS obtain from ULOQ
2.5.4 Linearity
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sample should be less than 5% of that obtained from the control sample.
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Linear calibration curves in rat plasma were generated by plotting the peak area
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ratio of liraglutide-IS versus the nominal concentration of calibration standards over the range 0.5-250 ng/mL. The calibration curves were fitted using a least square linear regression model y=ax + b, weighted by 1/x2 using Labsolution software. 2.5.5 Accuracy and precision The intra- and inter-day precision and accuracy were evaluated by parallel analytical runs performed on three consecutive days. Six replicates of liraglutide at four QC levels (0.5, 1.25, 25 and 200 ng/mL) were analyzed. Intra-day precision was
ACCEPTED MANUSCRIPT calculated using replicate (n=6) determinations for each concentration of the spiked plasma samples during a single analytical run. Inter-day precision and accuracy were calculated using replicate (n=18) determinations of each concentration made on three separate days. The precision was determined as the relative standard deviation (%
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RSD) and the accuracy was expressed as a percentage of the nominal concentration
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(relative error, % RE).
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2.5.6 Extraction recovery and matrix effect
The extraction recovery and matrix effect of the method were determined by
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comparing the peak areas of liraglutide at two concentrations (1.25, 25 and 200
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ng/mL) and those of IS at 10 ng/mL with six replicates, including standards, standards spiked before extraction and standards spiked after extraction. The extraction
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recoveries of liraglutide and IS were evaluated by comparing the peak areas of
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standards spiked before extraction with those of standards spiked after extraction. The matrix effects of liraglutide and IS were assessed by comparing the peak areas of
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standards with those of standards spiked after extraction.
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2.5.7 Stability.
The stability of QC samples for liraglutide were studied under different possible conditions, which included stability of three cycles of freeze/thaw (-80°C/ 22°C), stability of the analyte in the auto-sampler at 6°C for 24 h, stability of plasma samples at room temperature for 2.5 h. The stability of liraglutide was demonstrated by evaluating the relative error (% RE) and the relative standard deviation (% RSD) of the mean measured QC concentrations from their nominal concentrations.
ACCEPTED MANUSCRIPT 2.6. Pharmacokinetics study SD rats (weighted 200±20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The protocols for animal studies were carried out by the guidelines of the Animal Care and Use Committee of
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Tianjin Institute of Pharmaceutical Research, which was approved by Tianjin
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Municipal Science and Technology Commission (Tianjin, China). All animals were
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allowed free access to food and water during the experiments. Four rats (2 males and 2 females) were subcutaneously administered a single dose of 100 μg/kg liraglutide.
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200 μL of blood were collected from each rat at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 30, 48
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and 72 h after subcutaneous administration. Heparin was used as the anticoagulant to obtain the rat plasma. All samples were immediately centrifuged at 3000 ×g for 5 min
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3. Result and discussion
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at 4°C immediately and separated plasma samples were stored at -80°C until analysis.
3.1 LC-MS/MS conditions
CE
Initially, liraglutide was dissolved in solvent consisting of 30/70 ACN/ water.
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However, poor linearity was observed in the concentration range 0.5~250 ng/mL when analyzing the working solutions, and we suspect that this was due to strong peptides adsorption. Then we enhanced the elution strength of the solvent and replaced the initial solvent with 70/30 ACN/ 5mM ammonium acetate. The results showed that the linearity of the working solutions in the range 0.5~250 ng/mL was very good. Therefore, peptides adsorption is directly related to the elution strength of the solvent, and the presence of ammonium ions is beneficial for the stabilization of
ACCEPTED MANUSCRIPT the peptides and proteins. In this study, the Shimadzu 8060 mass spectrometer was selected to quantify liraglutide in rat plasma. Since the mass-to-charge ratio range of the Shimadzu 8060 was less than 2000, the multiply charged precursor of liraglutide (the 4+ precursor at
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m/z 938.4) was selected (Fig. 1A). Many ion fragments with similar intensities were
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produced under a certain collision energy (Fig. 1B). Due to the similar structure of the
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peptide bond in liraglutide, multiple cleavages of the peptide bond occur at a certain collision energy, which makes it difficult to confirm the specific ion fragments with a
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high intensity for quantification. In addition, the multiply charged form of precursors
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and product ions of liraglutide also increase the difficulty to confirm the specific ion fragments. Initially a number of possible ion fragments ( m/z 1064, m/z 1128, m/z
and
pre-analysis
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optimization
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1185, m/z 1353 and m/z 523) were selected for investigation. After careful MRM of
biological
samples,
the
transitions
m/z
938.4→1128.3, which was high intensity with minimum interference, was finally
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selected to quantify liraglutide in rat plasma. After careful analysis we speculate that
AC
m/z 1128 is the b11 ion fragment. The transitions from precursor ion to product ion are m/z 1162.2→143.2 for human insulin (IS), and m/z 143.2 is the a2 ion fragment. Optimization of the chromatographic conditions was relatively simple, and focused mainly on evaluating the compositions of the mobile phase and the type of analytical column [26]. Based on our previous research experience, we found that ACN is more suitable than methanol as the organic phase, and the presence of FA was beneficial for the sensitivity of liraglutide. So the final mobile phases comprised 0.1%
ACCEPTED MANUSCRIPT FA in ACN (mobile phase A) and 0.1% FA in water (mobile phase B). The Diamonsil C18 column (Dikma Technologies Inc. 100 × 4.6 mm, 5 µm, pore diameter: 10 nm), the ZORBAX Eclipse Plus C8 column (Agilent, 100 × 4.6 mm, 5 µm, pore diameter: 10 nm) and the InertSustain Bio C18 column (GL Sciences, 100 × 2.1 mm, 1.9 µm,
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pore diameter: 20 nm) were tested to achieve the minimum matrix interference and
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symmetric peak shapes. Both the Diamonsil C18 column and the ZORBAX Eclipse
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Plus C8 column showed a poor peak shape and a serious peak tailing. In contrast, the InertSustain Bio C18 column provided minimum matrix interference and a good
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chromatographic resolution with sharp peaks. This difference was caused by the
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difference in the particle size of the analytical columns. The smaller the particle size, the higher the efficiency.
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As we all know, solid-phase extraction (SPE) is the preferred way for the
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biological sample preprocessing of peptides and proteins when using LC-MS/MS. In our study, the WondaSep MCX and Monospin C18 SPE cartridges were initially
CE
tested to separate and enrich liraglutide; however, only 15% of liraglutide was
AC
recovered because of a high plasma protein binding ratio. Then, protein precipitation was tested to both clean the sample and disrupt protein binding. To find the best precipitant, methanol, acetonitrile and the mixed solution of them were tested. When acetonitrile was used as a precipitant, coprecipitation of partial liraglutide with plasma proteins resulted in lower recovery. 55% recovery was achieved using an acetonitrile: plasma ratio of 1:1. When the above ratio became 2:1 and 3:1, the recoveries were 35% and 30%, respectively. Although methanol did not cause precipitation of the analyte,
ACCEPTED MANUSCRIPT it was not as effective as acetonitrile in removing interference. Fortunately, the ACN and methanol combination (70/30 ACN/methanol) provided the best sensitivity and recoveries (80~90%) for the liraglutide from rat plasma after continuous testing. 3.2 Method validation
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3.2.1 Sensitivity and specificity
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Representative chromatograms of liraglutide and IS are shown in Figure 2,
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including a blank plasma sample (Fig. 2A), a blank plasma sample spiked with liraglutide at 0.5 ng/mL (Fig. 2B) and a rat plasma sample obtained 6 h after
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subcutaneous administration (Fig. 2C). No interfering peaks were observed at the
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retention times of liraglutide and IS in the blank sample. The response intensity of liraglutide was sufficient for quantification at the concentration of 0.5 ng/ mL
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3.2.2 Carryover
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(LLOQ).
A blank sample was analyzed following the ULOQ to evaluate carryover. No
CE
peak was found at the retention time of liraglutide in the blank sample, indicating that
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the carryover of the method was acceptable. 3.2.3 Cross-interference No peak was found at the retention time of liraglutide in the control sample, and no peak was found at the retention time of IS in the ULOQ sample. The result showed that there was no cross-interference between the liraglutide and IS. 3.2.4 Linearity The plasma calibration curve was constructed by seven calibrators over the
ACCEPTED MANUSCRIPT concentration range of 0.5~250 ng/mL. A typical standard curve for liraglutide was y=0.0467x-0.00145 (r2=0.9978) with a weighting factor of 1/x2. The ratio of liraglutide to IS was calculated, and x represented the corresponding plasma concentrations of liraglutide.
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3.2.5 Accuracy and precision
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The accuracy and precision of liraglutide at four concentrations (0.5, 1.25, 25
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and 200 ng/mL) were calculated (Table 2). The intra- and inter-day precision (expressed as RSD) of liraglutide were in the range of 1.97~7.63% and 5.25~11.9,
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respectively. The accuracy (expressed as RE) ranged from -8.79~11.4%. The results
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indicate that the LC/MS/MS method had good accuracy and excellent precision. 3.2.6 Extraction recovery and matrix effect
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The matrix effect can be regarded as the ion suppression or enhancement of the
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analyte [27]. The extraction procedure and chromatographic conditions were optimized to decrease or even eliminate the unfavorable influence of the matrix effect
CE
[28]. In this study, the matrix values were 109±3.40%, 112±2.10% and 107±3.28% for
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liraglutide and 102±3.12% for IS, indicating that no matrix effect for liraglutide and IS existed in the method. Under these operating conditions, the detailed values of recoveries in rat plasma were 82.3±1.98%, 85.1±2.46% and 84.2±2.07% for liraglutide and 65.6±3.42% for IS.
ACCEPTED MANUSCRIPT 3.2.7 Stability The stability results, which are listed in Table 3, demonstrate that the analyte displayed excellent stability under all experimental conditions. The RSD ranged from 0.541% to 3.12%, and the RE ranged from -10.4~10.0%.
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3.2.8 Incurred sample reanalysis
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In this study, about 20% of the study sample size were selected for reanalysis.
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The result showed that 90% of the repeated sample results were within 20%. The detail results were shown in Table 4.
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3.3 Comparison of methods
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In recent years, a few LC-MS/MS methods [29-31], which are summarized in the Table 5, have been developed to explore the PK profiles of liraglutide as the
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shortcomings of LBA methods are gradually recognized. Compared with the
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published LC-MS/MS methods, we believe that three aspects of this manuscript will make it interesting to general readers. Firstly, the key technique of our LC-MS/MS
CE
method compared to the reported methods was the simplification and improvement of
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the sample preprocessing. In the published reports, the researchers failed to directly measure liraglutide in the biological samples after precipitating because the precipitant they used was not suitable. In Meng’s study, methanol was selected to disrupt the binding of liraglutide to albumin and to inactivate neutral endopeptidases. But methanol was not enough to remove all interference in plasma. Therefore, after the protein precipitation procedure, SPE was utilized for the sample preprocessing of liraglutide to enhance the sample cleanup. In Shah’s study, initially ice cold (-20ºC)
ACCEPTED MANUSCRIPT acetonitrile was selected as a plasma protein denaturing agent, but a strong interference and low recovery were observed at analyte RT in lower concentration. Therefore, protein precipitation was eventually replaced by SPE for plasma sample preprocessing. In this study, the new precipitant we formulated provided the best
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sensitivity and recoveries (80~90%) for the liraglutide from rat plasma, and the
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biological sample could be analyzed directly by the mass spectrometer after a simple
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step of precipitation. In addition, the sensitivity and linearity of our method are superior to Meng’s study. In Shah’s study and Kellie’s study, the linearity is quite
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unreasonable because the exposure level of liraglutide in vivo range from 1~200
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ng/mL [7,8,10,32]. Moreover, the plasma volume used for analysis is smaller in our study, which is a huge benefit to experimental animals or people.
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3.4 Pharmacokinetics study
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Our method was successfully applied to the PK study after the subcutaneous administration of 100 μg/kg liraglutide to rats. The mean plasma concentration-time
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profile of liraglutide with a single subcutaneous administration to four rats is
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illustrated in Fig. 3. The mean PK parameters, including Cmax, Tmax, t1/2, AUC0-12h, AUC0-∞, Vd, CL and MRT, obtained after the subcutaneous administration of liraglutide are summarized in Table 6. Liraglutide could balance blood glucose for a long time, and the elimination of the drug was slow in rats. The level of liraglutide was lower than the LLOQ in rat plasma 48 h after dosing. The successful application of the LC–MS/MS method indicated that this analytical method was suitable and sufficient for the PK study of liraglutide.
ACCEPTED MANUSCRIPT 4. Conclusion In this manuscript, a simple, specific and high-throughput LC-MS/MS analytical method was successfully developed and fully validated to quantify liraglutide in rat plasma. This method was successfully applied to the pharmacokinetics study of
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liraglutide after subcutaneous administration to rats. In addition, a simple precipitation
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method was developed for the biological sample pretreatment, which was simpler,
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more efficient and more inexpensive than previously reported methods. The biological samples could be analyzed directly by the mass spectrometer after a simple protein
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precipitation step. Moreover, the sensitivity was reasonable, and the LLOQ of this
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method was 0.5 ng/ mL. The plasma volume used for analysis is smaller in our study, which is a huge benefit to experimental animals or people. I believe our method will
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dramatically enhance the analytical efficiency of liraglutide and greatly reduce the
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costs caused by time-consuming method development, sample analysis and critical reagents, such as antibodies. We hope that this study will serve as a valuable tool to
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accelerate the development of liraglutide and its analogues.
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Acknowledgments
The work was supported by “National Natural Science Foundation of China” [grant number 81503154].
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Analytical Chemistry 85 (2013) 9528-9535.
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[30] X. Meng, H. Xu, Z. Zhang, J.P. Fawcett, J. Li, Y.Yang, J. Gu, Differential
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ACCEPTED MANUSCRIPT
Table 1 Mass Spectrometry Conditions for liraglutide and IS
Compound name
MRM
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Q1 pre bias
Interface
DL
Heat block
Drying
gas flow
gas flow
temperature
temperature
temperature
gas flow
(L/min)
(℃)
(℃)
(℃)
(L/min)
10
300
250
400
10
10
300
250
400
10
N A
(eV)
(V)
(V)
(L/min)
M
-29
-40
-32
IS
1162.2→143.2
-58
-44
D E
T P E
A
C C
C S U
Heating
transition
938.4→1128.3
-29
I R
Nebulizing Q3 pre bias
liraglutide
T P
3 3
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Table 2 Intra- and inter-day precision and accuracy of the method for the determination of liraglutide in rat plasma
Spiked concentration
Measured concentration
Intra-day precision (n=6)
(ng/mL)
(ng/mL)
(%, RSD)
0.5
0.510±0.0391
7.63
1.25
1.14±0.05
4.19
25
24.4±1.10
1.97
200
223±7.33
T P E
D E
A
C C
T P
I R
Inter-day precision (n=18)
C S U
Accuracy (n=18)
(%, RSD)
(%, RE)
7.91
2.02
5.25
-8.79
M
11.9
-2.30
2.33
7.16
11.4
N A
ACCEPTED MANUSCRIPT Table 3 Stability of liraglutide in rat plasma under different storage conditions (n=6)
Concentration levels (ng/mL)
Precision
Accuracy
Storage conditions Measured (mean ± SD)
RSD (%)
RE (%)
Stability for 2.5 h at room
1.25
1.28 ± 0.0398
3.12
2.21
temperature
200
220 ± 1.33
0.571
10.0
Stability for extracted samples in
1.25
1.12 ± 0.0237
2.31
-10.4
auto-sample for 24 h
200
189 ± 3.66
1.94
-5.55
Stability for three freeze-thaw
1.25
1.30 ± 0.007
0.541
3.60
211 ± 3.84
1.82
5.34
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Cycles (-80oC /22oC）
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Spiked
200
ACCEPTED MANUSCRIPT Table 4 The result of incurred sample reanalysis
Difference Number
Sample name
Original
Repeat
Mena (%)
124
118
2
ISR-Dog-SC-1#-24h
19.5
20.2
3
ISR-Dog-SC-2#-10h
61.5
85
4
ISR-Dog-SC-2#-12h
61.8
5
ISR-Dog-SC-2#-24h
6.29
6
ISR-Dog-SC-3#-10h
101
7
ISR-Dog-SC-3#-24h
8
ISR-Dog-SC-4#-8h
9 10
121
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ISR-Dog-SC-1#-10h
19.9
-4.96 3.53 32.1
60.4
61.1
-2.29
6.44
6.365
2.36
103
102
1.96
22.1
20.8
21
-6.06
101
100.0
100.5
-1.00
ISR-Dog-SC-4#-10h
102
104
103
1.94
ISR-Dog-SC-4#-24h
9.45
10.2
9.8
7.63
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ISR number
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Accept ISR number
Accept ISR percentage (%)
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73
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1
10 9 90
ACCEPTED MANUSCRIPT Table 5 The comparison of our LC-MS/MS method with other published LC-MS/MS methods for the bioanalysis of liraglutide
Plasma Publication date
Sample preparation
Sensitivity
Linearity
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volume ng/mL
ng/mL
µL
100
100~10000
25
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year 2015
Incubation + precipitation
Meng’s study
2017
Precipitation + SPE
1.0
1.0~100
400
Shah’s study
2017
SPE
0.0005
0.0005~0.1
1000
0.5
0.5~250
50
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Precipitation
CE
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My manuscript
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Kellie’ study
ACCEPTED MANUSCRIPT Table 6 Pharmacokinetic parameters of liraglutide in rats after subcutaneous administration
subcutaneous Pharmacokinetic parameter
T1/2
h
4.01 ± 0.227
Cmax
ng/ml
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administration (100 μg/kg)
Tmax
h
AUC0-t
ng·h/ml
AUC0-∞
ng·h/ml
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10 ± 0.476
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Vd
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1361 ± 21.2 1385 ± 19.6
mL
100 ± 10.4
mL/h
84 ± 2.12
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CL MRT
103 ± 31.1
h
11 ± 0.289
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T1/2, Elimination half-life; Cmax, The peak plasma concentration; Tmax, Time to reach Cmax; AUC0-t,
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Area under the curve the concentration-time curve from 0 to 48h; AUC0-∞, Area under the curve from 0 to infinity; Vd, Volume of distribution; CL, Clearance; MRT, Mean residence time.
ACCEPTED MANUSCRIPT
Legend of figure Fig.1. Full scan (A) and product ion (B) spectra of liraglutide
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Fig.2. Representative chromatograms of liragultide (4.08 min) and IS (3.17 min). (A)
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Blank rat plasma sample. (B) Blank plasma sample spiked with 0.5 ng/mL of
SC
liraglutide (LLOQ) and 10 ng/mL of IS. (C) A plasma sample collected at 6 h after subcutaneous administration of 100 μg/kg liraglutide.
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Fig.3. The mean plasma concentration–time profile of liraglutide in rat plasma after
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subcutaneous administration of 100 μg/kg liraglutide (n=4).
ACCEPTED MANUSCRIPT Highlights (1) A simple, specific and high-throughput LC-MS/MS analytical method was successfully developed and validated to quantify liraglutide in rat plasma. (2) The sample preprocessing is simple and the biological samples can be analyzed
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directly by the mass spectrometer after a simple protein precipitation step.
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more reasonable than previously reported methods.
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(3) The calibration curve range and the sensitivity of our LC-MS/MS method are
(4) The plasma volume used for analysis is smaller in our study, which is a huge
AC
CE
PT E
D
MA
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benefit to experimental animals or people.
Figure 1
Figure 2
Figure 3