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MS method for the determination of talazoparib in rat plasma and its pharmacokinetic study
Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112850
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
UPLC-MS/MS method for the determination of talazoparib in rat plasma and its pharmacokinetic study Lei Ye a,1 , Jingjing Chen a,1 , Shuang-long Li b , Yong-liang Zhu b , Saili Xie a,∗ , Xiaoxiang Du a,∗ a b
The First Affiliated Hospital of Wenzhou Medical University, 325000 Wenzhou, PR China Medical College of Henan University of Science and Technology, 471003 Luoyang, PR China
a r t i c l e
i n f o
Article history: Received 7 July 2019 Received in revised form 26 August 2019 Accepted 29 August 2019 Available online 31 August 2019 Keywords: Talazoparib Method UPLC-MS/MS Rat plasma Pharmacokinetics
a b s t r a c t In the present study, an accurate and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of plasma talazoparib concentration in rats was developed and established. The purpose of chromatographic separation of talazoparib and the internal standard (bosutinib) was achieved on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 m) column with a flow rate of 0.40 mL/min, using a gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. The detection was performed on a XEVO TQ-S triple quadrupole tandem mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions of m/z 381.3 → 285.2 for talazoparib and m/z 530.2 → 141.2 for bosutinib (IS), respectively. The method was linear over the range of 0.5–200 ng/mL for talazoparib. The accuracies and precisions of intra- and inter-day were all within the acceptance limits, and no matrix effect was observed in this method. The validated method was further employed to a pharmacokinetic study of talazoparib after oral treatment with 0.2 mg/kg talazoparib to rats. © 2019 Elsevier B.V. All rights reserved.
1. Introduction As an oral polyadenosine 5 -diphosphoribose polymerase (PARP) inhibitor, talazoparib (Fig. 1A) has recently emerged as a promising anticancer therapy [1–4]. In 2018, talazoparib was received its first approval in the USA for the treatment of adults with deleterious or suspected deleterious germline BRCA-mutated, human epidermal growth factor receptor 2 (HER2)-negative, locally advanced or metastatic breast cancer [5]. Interesting, talazoparib has no clinically relevant effect on the QT interval in 37 patients with advanced solid tumors [6]. After oral administration of talazoparib, a maximum concentration (Cmax ) was generally achieved at 1–2 h post-dose [5]. Although food could delay the median time to Cmax and decrease mean Cmax , the area under the concentration-time curve (AUC) was no change. Therefore, food had no particular effect on the metabolism of talazoparib [7]. In vitro study, it was found that talazoparib did not inhibit any enzymes, like CYP1A2, CYP2C9, CYP2C19, CYP2D6 and
∗ Corresponding authors. E-mail addresses: [email protected] (S. Xie), [email protected] (X. Du). 1 The work has been contributed by these authors equally. https://doi.org/10.1016/j.jpba.2019.112850 0731-7085/© 2019 Elsevier B.V. All rights reserved.
CYP3A4, even the concentration reached to 10 mol/L [8]. However, P-glycoprotein (P-gp) inhibitors and breast cancer resistance protein inhibitors could increase the exposure of talazoparib when they were taken at the same time [9]. An accurate and simple bioanalytical assay is necessary for the detection of a drug to support the upcoming clinical pharmacokinetic or drug-drug interaction study in bio-samples. As we know, due to the high selectivity and sensitivity of liquid chromatography tandem mass spectrometry (LC–MS/MS) method, it has been proved to be one of the most powerful tools for the determination of trace amount of drugs [10,11]. To the best of our knowledge, no publication regarding the bioanalytical method of talazoparib has been described until now. Therefore, in this present study, an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of talazoparib was fully developed and established according to the latest guidelines of the US Food and Drug Administration (FDA) [12]. We demonstrated the applicability of the validated method by analyzing rat plasma samples, to support a pharmacokinetic study.
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L. Ye, J. Chen, S.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112850
Fig. 1. The chemical structures of the analyte and IS in the present study: (A) talazoparib; (B) bosutinib (IS).
2. Experimental
2.4. Sample preparation
2.1. Chemicals materials
To 100 L plasma, an aliquot of 400 L acetonitrile solution (IS in acetonitrile 50 ng/mL) was added for protein precipitation in a 1.5 mL centrifuge tube. The mixture was vortexed for 1.0 min and then centrifugated at 13,000 g for 10 min. The clear supernatant (5.0 L) was injected into the UPLC-MS/MS system for analysis.
Talazoparib (purity > 98%) and bosutinib (internal standard, IS, purity > 98%, Fig. 1B) were purchased from Beijing sunflower and technology development CO., LTD (Beijing, China). HPLC grade acetonitrile and methanol were supplied by Merck Company (Darmstadt, Germany). HPLC grade water was prepared using a Milli Q system (Millipore, Bedford, USA).
2.2. UPLC-MS/MS conditions The liquid chromatography was carried out on an Acquity ultra performance liquid chromatography (UPLC) system (Milford, MA, USA) interfaced to a XEVO TQ-S triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Milford, MA, USA). Chromatographic separation was achieved by gradient elution on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m) maintained at 40 ◦ C and inline 0.2 m stainless steel frit filter (Milford, MA, USA). The mobile phase was consisted of acetonitrile (solvent A), and 0.1% formic acid in water (solvent B) with a flow rate of 0.40 mL/min, and the linear gradient elution program was employed as follows: 0-0.5 min (10-10% A), 0.5–1.0 min (10–90% A), 1.0–2.0 min (90-90% A), 2.0–2.1 min (90-10% A), 2.1–3.0 min (1010% A). The injection volume was 5.0 L and the entire run time was 3.0 min. Quantification analysis was operated in the multiple reaction monitoring (MRM) mode in the mass analyzers. The MRM transitions of talazoparib and IS were m/z 381.3 → 285.2 and m/z 530.2 → 141.2, respectively. The Masslynx 4.1 software (Milford, MA, USA) was conducted for data acquisition and instrument control.
2.3. Standard solutions, calibration standards and quality control (QC) sample Talazoparib stock solution was prepared at a concentration of 1.00 mg/mL in methanol. A series of calibration standard and quality control (QC) working solutions were gradiently diluted from its stock solution with methanol. The calibration standards in plasma were prepared by spiking 10 L of the corresponding working solutions into 90 L blank rat plasma to obtain 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 ng/mL for talazoparib. In the same manner, QC samples were prepared at low, medium and high concentrations: 1, 80, 160 ng/mL for talazoparib. Similarly, the working solution of IS (50 ng/mL) was made from its stock solution (1.00 mg/mL) using acetonitrile for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C.
2.5. Method validation Bioanalytical method validation was conducted to evaluate the selectivity, linearity, precision, accuracy, matrix effect, recovery and stability according to the principles of Guidance for Industry Bioanalytical Method Validation by the US FDA [12]. 2.5.1. Selectivity Selectivity of the method was assessed by comparing chromatograms of blank rat plasma samples collected from six different lots, with the blank plasma spiked with talazoparib and IS, and a real rat plasma sample. 2.5.2. Linearity of calibration curve and lower limit of quantification Calibration curves at nine concentrations from 0.5 to 200 ng/mL for talazoparib in rat plasma were pretreated in duplicate and analyzed by UPLC-MS/MS in three consecutive runs. The linearity of the calibration curve was fitted using a weighted (1/x2 ) least-squares linear regression method by plotting the peak area ratio (the analyte/IS) versus the nominal concentration. The sensitivity of the method was calculated by the lower limit of quantification (LLOQ), for which should be within a deviation of ± 20%. 2.5.3. Precision and accuracy The intra-day precision and accuracy were determined through the performance of six replicates QC samples at three concentration levels during a single analytical run. The inter-day precision and accuracy were measured using six replicates determinations of three concentration levels of QC samples on three separate days. The precision was illustrated as the relative standard deviation (RSD%), which should be required not to exceed 15%, whereas the accuracy was illustrated as the relative error (RE%), which should be within ± 15%. 2.5.4. Extraction recovery and matrix effect The extraction recovery of talazoparib from plasma was assessed by calculating the ratio of peak areas of samples spiked before to after extraction at three different concentrations. The matrix effect was evaluated by comparing peak areas of spiked samples with extracted matrix to the pure reference standard solution at equivalent concentrations.
L. Ye, J. Chen, S.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112850
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Fig. 2. Representative chromatograms of blank plasma (A), blank plasma spiked with standard solution (B) and real plasma sample of talazoparib in rats after 1.0 h oral administration (C).
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L. Ye, J. Chen, S.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112850
Table 1 Intra- and Inter-day accuracy and precision of talazoparib in rat plasma (n = 6). Compound
Talazoparib
Concentration (ng/mL)
1 80 160
Inter-day
Intra-day RSD%
RE%
RSD%
RE%
10.9 3.4 2.9
−8.0 7.5 2.7
12.0 4.4 3.2
−3.2 3.4 0.1
3. Results and discussion 3.1. Method development and optimization
Fig. 3. Mean plasma concentration-time profiles of talazoparib in rats after a single oral dose of 0.2 mg/kg of talazoparib. Data are expressed as mean ± SD (n = 8).
2.5.5. Stability Low, medium, and high concentration levels of QC samples (n = 5) were determined to evaluate the stability of the analyte in rat plasma. For each concentration, the short term stabilities of QC samples were assessed after storage at room temperature for 6 h and after preparation in an auto-sampler for 18 h at 4 ◦ C, and the long term stability was also measured for 31 days at −20 ◦ C. In addition, three complete freeze-thaw cycles from −20 ◦ C to room temperature was detected. The analyte was considered to be stable in plasma when 85–115% of the initial concentrations were found.
2.6. Pharmacokinetic study The analytical method was used to estimate the concentration and pharmacokinetic study of talazoparib in eight male SpragueDawley rats (180–220 g) purchased from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China). All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University and were in accordance with the Guide for the Care and Use of Laboratory Animals. Prior the study, diet was prohibited for 12 h but water was freely available. 0.3 mL of blood samples were drawn from the tail vein at 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h after oral administration of talazoparib (0.2 mg/kg) into heparinized 1.5 mL polythene tubes. The obtained blood samples were immediately subjected to centrifugation at 4000 g for 8 min to allow for separation of plasma, whose volumn was 100 L and stored at −20 ◦ C until analysis. Following determination of analyte concentrations, plasma talazoparib concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Shanghai University of Traditional Chinese Medicine, China) in non-compartmental mode.
Different chromatographic conditions, including the different types of columns and the composition of mobile phase were optimized to achieve a good separation, short running time and negligible matrix effect from endogenous components of plasma. After testing several different kinds of columns, it turned out that the Acquity BEH C18 (2.1 mm × 50 mm, 1.7 m) column offered a better separation and peak shape, and promoted the retention time. In order to obtain adequate peak responses and sharp peak shape in a short run time, two organic phases (acetonitrile and methanol) were compared as the chromatographic mobile phase for the analyte and IS, which was found that acetonitrile performed a sharper peak than methanol. Moreover, the addition of 0.1% formic acid in water increased the ion intensity and the reproducibility of the analyte in chromatography. In addition, gradient elution of the mobile phase was superior to isocratic elution, which made no interference from endogenous substances of plasma. Finally, a gradient mobile phase was adopted, which was consisted of acetonitrile and 0.1% formic acid in water with a flow rate of 0.40 mL/min. In the running time of this study, the total time was only 3.0 min, including column cleaning, chromatographic separation, and equilibration time. 3.2. Method validation 3.2.1. Selectivity The selectivity of the method was demonstrated as shown in Fig. 2, and there were no interfering peaks from rat plasma detected at the retention times of the analyte and IS. The retention times of talazoparib and IS were 1.26, and 1.19 min, respectively. 3.2.2. Linearity of calibration curve and LLOQ In the range of 0.5–200 ng/ml for talazoparib, calibration curve showed excellent linearity. The regression equation obtained by least squared regression was Y = 0.695706 × X ± 0.0612487 ( r2 = 0.9998) for talazoparib, where Y indicates the peak area ratio of the analyte to its IS and X indicates the plasma concentration of the analyte. The lower limit of quantification (LLOQ) achieved in this study was established as 0.5 ng/mL for talazoparib, with RSD values < 11.5% and RE values within 10.9%. 3.2.3. Precision and accuracy The results listed in Table 1 showed a summary of the accuracy and precision of the method determined at three concentrations of the analyte spiked in blank plasma. For the low, medium, and high QC concentrations, the accuracy (RE%) of the analyte was within ± 8.0%. The intra- and inter-day precision (RSD%) of the analyte ranged from 2.9% to 12.0% for talazoparib. These data exhibited that the present analytical method was precise and accurate. 3.2.4. Recovery and matrix effect The recovery and matrix effect data were summarized in Table 2. The recovery from plasma was (87.4 ± 9.7)%, (88.7 ± 4.8)% and
L. Ye, J. Chen, S.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112850
5
Table 2 Recovery and matrix effect of talazoparib in rat plasma (n = 6). Analyte
Recovery (%)
Concentration added (ng/mL)
1 80 160
Talazoparib
Matrix effect (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
87.4 ± 9.7 88.7 ± 4.8 91.3 ± 6.0
11.1 5.4 6.6
87.8 ± 10.1 92.6 ± 4.7 94.8 ± 3.8
11.4 5.0 4.0
Table 3 Stability results of talazoparib in rat plasma in different conditions (n = 5). Analyte
Added (ng/mL)
Talazoparib
1 80 160
Room temperature, 6 h
Autosampler 4 ◦ C, 18 h
Three freeze-thaw
−20 ◦ C, 31 days
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
10.6 6.3 3.2
8.8 8.5 6.2
10.8 5.2 3.0
7.9 6.3 5.2
7.6 4.1 4.0
8.3 7.3 5.4
9.8 8.6 3.4
9.3 7.4 5.3
Table 4 The pharmacokinetic parameters of talazoparib in rat plasma after oral administration 0.2 mg/kg talazoparib (n = 8, Mean ± SD). Parameters
Talazoparib
t1/2 (h) Tmax (h) Cmax (ng/mL) AUC0→t (ng/mL h) AUC0→∞ (ng/mL h) Vd (L/kg) CL (L/h) MRT0→t (h) MRT0→∞ (h)
7.82 ± 1.57 5.67 ± 1.51 123.29 ± 38.11 1797.41 ± 340.74 1829.66 ± 334.07 1.29 ± 0.43 0.11 ± 0.02 13.48 ± 1.49 13.94 ± 1.50
(91.3 ± 6.0)% for talazoparib at their corresponding QC concentrations, respectively (n = 6). The matrix effect was (87.8 ± 10.1)%, (92.6 ± 4.7)% and (94.8 ± 3.8)% for talazoparib at three QC concentrations, respectively (n = 6). In addition, mean recovery and the matrix effect for the IS were 81.5 ± 7.2% and 108.2 ± 7.6%. These results illustrated that this developed method had high recovery and no matrix effect under the tested conditions. 3.2.5. Stability Under a variety of storage and process conditions, stability was investigated by evaluating three concentrations of QC samples (Table 3). Sample extracts were stable at room temperature for 6 h and in the auto-sampler (4 ◦ C) for at least 18 h. Moreover, the data of the three complete freeze-thaw cycles (at −20 ◦ C to room temperature) and long-term storage at −20 ◦ C for up to 31 days showed that, the analyte was also stable. 3.3. Pharmacokinetic study The analytical method was used to the determination of plasma talazoparib concentration in eight rats for the pharmacokinetic study after a single oral administration of 0.2 mg/kg talazoparib. The mean plasma concentration-time curves were exhibited in Fig. 3 and the pharmacokinetic parameters from non-compartment model analysis were listed in Table 4. As shown in Table 4, talazoparib reached peak concentration (Cmax ) of 123.29 ± 38.11 ng/mL at approximately 5.67 h. The pharmacokinetic value of half-life (t1/2 ) for talazoparib was 7.82 ± 1.57 h in rats, which was not in agreement with previous published study in patients with advanced solid tumors [4]. The reasons for this difference may be explained by individual differences in rats and racial differences between rats and humans.
4. Conclusions For the first time, we developed and validated a sensitive and rapid UPLC-MS/MS method for the measurement of talazoparib in rat plasma. This method was shown to be of great precision and accuracy, and was also successfully applied to the pharmacokinetic investigations of talazoparib in rats after oral administration of 0.2 mg/kg talazoparib. References [1] J. de Bono, R.K. Ramanathan, L. Mina, R. Chugh, J. Glaspy, S. Rafii, S. Kaye, J. Sachdev, J. Heymach, D.C. Smith, J.W. Henshaw, A. Herriott, M. Patterson, N.J. Curtin, L.A. Byers, Z.A. Wainberg, I. Phase, Dose-Escalation, Two-Part Trial of the PARP Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 Mutations and Selected Sporadic Cancers, Cancer Discov. 7 (2017) 620–629. [2] N.C. Turner, M.L. Telli, H.S. Rugo, A. Mailliez, J. Ettl, E.M. Grischke, L.A. Mina, J. Balmana, P.A. Fasching, S.A. Hurvitz, A.M. Wardley, C. Chappey, A.L. Hannah, M.E. Robson, A.S. Group, A phase II study of talazoparib after platinum or cytotoxic nonplatinum regimens in patients with advanced breast Cancer and germline BRCA1/2 mutations (ABRAZO), Clin. Cancer Res. 25 (2019) 2717–2724. [3] J.K. Litton, H.S. Rugo, J. Ettl, S.A. Hurvitz, A. Goncalves, K.H. Lee, L. Fehrenbacher, R. Yerushalmi, L.A. Mina, M. Martin, H. Roche, Y.H. Im, R.G.W. Quek, D. Markova, I.C. Tudor, A.L. Hannah, W. Eiermann, J.L. Blum, Talazoparib in patients with advanced breast Cancer and a germline BRCA mutation, N. Engl. J. Med. 379 (2018) 753–763. [4] Y. Yu, C.H. Chung, A. Plotka, K. Quinn, H. Shi, Z. Papai, L. Nguyen, D. Wang, A Phase 1 Mass Balance Study of (14) C-Labeled Talazoparib in Patients With Advanced Solid Tumors, J. Clin. Pharmacol. 59 (2019) 1195–1203. [5] S.M. Hoy, Talazoparib: first global approval, Drugs 78 (2018) 1939–1946. [6] J. Hoffman, J. Chakrabarti, A. Plotka, A.M. Naraine, D. Kanamori, R. Moroose, L. Nguyen, D. Wang, Z.A. Wainberg, Talazoparib has no clinically relevant effect on QTc interval in patients with advanced solid tumors, Anticancer Drugs 30 (2019) 523–532. [7] A.S. Zimmer, M. Gillard, S. Lipkowitz, J.M. Lee, Update on PARP inhibitors in breast Cancer, Curr. Treat. Options Oncol. 19 (2018) 21. [8] Y. Shen, F.L. Rehman, Y. Feng, J. Boshuizen, I. Bajrami, R. Elliott, B. Wang, C.J. Lord, L.E. Post, A. Ashworth, BMN 673, a novel and highly potent PARP1/2 inhibitor for the treatment of human cancers with DNA repair deficiency, Clin. Cancer Res. 19 (2013) 5003–5015. [9] Y. Yu, C. Durairaj, H. Shi, D.D. Wang, Population pharmacokinetic analyses for talazoparib (TALA) in cancer patients [abstract no. 432P], Ann. Oncol. 29 (Suppl 8) (2018) viii133–viii148. [10] X. Qiu, S. Xie, L. Ye, R.A. Xu, UPLC-MS/MS method for the quantification of ertugliflozin and sitagliptin in rat plasma, Anal. Biochem. 567 (2019) 112–116. [11] R.A. Xu, Q. Lin, X. Qiu, J. Chen, Y. Shao, G. Hu, G. Lin, UPLC-MS/MS method for the simultaneous determination of imatinib, voriconazole and their metabolites concentrations in rat plasma, J. Pharm. Biomed. Anal. 166 (2019) 6–12. [12] Center for Drug Evaluation and Research of the U.S. Department of Health and Human Services Food and Drug Administration, Guidance for Industry; Bioanalytical Method Validation, 2018, Accessed: August 2, 2018 http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm064964.htm.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
UPLC-MS/MS method for the determination of talazoparib in rat plasma and its pharmacokinetic study Lei Ye a,1 , Jingjing Chen a,1 , Shuang-long Li b , Yong-liang Zhu b , Saili Xie a,∗ , Xiaoxiang Du a,∗ a b
The First Affiliated Hospital of Wenzhou Medical University, 325000 Wenzhou, PR China Medical College of Henan University of Science and Technology, 471003 Luoyang, PR China
a r t i c l e
i n f o
Article history: Received 7 July 2019 Received in revised form 26 August 2019 Accepted 29 August 2019 Available online 31 August 2019 Keywords: Talazoparib Method UPLC-MS/MS Rat plasma Pharmacokinetics
a b s t r a c t In the present study, an accurate and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of plasma talazoparib concentration in rats was developed and established. The purpose of chromatographic separation of talazoparib and the internal standard (bosutinib) was achieved on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 m) column with a flow rate of 0.40 mL/min, using a gradient elution with acetonitrile and 0.1% formic acid in water as the mobile phase. The detection was performed on a XEVO TQ-S triple quadrupole tandem mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions of m/z 381.3 → 285.2 for talazoparib and m/z 530.2 → 141.2 for bosutinib (IS), respectively. The method was linear over the range of 0.5–200 ng/mL for talazoparib. The accuracies and precisions of intra- and inter-day were all within the acceptance limits, and no matrix effect was observed in this method. The validated method was further employed to a pharmacokinetic study of talazoparib after oral treatment with 0.2 mg/kg talazoparib to rats. © 2019 Elsevier B.V. All rights reserved.
1. Introduction As an oral polyadenosine 5 -diphosphoribose polymerase (PARP) inhibitor, talazoparib (Fig. 1A) has recently emerged as a promising anticancer therapy [1–4]. In 2018, talazoparib was received its first approval in the USA for the treatment of adults with deleterious or suspected deleterious germline BRCA-mutated, human epidermal growth factor receptor 2 (HER2)-negative, locally advanced or metastatic breast cancer [5]. Interesting, talazoparib has no clinically relevant effect on the QT interval in 37 patients with advanced solid tumors [6]. After oral administration of talazoparib, a maximum concentration (Cmax ) was generally achieved at 1–2 h post-dose [5]. Although food could delay the median time to Cmax and decrease mean Cmax , the area under the concentration-time curve (AUC) was no change. Therefore, food had no particular effect on the metabolism of talazoparib [7]. In vitro study, it was found that talazoparib did not inhibit any enzymes, like CYP1A2, CYP2C9, CYP2C19, CYP2D6 and
∗ Corresponding authors. E-mail addresses: [email protected] (S. Xie), [email protected] (X. Du). 1 The work has been contributed by these authors equally. https://doi.org/10.1016/j.jpba.2019.112850 0731-7085/© 2019 Elsevier B.V. All rights reserved.
CYP3A4, even the concentration reached to 10 mol/L [8]. However, P-glycoprotein (P-gp) inhibitors and breast cancer resistance protein inhibitors could increase the exposure of talazoparib when they were taken at the same time [9]. An accurate and simple bioanalytical assay is necessary for the detection of a drug to support the upcoming clinical pharmacokinetic or drug-drug interaction study in bio-samples. As we know, due to the high selectivity and sensitivity of liquid chromatography tandem mass spectrometry (LC–MS/MS) method, it has been proved to be one of the most powerful tools for the determination of trace amount of drugs [10,11]. To the best of our knowledge, no publication regarding the bioanalytical method of talazoparib has been described until now. Therefore, in this present study, an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of talazoparib was fully developed and established according to the latest guidelines of the US Food and Drug Administration (FDA) [12]. We demonstrated the applicability of the validated method by analyzing rat plasma samples, to support a pharmacokinetic study.
2
L. Ye, J. Chen, S.-l. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112850
Fig. 1. The chemical structures of the analyte and IS in the present study: (A) talazoparib; (B) bosutinib (IS).
2. Experimental
2.4. Sample preparation
2.1. Chemicals materials
To 100 L plasma, an aliquot of 400 L acetonitrile solution (IS in acetonitrile 50 ng/mL) was added for protein precipitation in a 1.5 mL centrifuge tube. The mixture was vortexed for 1.0 min and then centrifugated at 13,000 g for 10 min. The clear supernatant (5.0 L) was injected into the UPLC-MS/MS system for analysis.
Talazoparib (purity > 98%) and bosutinib (internal standard, IS, purity > 98%, Fig. 1B) were purchased from Beijing sunflower and technology development CO., LTD (Beijing, China). HPLC grade acetonitrile and methanol were supplied by Merck Company (Darmstadt, Germany). HPLC grade water was prepared using a Milli Q system (Millipore, Bedford, USA).
2.2. UPLC-MS/MS conditions The liquid chromatography was carried out on an Acquity ultra performance liquid chromatography (UPLC) system (Milford, MA, USA) interfaced to a XEVO TQ-S triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Milford, MA, USA). Chromatographic separation was achieved by gradient elution on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m) maintained at 40 ◦ C and inline 0.2 m stainless steel frit filter (Milford, MA, USA). The mobile phase was consisted of acetonitrile (solvent A), and 0.1% formic acid in water (solvent B) with a flow rate of 0.40 mL/min, and the linear gradient elution program was employed as follows: 0-0.5 min (10-10% A), 0.5–1.0 min (10–90% A), 1.0–2.0 min (90-90% A), 2.0–2.1 min (90-10% A), 2.1–3.0 min (1010% A). The injection volume was 5.0 L and the entire run time was 3.0 min. Quantification analysis was operated in the multiple reaction monitoring (MRM) mode in the mass analyzers. The MRM transitions of talazoparib and IS were m/z 381.3 → 285.2 and m/z 530.2 → 141.2, respectively. The Masslynx 4.1 software (Milford, MA, USA) was conducted for data acquisition and instrument control.
2.3. Standard solutions, calibration standards and quality control (QC) sample Talazoparib stock solution was prepared at a concentration of 1.00 mg/mL in methanol. A series of calibration standard and quality control (QC) working solutions were gradiently diluted from its stock solution with methanol. The calibration standards in plasma were prepared by spiking 10 L of the corresponding working solutions into 90 L blank rat plasma to obtain 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 ng/mL for talazoparib. In the same manner, QC samples were prepared at low, medium and high concentrations: 1, 80, 160 ng/mL for talazoparib. Similarly, the working solution of IS (50 ng/mL) was made from its stock solution (1.00 mg/mL) using acetonitrile for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C.
2.5. Method validation Bioanalytical method validation was conducted to evaluate the selectivity, linearity, precision, accuracy, matrix effect, recovery and stability according to the principles of Guidance for Industry Bioanalytical Method Validation by the US FDA [12]. 2.5.1. Selectivity Selectivity of the method was assessed by comparing chromatograms of blank rat plasma samples collected from six different lots, with the blank plasma spiked with talazoparib and IS, and a real rat plasma sample. 2.5.2. Linearity of calibration curve and lower limit of quantification Calibration curves at nine concentrations from 0.5 to 200 ng/mL for talazoparib in rat plasma were pretreated in duplicate and analyzed by UPLC-MS/MS in three consecutive runs. The linearity of the calibration curve was fitted using a weighted (1/x2 ) least-squares linear regression method by plotting the peak area ratio (the analyte/IS) versus the nominal concentration. The sensitivity of the method was calculated by the lower limit of quantification (LLOQ), for which should be within a deviation of ± 20%. 2.5.3. Precision and accuracy The intra-day precision and accuracy were determined through the performance of six replicates QC samples at three concentration levels during a single analytical run. The inter-day precision and accuracy were measured using six replicates determinations of three concentration levels of QC samples on three separate days. The precision was illustrated as the relative standard deviation (RSD%), which should be required not to exceed 15%, whereas the accuracy was illustrated as the relative error (RE%), which should be within ± 15%. 2.5.4. Extraction recovery and matrix effect The extraction recovery of talazoparib from plasma was assessed by calculating the ratio of peak areas of samples spiked before to after extraction at three different concentrations. The matrix effect was evaluated by comparing peak areas of spiked samples with extracted matrix to the pure reference standard solution at equivalent concentrations.
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Fig. 2. Representative chromatograms of blank plasma (A), blank plasma spiked with standard solution (B) and real plasma sample of talazoparib in rats after 1.0 h oral administration (C).
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Table 1 Intra- and Inter-day accuracy and precision of talazoparib in rat plasma (n = 6). Compound
Talazoparib
Concentration (ng/mL)
1 80 160
Inter-day
Intra-day RSD%
RE%
RSD%
RE%
10.9 3.4 2.9
−8.0 7.5 2.7
12.0 4.4 3.2
−3.2 3.4 0.1
3. Results and discussion 3.1. Method development and optimization
Fig. 3. Mean plasma concentration-time profiles of talazoparib in rats after a single oral dose of 0.2 mg/kg of talazoparib. Data are expressed as mean ± SD (n = 8).
2.5.5. Stability Low, medium, and high concentration levels of QC samples (n = 5) were determined to evaluate the stability of the analyte in rat plasma. For each concentration, the short term stabilities of QC samples were assessed after storage at room temperature for 6 h and after preparation in an auto-sampler for 18 h at 4 ◦ C, and the long term stability was also measured for 31 days at −20 ◦ C. In addition, three complete freeze-thaw cycles from −20 ◦ C to room temperature was detected. The analyte was considered to be stable in plasma when 85–115% of the initial concentrations were found.
2.6. Pharmacokinetic study The analytical method was used to estimate the concentration and pharmacokinetic study of talazoparib in eight male SpragueDawley rats (180–220 g) purchased from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China). All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University and were in accordance with the Guide for the Care and Use of Laboratory Animals. Prior the study, diet was prohibited for 12 h but water was freely available. 0.3 mL of blood samples were drawn from the tail vein at 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h after oral administration of talazoparib (0.2 mg/kg) into heparinized 1.5 mL polythene tubes. The obtained blood samples were immediately subjected to centrifugation at 4000 g for 8 min to allow for separation of plasma, whose volumn was 100 L and stored at −20 ◦ C until analysis. Following determination of analyte concentrations, plasma talazoparib concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Shanghai University of Traditional Chinese Medicine, China) in non-compartmental mode.
Different chromatographic conditions, including the different types of columns and the composition of mobile phase were optimized to achieve a good separation, short running time and negligible matrix effect from endogenous components of plasma. After testing several different kinds of columns, it turned out that the Acquity BEH C18 (2.1 mm × 50 mm, 1.7 m) column offered a better separation and peak shape, and promoted the retention time. In order to obtain adequate peak responses and sharp peak shape in a short run time, two organic phases (acetonitrile and methanol) were compared as the chromatographic mobile phase for the analyte and IS, which was found that acetonitrile performed a sharper peak than methanol. Moreover, the addition of 0.1% formic acid in water increased the ion intensity and the reproducibility of the analyte in chromatography. In addition, gradient elution of the mobile phase was superior to isocratic elution, which made no interference from endogenous substances of plasma. Finally, a gradient mobile phase was adopted, which was consisted of acetonitrile and 0.1% formic acid in water with a flow rate of 0.40 mL/min. In the running time of this study, the total time was only 3.0 min, including column cleaning, chromatographic separation, and equilibration time. 3.2. Method validation 3.2.1. Selectivity The selectivity of the method was demonstrated as shown in Fig. 2, and there were no interfering peaks from rat plasma detected at the retention times of the analyte and IS. The retention times of talazoparib and IS were 1.26, and 1.19 min, respectively. 3.2.2. Linearity of calibration curve and LLOQ In the range of 0.5–200 ng/ml for talazoparib, calibration curve showed excellent linearity. The regression equation obtained by least squared regression was Y = 0.695706 × X ± 0.0612487 ( r2 = 0.9998) for talazoparib, where Y indicates the peak area ratio of the analyte to its IS and X indicates the plasma concentration of the analyte. The lower limit of quantification (LLOQ) achieved in this study was established as 0.5 ng/mL for talazoparib, with RSD values < 11.5% and RE values within 10.9%. 3.2.3. Precision and accuracy The results listed in Table 1 showed a summary of the accuracy and precision of the method determined at three concentrations of the analyte spiked in blank plasma. For the low, medium, and high QC concentrations, the accuracy (RE%) of the analyte was within ± 8.0%. The intra- and inter-day precision (RSD%) of the analyte ranged from 2.9% to 12.0% for talazoparib. These data exhibited that the present analytical method was precise and accurate. 3.2.4. Recovery and matrix effect The recovery and matrix effect data were summarized in Table 2. The recovery from plasma was (87.4 ± 9.7)%, (88.7 ± 4.8)% and
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Table 2 Recovery and matrix effect of talazoparib in rat plasma (n = 6). Analyte
Recovery (%)
Concentration added (ng/mL)
1 80 160
Talazoparib
Matrix effect (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
87.4 ± 9.7 88.7 ± 4.8 91.3 ± 6.0
11.1 5.4 6.6
87.8 ± 10.1 92.6 ± 4.7 94.8 ± 3.8
11.4 5.0 4.0
Table 3 Stability results of talazoparib in rat plasma in different conditions (n = 5). Analyte
Added (ng/mL)
Talazoparib
1 80 160
Room temperature, 6 h
Autosampler 4 ◦ C, 18 h
Three freeze-thaw
−20 ◦ C, 31 days
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
10.6 6.3 3.2
8.8 8.5 6.2
10.8 5.2 3.0
7.9 6.3 5.2
7.6 4.1 4.0
8.3 7.3 5.4
9.8 8.6 3.4
9.3 7.4 5.3
Table 4 The pharmacokinetic parameters of talazoparib in rat plasma after oral administration 0.2 mg/kg talazoparib (n = 8, Mean ± SD). Parameters
Talazoparib
t1/2 (h) Tmax (h) Cmax (ng/mL) AUC0→t (ng/mL h) AUC0→∞ (ng/mL h) Vd (L/kg) CL (L/h) MRT0→t (h) MRT0→∞ (h)
7.82 ± 1.57 5.67 ± 1.51 123.29 ± 38.11 1797.41 ± 340.74 1829.66 ± 334.07 1.29 ± 0.43 0.11 ± 0.02 13.48 ± 1.49 13.94 ± 1.50
(91.3 ± 6.0)% for talazoparib at their corresponding QC concentrations, respectively (n = 6). The matrix effect was (87.8 ± 10.1)%, (92.6 ± 4.7)% and (94.8 ± 3.8)% for talazoparib at three QC concentrations, respectively (n = 6). In addition, mean recovery and the matrix effect for the IS were 81.5 ± 7.2% and 108.2 ± 7.6%. These results illustrated that this developed method had high recovery and no matrix effect under the tested conditions. 3.2.5. Stability Under a variety of storage and process conditions, stability was investigated by evaluating three concentrations of QC samples (Table 3). Sample extracts were stable at room temperature for 6 h and in the auto-sampler (4 ◦ C) for at least 18 h. Moreover, the data of the three complete freeze-thaw cycles (at −20 ◦ C to room temperature) and long-term storage at −20 ◦ C for up to 31 days showed that, the analyte was also stable. 3.3. Pharmacokinetic study The analytical method was used to the determination of plasma talazoparib concentration in eight rats for the pharmacokinetic study after a single oral administration of 0.2 mg/kg talazoparib. The mean plasma concentration-time curves were exhibited in Fig. 3 and the pharmacokinetic parameters from non-compartment model analysis were listed in Table 4. As shown in Table 4, talazoparib reached peak concentration (Cmax ) of 123.29 ± 38.11 ng/mL at approximately 5.67 h. The pharmacokinetic value of half-life (t1/2 ) for talazoparib was 7.82 ± 1.57 h in rats, which was not in agreement with previous published study in patients with advanced solid tumors [4]. The reasons for this difference may be explained by individual differences in rats and racial differences between rats and humans.
4. Conclusions For the first time, we developed and validated a sensitive and rapid UPLC-MS/MS method for the measurement of talazoparib in rat plasma. This method was shown to be of great precision and accuracy, and was also successfully applied to the pharmacokinetic investigations of talazoparib in rats after oral administration of 0.2 mg/kg talazoparib. References [1] J. de Bono, R.K. Ramanathan, L. Mina, R. Chugh, J. Glaspy, S. Rafii, S. Kaye, J. Sachdev, J. Heymach, D.C. Smith, J.W. Henshaw, A. Herriott, M. Patterson, N.J. Curtin, L.A. Byers, Z.A. Wainberg, I. Phase, Dose-Escalation, Two-Part Trial of the PARP Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 Mutations and Selected Sporadic Cancers, Cancer Discov. 7 (2017) 620–629. [2] N.C. Turner, M.L. Telli, H.S. Rugo, A. Mailliez, J. Ettl, E.M. Grischke, L.A. Mina, J. Balmana, P.A. Fasching, S.A. Hurvitz, A.M. Wardley, C. Chappey, A.L. Hannah, M.E. Robson, A.S. Group, A phase II study of talazoparib after platinum or cytotoxic nonplatinum regimens in patients with advanced breast Cancer and germline BRCA1/2 mutations (ABRAZO), Clin. Cancer Res. 25 (2019) 2717–2724. [3] J.K. Litton, H.S. Rugo, J. Ettl, S.A. Hurvitz, A. Goncalves, K.H. Lee, L. Fehrenbacher, R. Yerushalmi, L.A. Mina, M. Martin, H. Roche, Y.H. Im, R.G.W. Quek, D. Markova, I.C. Tudor, A.L. Hannah, W. Eiermann, J.L. Blum, Talazoparib in patients with advanced breast Cancer and a germline BRCA mutation, N. Engl. J. Med. 379 (2018) 753–763. [4] Y. Yu, C.H. Chung, A. Plotka, K. Quinn, H. Shi, Z. Papai, L. Nguyen, D. Wang, A Phase 1 Mass Balance Study of (14) C-Labeled Talazoparib in Patients With Advanced Solid Tumors, J. Clin. Pharmacol. 59 (2019) 1195–1203. [5] S.M. Hoy, Talazoparib: first global approval, Drugs 78 (2018) 1939–1946. [6] J. Hoffman, J. Chakrabarti, A. Plotka, A.M. Naraine, D. Kanamori, R. Moroose, L. Nguyen, D. Wang, Z.A. Wainberg, Talazoparib has no clinically relevant effect on QTc interval in patients with advanced solid tumors, Anticancer Drugs 30 (2019) 523–532. [7] A.S. Zimmer, M. Gillard, S. Lipkowitz, J.M. Lee, Update on PARP inhibitors in breast Cancer, Curr. Treat. Options Oncol. 19 (2018) 21. [8] Y. Shen, F.L. Rehman, Y. Feng, J. Boshuizen, I. Bajrami, R. Elliott, B. Wang, C.J. Lord, L.E. Post, A. Ashworth, BMN 673, a novel and highly potent PARP1/2 inhibitor for the treatment of human cancers with DNA repair deficiency, Clin. Cancer Res. 19 (2013) 5003–5015. [9] Y. Yu, C. Durairaj, H. Shi, D.D. Wang, Population pharmacokinetic analyses for talazoparib (TALA) in cancer patients [abstract no. 432P], Ann. Oncol. 29 (Suppl 8) (2018) viii133–viii148. [10] X. Qiu, S. Xie, L. Ye, R.A. Xu, UPLC-MS/MS method for the quantification of ertugliflozin and sitagliptin in rat plasma, Anal. Biochem. 567 (2019) 112–116. [11] R.A. Xu, Q. Lin, X. Qiu, J. Chen, Y. Shao, G. Hu, G. Lin, UPLC-MS/MS method for the simultaneous determination of imatinib, voriconazole and their metabolites concentrations in rat plasma, J. Pharm. Biomed. Anal. 166 (2019) 6–12. [12] Center for Drug Evaluation and Research of the U.S. Department of Health and Human Services Food and Drug Administration, Guidance for Industry; Bioanalytical Method Validation, 2018, Accessed: August 2, 2018 http://www. fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ ucm064964.htm.