Translate this page into:
Simultaneous determination of tofacitinib and its principal metabolite in beagle dog plasma by UPLC-MS/MS and its application in pharmacokinetics
⁎Corresponding authors. 651082@hospital.cqmu.edu.cn (Wei Tan), 16336596@qq.com (Xiaoxiang Du)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
The main objective of our current study is to develop and validate an accurate and direct ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to simultaneously detect plasma concentrations of tofacitinib and its metabolite M9, and to study the pharmacokinetic profiles of the two compounds in beagle dogs. After rapid precipitation of protein by adding acetonitrile, the chromatographic separation of tofacitinib was completed, as well as M9 and upadacitinib (internal standard, IS) by using an Acquity BEH C18 (1.7 μm, 2.1 mm × 50 mm) column. A Xevo TQ-S triple quadrupole tandem mass spectrometer was employed to determine their concentrations under the positive ion pattern. Selective reaction monitoring (SRM) was used with ion transitions at m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 137.03 for M9, and m/z 380.95 → 255.97 for IS, respectively. This assay demonstrated excellent linearity, and the ranges of calibration curves for both tofacitinib and M9 were 0.5–400 ng/mL. The new UPLC-MS/MS assay can reach the values (0.5 ng/mL) of lower limit of quantification (LLOQ) for both tofacitinib and M9. Both intra-day and inter-day accuracy of all analytes ranged from −12.0% to 14.3%, while the precision was ≤13.2%. The recovery rate of all analytes was >88.5%, and more importantly there was no conspicuous matrix effect. In addition, the stability was consistent with the quantificative requirements of plasma samples under all conditions. Finally, the assay on UPLC-MS/MS is able to be employed to determine the pharmacokinetic characteristics of tofacitinib and its metabolite M9 in the plasma of beagle dogs after taking orally a dose of tofacitinib at 2 mg/kg.
Keywords
Tofacitinib
Beagle dog
Pharmacokinetics
UPLC-MS/MS
Plasma
1 Introduction
Rheumatoid arthritis (RA), a chronic, systemic and autoimmune disease, affects 0.5 ∼ 1% of the above-18-years-old population in the industrialized countries, and the ratio of male to female is close to 1:3 (Bannwarth et al., 2013). At present, disease-modifying antirheumatic drugs are the mainstay of treatment for RA, especially the inhibitors of the Janus kinase (JAK) enzymes (Tan et al., 2021). Tofacitinib (Fig. 1A) is a new-type, effective and alternative inhibitor of JAK kinase family. It is considered as the best inhibitor for JAK3, and also effective for JAK1 and JAK2 (Traves et al., 2021). Until now, a numbers of countries have already given permission to it for the treatment of moderate to severe RA (Kerschbaumer et al., 2020; Miyazaki et al., 2021; Roskoski, 2021). Cytochrome P450 (CYP450) profiling illustrated that CYP3A4 played a chief role in the contribution to metabolize tofacitinib, while CYP2C19 contributed just a little (Dowty et al., 2014). And, the M9 (Fig. 1B) metabolite in urine was the most sufficient, at 20% or so (Dowty et al., 2014).
Mass spectra of tofacitinib (A), M9 (B), and upadacitinib (IS, C) in the present study.
As far as we know, several analytical assays are reported to evaluate tofacitinib concentrations as a single analyte (Dowty et al., 2014; Paniagua et al., 2005; S et al., 2015), combining with other kinase inhibitors (Abdelhameed et al., 2017; Kadi et al., 2016; Koller et al., 2020) or with methotrexate, an drug for treatment of rheumatic disease (Sharma et al., 2015) in biological samples. However, there is no bioanalytical approach at present on the strength of liquid chromatography tandem mass spectrometry (LC-MS/MS) technique for the detection of tofacitinib and M9 (a metabolite of tofacitinib) in biological samples at the same time. All those substances came into being during either the pharmacokinetic studies or pharmacokinetic drug-drug interaction studies.
For this purpose, the target of this paper is to validate a rapid and accurate ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) approach to detect tofacitinib and M9 (a metabolite of tofacitinib) in beagle dog plasma in the meantime, and to study the pharmacokinetic characteristics of tofacitinib as well as its metabolite M9 in beagle dogs.
2 Experimental
2.1 Materials chemicals and reagents
Reference substances of tofacitinib, M9 and upadacitinib (internal standard, IS, Fig. 1C) provided by Shanghai Chuangsai Technology Co., Ltd. (Shanghai, China) were absolutely claimed purity > 98%. Methanol and acetonitrile supplied by Merck Company (Darmstadt, Germany) were LC-grade. The water was filtered by a system called Milli-Q Reagent System (Millipore, Bedford, USA).
2.2 Pharmacokinetic study
Six beagle dogs (weight 10 ± 0.5 kg), fed but drank freely in the qualified laboratory for more than one week, were from the Laboratory Animal Center of Henan University of Science and Technology (Luoyang, China). From beginning to the end, animal studies were carried out under the National Institutes of Health (NIH) Guidelines for the welfare and use of animals (Clark et al., 1997).
Fasting almost 12 h, 2 mg/kg of tofacitinib prepared in 0.5% carboxymethyl cellulose sodium (CMC-Na) was orally administered to each beagle dog. The vein blood was collected (about 1.0 mL) at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h and placed in a 1.5 mL polyethylene tube containing heparin. Afterward, blood samples were centrifuged at 8000 × g for 10 min at 4 °C. Immediately after centrifugation, the supernatants were obtained, and next step is to store them at −80 °C. After monitoring the analyte concentration in plasma by UPLC-MS/MS, the non-compartmental analysis was carried out by using Drug and Statistics (DAS) 3.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China), and finally the main pharmacokinetic parameters were obtained what we wanted.
2.3 Instrumentations and analytical conditions
The instrument used in this assay is Waters Acquity ultra performance liquid chromatography (UPLC) (Milford, Massachusetts, USA). The system has a binary solvent with I-CLASS delivery manager, a constant temperature column set 40 °C and an automatic sampler (FTN, set 10 °C). The Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column fitting with a pre-column was used for executing the chromatographic separation. Then, the analytes were thoroughly separated via the optimized gradient program using not only mobile phase A (acetonitrile) but also mobile phase B (0.1% formic acid aqueous solution). The overall process gradient was set a constant flow rate at 0.40 mL/min, which started with 10% acetonitrile increased to 90% within 0.5 min; maintained at 90% acetonitrile from 0.5 to 0.8 min; declined to the pre-transitional stage from 0.8 to 1.0 min and remained unchanged during 1.0 min to 2.0 min. The circulation time was 2.0 min, completing a 2.0 µL injected sample.
Combined with an electrospray ion source (ESI), the MS/MS triple quadrupole system Xevo TQ-S (Milford, Massachusetts, USA) was carried out in the selective reaction monitoring (SRM) mode by means of positive ionization mode. The MS/MS system control parameters and data-collection, through Masslynx 4.1 (Milford, Massachusetts, USA), were shown in Table 1.
Analytes
Precursor ion
Product ion
CV (V)
CE (eV)
RT (min)
Tofacitinib
313.12
148.97
10
20
0.61
M9
329.10
137.03
30
30
0.65
IS
380.95
255.97
30
25
0.65
2.4 Preparation of standard and quality control (QC) samples
The experimental standards including IS at the concentration of 1.00 mg/mL as the stock solution was dissolved in methanol, respectively. To get the working solution mixture, the stock solutions were diluted with methanol, and the IS working solution was similarly diluted to 200 ng/mL. The non-zero eight points were prepared for calibration standards by adding 10 µL mixed-working solutions into 90 µL blank beagle dog plasma. The nominal concentrations of the calibration curves for both tofacitinib and M9 were: 0.5, 1, 5, 10, 50, 100, 200 and 400 ng/mL. Four quality control (QC) samples of low limit of quantification (LLOQ), low quality control (LQC), medium quality control (MQC) and high quality control (HQC) were prepared in blank samples. They were equipped with the concentration levels of 0.5, 1.0, 80, 320 ng/mL for both tofacitinib and M9. All the work samples and stock fluids for further studies were placed at −80 °C.
2.5 Sample operation
Calibration standards, plasma samples, and QC samples were operated properly as follows: adding 100 µL plasma sample, 20 µL IS working fluid (200 ng/mL), and 300 µL acetonitrile into a 1.5 mL centrifuge tube. The mixture of all three things was then rotated for 2.0 min and centrifugated for 10 min (13,000 × g, 4 °C). Subsequently, a tool called pipette gun could help us to transfer 100 µL supernatant to the auto-sampler vials. Then a 2.0 µL supernatant injection was made for the purpose of UPLC-MS/MS study.
2.6 Method validation
On the basis of the principles of FDA bioanalysis method validation, the optimized UPLC-MS/MS approach was verified that it could be able to simultaneously as well as quantitatively detect the levels of tofacitinib and its metabolite M9 in beagle dog plasma. The parameters of method validation mainly included selectivity, matrix effect, LLOQ, accuracy and precision, calibration curve, recovery as well as stability when situation changed (Tang et al., 2020; Xu et al., 2019).
In order to determine the selectivity of this method (endogenous interference from the plasma medium), three different kinds of beagle dog samples were evaluated and investigated: six blank beagle dog plasma sample (from different batchs), sample spiked with tofacitinib and M9 standard solutions with IS, and authentic specimen obtained from studies in the pharmacokinetics.
The linearity of the analysis program made it possible to obtain test results proportional to the sample concentration. The calibration curves were established by plotting the relative peak area ratios of the analyte to IS versus the plasma concentrations of each analyte using a weight factor of 1/x2 at eight different concentrations (0.5–400 ng/mL) of both tofacitinib and M9. The LLOQ was used to represent the sensitivity of the approach and its working theory was to determine the lowest concertation of the calibration curves whose peak area was expected to be at least 5 times more than blank beagle dog samples. The accuracy and precision of inter-day and intra-day should be within ± 20% and 20%, respectively.
Precision and accuracy were calculated and expressed as the relative standard deviation (RSD) and the relative error (RE), respectively, including three different concentrations of QC samples and LLOQ plasma samples for six replicates. They were assessed based on the analysis of QC samples on 3 different days. The acceptance criterion for within-day and between-day precision for each concentration should not exceed 15%, and the accuracy should be within ±15% of the nominal value.
Extraction recovery and matrix effect were evaluated at three QC levels (LQC, MQC and HQC). Six replicate analysis of QC samples before extraction (A), analytes added after extraction of blank plasma samples (B), and neat standard solutions (C) were prepared and performed. The obtained peak responses were compared as A/B*100 for extraction recovery, whereas B/C*100 for matrix effect.
The stability of tofacitinib and its metabolite M9 in different storage conditions were evaluated at LQC, MQC and HQC concentrations in five replicates using the freshly prepared calibration curve. Before processing, the samples were placed at indoor temperature for 4 h to determine the stability of short-term, whereas long-term stability for 60 days at −80 °C was also evaluated. The freeze and thaw stability of the analytes in plasma was determined in the process of three freeze–thaw cycles. Finally, the autosampler stability was investigated by keeping the extracted samples for 8 h after storage in the sample manager (10 °C). The values of the results should be within ± 15% of the concentration at the initial analysis.
3 Results and discussions
3.1 Method development and optimization
A good chromatographic condition should have ability to efficiently elute the analytes and separate them from the co-eluting endogenous substances and impurities at the lowest possible levels of the concentrations. By continually optimizing the separation conditions, we obtained high-class sensitivity, short period of retention time, high resolution and symmetrical peak pattern. Finally, it came out that an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column was suitable. In the light of our earlier work (Wang et al., 2021), it is the choice to take 0.1% formic acid aqueous and acetonitrile solution for the mobile phases of UPLC-MS/MS, and satisfactory results were obtained. Under existing limited chromatographic separation technology, we screened the chromatographic elution by gradient programing mode using different organic modifiers (methanol, acetonitrile) and aqueous buffers (formic acid, ammonium acetate), and found that the gradient elution of acetonitrile and water which we added extra 0.1% formic acid aqueous solution was effective but selective.
As a mature extraction method, protein precipitation (PPT) is straightforward, simple, one step and cost effective approach for sample preparation and used for generating clean extracts for LC-MS/MS quantitation from samples. Acetonitrile and methanol which were known as organic solvents, were indispensable in PPT production. In comparison with methanol, acetonitrile had higher extraction recovery rate and no evident endogenous interferences. Consequently, acetonitrile was selected for PPT.
3.2 Method validation
3.2.1 Selectivity
In order to verify the selectivity of the method, the comparisons between those chromatograms of blank plasma samples provided by six beagle dogs with those of standard solution spiked to plasma samples and plasma samples after oral operation were conducted. As seen in Fig. 2, blank plasma sample did not influence the chromatograms of tofacitinib, M9 and IS at the retention times, with the corresponding time of 0.61, 0.65 and 0.65 min, respectively. The result showed this UPLC-MS/MS study was selective and specific.
Representative SRM chromatograms of tofacitinib, M9 and IS in beagle dog samples: blank plasma (A), blank plasma spiked with standard solutions (B) and real plasma sample collected from a beagle dog after oral administration of 2 mg/kg tofacitinib (C).
3.2.2 LLOQ and linearity of calibration curve
In the calibration curves, when the ranges of both tofacitinib and M9 were 0.5–400 ng/mL, the curve of each analyte was high linear. In the validation tests, the determination coefficient (r2) of linear regression analysis was kept greater than 0.99 all the time. The equation of regression validated in this research was Y = 0.140042 × X + 0.177986 (r2 = 0.9993) for tofacitinib, and Y = 0.0537334 × X + 0.00182131 (r2 = 0.9997) for M9, respectively. As for LLOQ, the measured value of both tofacitinib and M9 right now by using UPLC-MS/MS method was 0.5 ng/mL. The relative precision and accuracy did met the bioanalytical verification guidelines’ demands (within 20%, Table 2).
Analytes
Concentration (ng/mL)
Intra-day
Inter-day
RSD%
RE%
RSD%
RE%
Tofacitinib
0.5
12.9
14.3
13.2
4.3
1.0
12.0
4.5
12.5
−2.2
80
2.2
−3.9
2.3
−3.9
320
0.8
−12.0
2.1
−11.7
M9
0.5
6.4
13.9
7.1
12.8
1.0
6.1
−8.4
7.0
−7.1
80
3.4
0.5
3.6
1.6
320
2.0
−3.9
2.1
−3.1
3.2.3 Accuracy and precision
Table 2 revealed both intra-day and inter-day accuracy and precision of each analyte, which were analyzed deeply and thoughly under LLOQ and three QC samples (LQC, MQC and HQC). As seen in Table 2, the range of accuracy and precision was within ±15%. The UPLC-MS/MS method, used to simultaneous determine the concentrations of tofacitinib and M9 in beagle dog plasma, was taken for truly reliable and highly reproducible according to our study.
3.2.4 Recovery and matrix effect
It was clearly exhibited in Table 3 that the average rate of extraction recoveries of QC samples at three different concentration levels for the two analytes in beagle dog plasma were 88.5%-98.3%, indicating that the method has high repeatability. The matrix effects of all analytes in our experiment were 95.1%–104.0%, and no conspicuous matrix effect was presented.
Analytes
Concentration (ng/mL)
Recovery (%)
Matrix effect (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
1.0
92.2 ± 9.8
10.6
99.4 ± 14.6
14.7
Tofacitinib
80
96.8 ± 5.5
5.6
102.3 ± 7.2
7.0
320
98.3 ± 2.1
2.1
100.7 ± 2.8
2.8
1.0
88.5 ± 7.5
8.5
104.0 ± 15.0
14.4
M9
80
92.9 ± 0.9
0.9
97.4 ± 4.4
4.6
320
93.7 ± 3.4
3.7
95.1 ± 5.0
5.3
3.2.5 Stability
The stability of research samples was tested and verified at different concentrations of LQC, MQC and HQC (Table 4). It turned out that the results were stable when the process time at room temperature was greater than 4 h, at −80 °C greater than 60 days, at the autosampler (10 °C) greater than 8 h, or in three complete freeze–thaw periods.
Analyte
Added (ng/mL)
Room temperature, 4 h
Autosampler 10 °C, 8 h
Three freeze–thaw
−80 °C, 60 days
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD(%)
RE(%)
RSD(%)
RE(%)
1.0
12.9
−5.0
13.4
−6.8
9.2
−6.9
13.5
−3.6
Tofacitinib
80
4.2
−0.7
2.6
−1.4
3.5
−1.0
3.2
−1.2
320
2.6
−9.3
1.9
−9.2
3.2
−9.5
1.6
−9.1
1.0
12.5
−1.5
8.8
5.6
9.8
2.6
7.8
6.1
M9
80
2.4
3.6
2.4
3.4
3.7
3.3
3.1
3.1
320
2.1
−0.8
2.1
−0.5
1.6
−0.8
1.9
−0.2
3.3 Animal experiments
After an oral dose of 2 mg/kg tofacitinib, the up-to-date inaugurated UPLC-MS/MS method was employed to measure the concentrations of plasma of tofacitinib and its metabolite M9 in beagle dogs. Fig. 3 shows average plasma concentrations of tofacitinib and M9 over time, and Table 5 shows the primary pharmacokinetic parameters worked out by DAS 3.0 which used the non-compartment model analysis method.
Mean plasma concentration–time curves of tofacitinib (A) and M9 (B) in beagle dogs after orally administrated of 2 mg/kg tofacitinib. (n = 6, Mean ± SD).
Parameters
Tofacitinib
M9
AUC0→t (ng/mL·h)
868.11 ± 276.66
1030.14 ± 281.30
AUC0→∞ (ng/mL·h)
869.70 ± 276.95
1045.07 ± 288.66
MRT0→t (h)
3.05 ± 0.67
5.74 ± 1.11
MRT0→∞ (h)
3.18 ± 0.87
6.62 ± 1.77
t1/2 (h)
4.68 ± 1.13
9.45 ± 2.98
Tmax (h)
1.60 ± 0.55
3.60 ± 0.89
CLz/F (L/h/kg)
2.54 ± 1.01
2.11 ± 0.88
Cmax (ng/mL)
280.88 ± 43.66
197.35 ± 46.40
In this study, after taking tofacitinib orally, it was slowly absorbed which reached for its maximum concentration (Cmax) within 1.60 ± 0.55 h post-dose. Moreover, the half-life (t1/2) of tofacitinib was 4.68 ± 1.13 h, which drew near to the previous study in rats (Sharma et al., 2015). Although a number of LC-MS/MS methods for the measurement of tofacitinib in biological fluids had been discovered (Abdelhameed et al., 2017; Kadi et al., 2016; Paniagua et al., 2005; Sharma et al., 2015), detail pharmacokinetic profiles and parameters of its metabolite M9 had not been found. Therefore, the pharmacokinetic parameters of M9 in beagle dogs were described for the first time. The t1/2 and the peak time (Tmax) were 9.45 ± 2.98 h and 3.60 ± 0.89 h, respectively. Because of different species characteristics between humans, beagle dogs and rats, along with individual differences between samples and samples in our research, it is a must to further explore the precise and receivable pharmacokinetic curves and also parameters of those two analytes in our human beings.
4 Conclusions
To sum up, it was the first instance of establishing a UPLC-MS/MS method to simultaneously determinate tofacitinib and its metabolite M9 levels in plasma, and to describe the pharmacokinetics of them in beagle dogs. In addition, the optimized UPLC-MS/MS approach for simultaneous detection of two analytes in plasma was more rapid as well as reliable, which was able to shorten the time of retention, enhance sensitivity and accuracy. At present, this method can be served as a way to study the pharmacokinetics of tofacitinib and its metabolite M9 in beagle dogs and further obtain the pharmacokinetic parameters.
Acknowledgements
This study was supported by grant from Wenzhou City Science and Technology Bureau (Grant No. Y2020840).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
- An LC-MS/MS method for rapid and sensitive high-throughput simultaneous determination of various protein kinase inhibitors in human plasma. Biomed. Chromatography. 2017;31(2)
- [Google Scholar]
- A pharmacokinetic and clinical assessment of tofacitinib for the treatment of rheumatoid arthritis. Expert Opin. Drug Metab. Toxicol.. 2013;9(6):753-761.
- [CrossRef] [Google Scholar]
- Special Report: The 1996 Guide for the Care and Use of Laboratory Animals. ILAR J.. 1997;38(1):41-48.
- [CrossRef] [Google Scholar]
- The pharmacokinetics, metabolism, and clearance mechanisms of tofacitinib, a janus kinase inhibitor, in humans. Drug Metab. Dispos.. 2014;42(4):759-773.
- [CrossRef] [Google Scholar]
- Liquid chromatographic-tandem mass spectrometric assay for simultaneous quantitation of tofacitinib, cabozantinib and afatinib in human plasma and urine. Trop. J. Pharm. Res.. 2016;15(12):2683-2692.
- [CrossRef] [Google Scholar]
- Efficacy of pharmacological treatment in rheumatoid arthritis: a systematic literature research informing the 2019 update of the EULAR recommendations for management of rheumatoid arthritis. Ann. Rheum. Dis.. 2020;79(6):744-759.
- [CrossRef] [Google Scholar]
- Effective quantification of 11 tyrosine kinase inhibitors and caffeine in human plasma by validated LC-MS/MS method with potent phospholipids clean-up procedure. Application to therapeutic drug monitoring. Talanta. 2020;208
- [Google Scholar]
- Efficacy and safety of tofacitinib versus baricitinib in patients with rheumatoid arthritis in real clinical practice: analyses with propensity score-based inverse probability of treatment weighting. Ann. Rheum. Dis. 2021
- [CrossRef] [Google Scholar]
- Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column-switching high-performance liquid chromatography and mass spectrometry detection. Ther. Drug Monit.. 2005;27(5):608-616.
- [CrossRef] [Google Scholar]
- Properties of FDA-approved small molecule protein kinase inhibitors: A 2021 update. Pharmacol. Res.. 2021;165:105463.
- [CrossRef] [Google Scholar]
- S, V. K., Dhiman, V., Giri, K. K., Sharma, K., Zainuddin, M., Mullangi, R., 2015. Development and validation of a RP-HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study. Biomed. Chromatogr, 29(9), 1325–1329. https://doi.org/10.1002/bmc.3426.
- A validated LC-MS/MS assay for simultaneous quantification of methotrexate and tofacitinib in rat plasma: application to a pharmacokinetic study. Biomed. Chromatogr.. 2015;29(5):722-732.
- [CrossRef] [Google Scholar]
- Tofacitinib in the Treatment of Moderate-to-Severe Rheumatoid Arthritis in China: A Cost-Effectiveness Analysis Based on a Mapping Algorithm Derived from a Chinese Population. Adv. Ther.. 2021;38(5):2571-2585.
- [CrossRef] [Google Scholar]
- In vivo Pharmacokinetic Drug-Drug Interaction Studies Between Fedratinib and Antifungal Agents Based on a Newly Developed and Validated UPLC/MS-MS Method. Front. Pharmacol.. 2020;11:626897.
- [CrossRef] [Google Scholar]
- JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib. Ann. Rheum. Dis. 2021
- [CrossRef] [Google Scholar]
- Analytical methodology and pharmacokinetic study of elagolix in plasma of rats using a newly developed UPLC-MS/MS assay. Arabian J. Chem.. 2021;14(7)
- [Google Scholar]
- UPLC-MS/MS method for the simultaneous determination of imatinib, voriconazole and their metabolites concentrations in rat plasma. J. Pharm. Biomed. Anal.. 2019;166:6-12.
- [CrossRef] [Google Scholar]