Journal of Pharmaceutical and Biomedical Analysis

A novel bioanalytical method for the quantification of pemigatinib in rat plasma by UPLC-MS/MS
Qinghua Wenga,1, Wei Tanb,1, Ren-yue Yuc, Ren-ai Xuc,∗, Yu Chenc,∗
a The Third Affiliated Hospital of Shanghai University (Wenzhou People’s Hospital), 325000, Wenzhou, PR China b The Third Affiliated Hospital of Chongqing Medical University (Gener Hospital), 401120, Chongqing, PR China c The First Affiliated Hospital of Wenzhou Medical University, 325000, Wenzhou, PR China

Keywords: Pemigatinib Sample preparation Pharmacokinetic UPLC-MS/MS
Rat

Pemigatinib is an oral, selective, potent, competitive inhibitor acting on fibroblast growth factor receptor (FGFR)1, FGFR2, and FGFR3, which has obtained accelerated approval in the USA through a test approved by the USA FDA. It is not only significant in the therapy of adult recurrent, unresectable, metastatic or locally advanced cholangiocarcinoma, but also plays an important role in treating adult patients with FGFR2 fusion or other rearrangements. The aim of our research was to establish and verify a reliable and quick ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay to determine the level of pemigatinib in rat plasma. The analyte was prepared using a simple and convenient approach with acetonitrile for protein crash, and then separated from the matrix on a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) in a gradient elution program, where the mobile phase was consisted of acetonitrile and 0.1 % formic acid in water and was set at 0.40 mL/min flow rate. Selective reaction monitoring (SRM) was used to conducted for UPLC-MS/MS dectection with ion transitions at m/z 488.01 → 400.98 for pemigatinib and m/z 447.00 → 361.94 for erdafitinib (Internal standard, IS), respectively. This method had good linearity in a 0.5−1000 ng/mL calibration range for pemigatinib, where the lower limit of quantification (LLOQ) was validated at 0.5 ng/mL. The precision of pemigatinib for intra- and inter-day was less than 13.3 %, and the accuracy was determined to be from −4.8%–11.2%. During the assay in plasma samples, the analyte was found to be stable. Besides, matrix effect and recovery of the analyte and IS were acceptable. The novel optimized UPLC-MS/MS assay was also suitable for determining the concentration level of pemigatinib in a pharmacokinetic study after a single dose of
1.35 mg/kg pemigatinib orally to the rats.

1. Introduction

As a group of heterogeneous tumors, cholangiocarcinoma can be classified according to its location in the biliary tract as intrahepatic or extrahepatic (perihilar and distal) [1]. In patients with cholan- giocarcinoma, several potentially actionable oncogenic alterations have been identified by comprehensive genomic profiling, includ- ing in genes encoding FGFR [2]. And, FGFR inhibitors has attracted much attention as a potential treatment for cholangiocarcinoma [3]. Pemigatinib (Fig. 1A) is a selective, effective, oral competitive inhibitor of FGFR1, FGFR2, and FGFR3 [4], which has obtained accel-

∗ Corresponding authors at: The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang Street, Wenzhou, 325000, PR China.
E-mail addresses: [email protected] (R.-a. Xu), [email protected] (Y. Chen).
1 The work has been contributed by these authors equally.

erated approval in the USA through a test approved by the USA FDA. It is not only significant in the therapy of adult recurrent, unre- sectable, metastatic or locally advanced cholangiocarcinoma, but also plays an important role in treating adult patients with FGFR2 fusion or other rearrangements [5–8]. As a treatment for several other FGFR-driven malignancies (e.g. esophageal-gastric junction cancer), it also has been clinical developed in various countries around the world [9]. As reported previously, the pharmacoki- netic changes of the exposure of pemigatinib are not affected by factors such as age, sex, race, food, bodyweight, mild to moder- ate hepatic or renal impairment [5]. However, moderate or strong CYP3A4 inhibitors could elevate the area under the concentration- time curve and maximum concentration values of pemigatinib, and should be avoided of the concomitant use.
Given that patients with cancer are frequently treated with
many kinds of drugs, whether co-administrated pemigatinib with these drugs would lead to drug-drug interaction (DDI) must be

Fig. 1. Mass spectra of pemigatinib (A) and erdafitinib (IS, B) in this study.

explored. Thus, it is essential to invent and establish a quantitative assay for pemigatinib in order to investigate the pharmacokinetic profile and DDI for its clinic application. To date, there is no analyt- ical assay for the quantification of pemigatinib in biological media as per regulatory guideline requirement with higher sensitivity and commercially available internal standard (IS).
Therefore, in this experiment, the purpose of our research was to invent and test a sensitive and quick ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay to determine the concentration of pemigatinib in rat plasma. And, the novel developed UPLC-MS/MS assay was suitable to explore the pharmacokinetic profile of pemigatinib in rats.

2. Experimental

2.1. Chemicals materials

Shanghai Chuangsai Technology Co., Ltd. (Shanghai, China) pro- vided pemigatinib and erdafitinib (used as IS, Fig. 1B) with the purity of both were > 98 %. Methanol and acetonitrile in this study were LC grade, and were supplied by Merck Company (Darm- stadt, Germany). Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) supplied formic acid, which was analytical grade. The ultrapure water was prepared by Milli-Q Water Purifi- cation System (Millipore, Bedford, USA).

2.2. Animal experiments

The Laboratory Animal Center of Wenzhou Medical Univer- sity (Zhejiang, China) provided the experimental rats (weight
200 20 g), and all six of them were fed in the feeding room where the environment was under controlled (standard temperature
25 28 ◦C, humidity 50–60 % and 12 h light/12 h dark). Water and
food were supplied to them unlimited. The experimental behaviors and operations were all reviewed and approved by the Institutional Ethics Committee of Wenzhou Medical University (Zhejiang, China) in accordance with the National Institute of Health (NIH) guidelines for the welfare and use of animals [10].

Before experiment, the six rats would have a 12 h fasting, while they were allowed freely to the water. We gave each rat the oral administration of 1.35 mg/kg pemigatinib after formulated in the 0.5 % carboxymethyl cellulose sodium (CMC-Na) solution. At several different points at the time of 0, 0.333, 0.667, 1, 1.5,
2, 3, 4, 6, 8, 12, 24, and 48 h, we collected approximate 0.3 mL blood samples through the caudal vein and took them into 1.5 mL
heparin-containing polythene tubes. Before placed them at 80 ◦C
pending further analysis, we separated plasma samples by cen- trifuging them at 4000 g at 25 ◦C for 8 min immediately. After preparing the samples as “Sample preparation” section, we used the developed bioanalytical assay based on UPLC-MS/MS technique to assess the concentration levels of pemigatinib in rat plasma in our study, and Drug and Statistics (DAS) 3.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China) was used for non-compartmental analysis, through which we can examined and calculated the important pharmacokinetic parame- ters.

2.3. Instrumentations and analytical conditions

Waters Xevo TQ-S triple quadrupole tandem mass spectrometer and the Waters ACQUITY UPLC I-Class system (Milford, MA, USA) composed the UPLC-MS/MS system, which was also coupled to an electro-spray ionization (ESI) source (Milford, MA, USA). Quanlynx programme and Masslynx 4.1 software (Milford, MA, USA) were used to acquired and processed all experimental data.
The chromatographic separation of pemigatinib and IS was achieved by an Acquity UPLC BEH C18 column (2.1 mm 50 mm,
1.7 µm). Meanwhile, solvent A (acetonitrile) and solvent B (0.1 % formic acid in water) was used for the mobile phase in this study. Maintained 30 % A between 1.0 2.0 min for equilibration before linear gradient elution, which was conducted at a flow rate of
0.40 mL/min as follows: 0 0.9 min (A, 30–85 %) and 0.9–1.0 min (A,
85 30 %). The volume of each injection was 2.0 µL, and the time for each analysis was 2.0 min. For all samples, the autosampler was set at 10 ◦C, and the temperature of column was maintained at 40 ◦C.

An ACQUITY UPLC system equipped with a Xevo TQ-S triple quadrupole tandem mass spectrometer was used to detect pemi- gatinib and IS under the positive ion mode. The detection was performed under selective reaction monitoring (SRM) mode, in which the ion transitions of pemigatinib and IS were m/z 488.01
400.98 and m/z 447.00 361.94, respectively. The collision energy and cone voltage were respectively 30 eV and 15 V for pemi- gatinib and were respectively 30 eV and 20 V for IS. Desolvation
temperature 600 ◦C is the optimized general MS parameters, capil-
lary voltage 2.0 kV, collision gas 0.15 mL/min, cone gas 200 L/h, and desolvation gas 1000 L/h.

2.4. Standard solutions, calibration curves and quality control (QC) samples

To quality control (QC) samples and calibration curve, we need to separately dissolving the stock solutions of pemigatinib and IS with the corresponding concentration of 1.00 mg/mL in an appro- priate amount of methanol. The dilution of the stock solution of the analyte with methanol was used to prepare a range of working solu- tions. Then, to get final concentrations between 0.5 1000 ng/mL, calibration curves spiked with blank rat plasma (90 µL) were oper- ated by ten-fold dilution of the corresponding working solutions (10 µL). In the same way, the final concentrations of 0.5 (lower limit of quantification, LLOQ), 1 (LQC), 80 (MQC) and 800 (HQC) ng/mL for QC samples were also prepared. We diluted the IS stock solution with methanol to obtain the IS working solution at the concentra- tion of 200 ng/mL. All the stock and working solutions were ready
in advance and placed at −80 ◦C for further use.
2.5. Sample preparation

A simple protein precipitation approach with acetonitrile was used to prepared samples. Adding 200 ng/mL IS working solution with the volume of 20 µL–100 µL plasma sample in 1.5 mL EP cen- trifuge tube, and the mixture was mixed for 1.0 min. Then, proteins were precipitated by the addition of 300 µL acetonitrile after vor- texing for 1.0 min, and centrifugating at 13,000 g for 10 min at 4 ◦C. Finally, we transferred 100 µL of the clear supernatant into the new auto-sampler vial, and injected 2.0 µL aliquot of the supernatant into the chromatographic system for quantitative analysis.

2.6. Method validation

Fully method validation procedures for this analytical method, including the calibration curve, selectivity, LLOQ, recovery, matrix effect, precision and accuracy, and stability, were performed according to the FDA principle on the bioanalytical method vali- dation [11].
The selectivity of the assay was investigated by checking the absence of interferences from the blank (neither analyte nor IS from six different rats), stardard solutions (at the concentration of LLOQ) and real rat plasma at the corresponding retention times of pemigatinib and IS.
A weighted (1/x2) least square regression mode was used to plotting the ratio of peak area of analyte to peak area of IS against the nominal concentrations of the analyte in order to evaluate the calibration curves. The sensitivity of this method was performed in terms of LLOQ, which was identified as the lowest point on the calibration curve, and determined with accuracy within 20 % and precision below 20 % of the nominal value.
We performed sextuple detection on the QC samples at three different concentration levels over three consecutive days to esti- mate the precision and accuracy. Recovery from present approach of sample preparation was investigated by comparing the peak area ratios of the analytes before and after extraction, respectively.

Matrix effect (ME) was also analyzed in 6 replicates by compara- tive study of the responses of the analyte in plasma matrix after extraction and in the pure solution.
The stability of the spiked analyte in plasma was examined by detecting LQC, MQC and HQC samples (1, 80 and 800 ng/mL) under different storage conditions. Stability (short-term and long- term) evaluation was examined respectively at ambient conditions
temperature for 2 h and 80 ◦C for three weeks. In addition, the
stability after 4 h of preparation was tested in an autosampler at 10 ◦C. Moreover, three complete freez/thaw stability ( 80 ◦C to room temperature) was also studied.

3. Results and discussion

3.1. Method development and optimization

At the beginning of this study, we used the mass spectrometer to optimize the corresponding compound in order to obtained the MS settings of pemigatinib and IS. The MS spectrum for pemiga- tinib (A) and IS (B) was shown in Fig. 1. In the mass transition, we selected the product ion at m/z 400.98 for this bioanalytical method because the most abundant is the product ion of pemigatinib at m/z
400.98. In addition, the most abundant fragment with m/z 361.94 was selected as product ion for IS, because the low signal prevented detector saturation and the baseline obtained with the m/z 447.00
361.94 mass transition was stable.
In the method development, different mobile phase combina- tions, including water phase (such as water, 0.1 % acetic acid in water, 0.1 % formic acid in water and 1 mM ammonium acetate in water) and organic phase (such as acetonitrile and methanol) were evaluated to obtain high sensitivity and symmetric peaks of the analyte and IS. However, poor sensitivity and asymmetric peaks were produced by the supernatant of precipitated samples when acetonitrile and water were firstly selected to separate the analyte and IS as the mobile phase. Since it did not satisfy the requirement of the method validation, 0.1 % formic acid in water was adopted as water phase with acetonitrile as organic phase. Symmetric peaks and higher sensitivity were produced by the optimized condition, the ionization of the analyte was improved as well.

3.2. Method validation

3.2.1. Selectivity
As indicated in Fig. 2, the corresponding retention times of pemi- gatinib and IS were approximately 0.53 and 0.64 min, respectively. Comparison of the representative SRM chromatograms of blank rat plasma from 6 individual sources, blank plasma added with pemi- gatinib at the concentration of LLOQ and IS, and the actual plasma sample demonstrated that no potential interfering substances was found. It suggested that the method had a good selectivity in the determination of pemigatinib and IS in rat plasma.

3.2.2. Calibration curve and LLOQ
At the concentration range of 0.5 1000 ng/mL for pemigatinib, the representative linear regression equation of peak ratios (Y) versus the matching concentrations (X) for pemigatinib obtained was Y = 0.73903 X + 1.60911 ( r2 = 0.9962), which exhibited an excellent linearity. LLOQ was used to detected the sensitivity of the method, which was established as 0.5 ng/mL, and the precision was below 13.3 % whereas the accuracy was not more than 11.2 % (Table 1).

3.2.3. Precision and accuracy
The precision and accuracy of the developed UPLC-MS/MS assay were calculated by detecting QC samples over three separate days at HQC, MQC, LQC and LLOQ four different concentration levels

Fig. 2. Representative chromatograms of pemigatinib and IS in rat plasma: (A) blank plasma; (B) blank plasma spiked with LLOQ concentration of standard solutions; (C) sample obtained from a rat at 1.0 h after oral administration of 1.35 mg/kg pemigatinib.

Table 1
The precision and accuracy of pemigatinib in rat plasma (n = 6).

(n = 6). The accuracy of pemigatinib for intra- and inter-day, as listed in Table 1, was below 11.2 %, and the precision was below 13.3 % at four determined QC concentration levels. These data demonstrated that in the quantitative analysis of pemigatinib in rat plasma, the described UPLC-MS/MS method has both precision and accuracy.

3.2.4. Recovery and matrix effect
The mean recovery of pemigatinib was within the range of 89.3–92.2 % (Table 2), and the matrix effect values for pemigatinib were 89.5–97.1 % (Table 2) at LQC, MQC and HQC three different concentration levels, suggesting that no significant matrix effects were observed in rat plasma using the present optimized UPLC- MS/MS conditions.

3.2.5. Stability
Different experiments of stability were conducted to survey whether pemigatinib was still stable under different storage and processing conditions in rat plasma. As listed in Table 3, plasma

pemigatinib samples were found to be stable in a routine labora- tory when they were placed at −80 ◦C for at least 3 weeks, after three complete freeze (−80 ◦C)/thaw (room temperature) cycles, placed for at least 4 h in the autosampler (10 ◦C), and for at least 2 h
at room temperature.

3.3. Animal study

The novel established UPLC-MS/MS assay was suitable for determining the plasma concentrations of pemigatinib after the rats was orally given a single dose of 1.35 mg/kg pemigatinib in a pharmacokinetic study. Fig. 3 displayed the average plasma concentration-time curves of pemigatinib in rats, and Table 4 sum- marized the main pharmacokinetic parameters of pemigatinib after a non-compartment model analysis.
After oral administration of pemigatinib in rats, it was fastly absorbed with the maximum plasma concentration (Cmax) of

Table 2
Recovery and matrix effect of pemigatinib in rat plasma (n = 6).
Concentration
Analyte added(ng/mL)

Table 3
Stability results of pemigatinib in plasma under different conditions (n = 5).

Fig. 3. Mean plasma concentration-time curves of pemigatinib in rats after oral administration of pemigatinib at a single dose of 1.35 mg/kg. (n = 6).

Table 4
The main pharmacokinetic parameters of pemigatinib in rat plasma after oral admin- istration of pemigatinib at a single dose of 1.35 mg/kg. (n = 6,

521.55 ± 98.28 ng/mL. Moreover, it achieved to the value of the time to peak concentration (Tmax) at about 3.00 ± 0.63 h in rat plasma. In addition, its half-life (t1/2) in the rats was 3.96 0.61 h. Inter- estingly, similar result of distribution volume (Vz/F) was observed in another study from rats [12]. Considering our study was per- formed in rats and only has the animal number of six with a few, more researches should be done to confirm the pharmacokinetic profile of pemigatinib accurately.

4. Conclusions

In conclusions, our research fully optimized and firstly devel- oped a robust, quick and reliable UPLC-MS/MS assay for the determination of the concentration of pemigatinib in rats plasma. This optimized method offered significant advantages according

to short analysis time (only 2.0 min) and cost-effective sample preparation (a quick and simple protein precipitation with acetoni- trile). The optimized UPLC-MS/MS assay has shown its applicability in a pharmacokinetic study after a single oral administration of
1.35 mg/kg pemigatinib in rats.

Author statement

Qinghua Weng: Writing – original draft; Conceptualization; Data curation; Formal analysis;
Wei Tan: Visualization; Writing – original draft; Writing – review & editing;
Ren-yue Yu: Investigation; Methodology;
Ren-ai Xu: Writing – original draft; Conceptualization; Data curation; Formal analysis; Writing – review & editing;
Yu Chen: Project administration; Resources; Software; Supervi- sion; Writing – review & editing; Validation.

Declaration of Competing Interest

The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledge

This study was supported by grant from Wenzhou City Science and Technology Bureau (Grant No. R2020022).

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