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Determination of cinacalcet hydrochloride in human plasma by liquid chromatography–tandem mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 237–241
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Determination of cinacalcet hydrochloride in human plasma by liquid chromatography–tandem mass spectrometry Fen Yang 1 , Hongyun Wang 1 , Qian Zhao, Hongzhong Liu, Pei Hu, Ji Jiang ∗ Clinical Pharmacology Research Center, Peking Union Medical College Hospital and Chinese Academy of Medical Sciences, Beijing 100730, China
a r t i c l e
i n f o
Article history: Received 20 May 2011 Received in revised form 18 October 2011 Accepted 19 October 2011 Available online 25 October 2011 Keywords: Cinacalcet hydrochloride Calcimimetic agent SPE LC–MS/MS MRM
a b s t r a c t A sensitive and selective high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed to determine the cinacalcet hydrochloride in human plasma. The analyte was extracted from plasma samples using a 96-well plate automatic solid-phase extraction (SPE) device and chromatographed on an Inertsil SIL-150 (2.1 mm × 50 mm, i.d. 5 m) column using acetonitrile–water–formic acid (90:10:1) as the mobile phase with an isocratic flow rate of 0.35 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.1–25 ng/mL. The indicators of inter- and intra-day precision (RSD%) were all within 15.1%, and the accuracy (RE%) was within ±15%. The lower limit of quantitation (LLOQ) was 0.1 ng/mL. The average extraction recovery was 51.7%, and the detection was not affected by the matrix. The method was successfully applied to a pharmacokinetic study of cinacalcet hydrochloride in healthy Chinese volunteers. © 2011 Published by Elsevier B.V.
1. Introduction Patients with chronic kidney disease (CKD) often exhibit a combination of increased serum parathyroid hormone (PTH) levels, disordered calcium and phosphorus, and parathyroid hyperplasia commonly referred to as secondary hyperparathyroidism (SHPT) [1]. SHPT develops early and worsens as kidney function decreases [2]. HPT-associated disturbances in PTH and mineral metabolism in patients receiving dialysis contribute to an increased risk of mortality as well as vascular calcification and skeletal abnormalities collectively known as CKD-mineral and bone disorder. Traditional treatment options, including dietary phosphorus restriction, phosphorus-binding agents, calcium supplementation and 1,25-dihydroxy vitamin-D sterols, have proven inadequate in successfully controlling all aspects of SHPT [3]. Cinacalcet hydrochloride (hereinafter referred to as cinacalcet or KRN1493) is a calcimimetic agent that activates the calciumsensing receptor on the surface of parathyroid cells and that inhibits parathyroid hormone (PTH) secretion [4]. It was approved for the treatment of SHPT in patients with chronic kidney disease
(CKD) receiving dialysis and for the treatment of hypercalcaemia in patients with parathyroid carcinoma [5]. To our knowledge, there have been several reports [6–9] concerning the determination of KRN1493 in human plasma samples using the LC–MS/MS method. In these methods, KRN1493 was extracted using solid phase extraction (SPE) techniques [6,8] and 500 L of plasma sample was required [7]. A lower limit of quantitation of 0.1 ng/mL was used in all the above methods, which was sufficient for pharmacokinetic studies of cinacalcet hydrochloride. The present study describes an assay for the determination of KRN1493 using LC–MS/MS in MRM mode. This method requires 250 L of plasma sample, and the analyte was extracted using a 96-well plate automatic solid-phase extraction (SPE) device. The running time for each sample was only 2.2 min. In brief, the present method has advantages over the old methods that include the small plasma volume required, high-throughput sample preparation and a short running time. Furthermore, this method was validated according to FDA guidelines [10], and was successfully applied to pharmacokinetic studies of KRN1493 in healthy Chinese volunteers. 2. Materials and methods
∗ Corresponding author. Tel.: +86 10 8806 8357; fax: +86 10 8806 8864. E-mail address: [email protected] (J. Jiang). 1 These authors contributed equally to this work and should be considered as co-first authors. 0731-7085/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.jpba.2011.10.022
2.1. Chemical reagents KRN1493 (Fig. 1A) was supplied by Kirin Kunpeng (China) Biopharmaceutical Co., Ltd. Fendiline (internal standard, (IS), Fig. 1B)
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0.2 (LQC), 2 (MQC), 20 (HQC), 25 (ULOQ) and 1000 ng/mL. All stock solutions and working solutions were stored at 4 ◦ C, and all quality control samples were stored at −30 ◦ C. Calibration standards were freshly prepared for each experiment. 2.5. Samples preparation
Fig. 1. Chemical structure of KRN1493 (A, MW 357) and internal standard fendiline (B, MW 315).
was purchased from Sigma–Aldrich (USA). Methanol and acetonitrile (HPLC grade) were purchased from Honeywell Burdick & Jackson (Muskegon, ML, USA). Formic acid and aqueous ammonia (both of analytical grades) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). Drug-free human plasma was supplied by the Peking Union Medical College Hospital blood bank. 2.2. HPLC The chromatographic separation was carried out on a Waters Alliance 2690 liquid chromatography system (Waters, USA). An Inertsil SIL-150 column (2.1 mm × 50 mm, i.d. 5 m, GL Sciences, Japan) and a guard column, an Inertsil SIL-150 (3.0 mm × 50 mm, i.d. 5 m, GL Sciences, Japan) were used for all the separations. The mobile phase consisted of acetonitrile–water–formic acid (90:10:1, v/v/v). The samples were delivered at a flow rate of 0.35 mL/min, and the injection volume was 10 L. 2.3. MS A mass spectrometric analysis was performed using an API 3000 triple quadruple mass spectrometer from Applied Biosystems Sciex (Toronto, Canada) equipped with an electro-spray ionization (ESI) source operating in positive mode. Detection was achieved in multiple reaction monitoring (MRM) mode with a dwell time of 200 ms for each transition. The MRM transitions of KRN1493 and the IS were 358.1 → 155.1 and 316.0 → 212.1, respectively. The mass spectrometric conditions were optimized as follows: source temperature, 300 ◦ C; curtain gas, 6 units; nebulizer gas, 5 units; auxiliary gas (7000 units); collision gas, 8 units; ion spray voltage, 5000 V; declustering potential, 38 V for KRN1493 and 66 V for the IS; collision energy, 35 eV for KRN1493 and 30 eV for the IS. Ultrahighpurity nitrogen gas was used as the collision gas. Data acquisition and processing were performed using Analyst software (version 1.4). 2.4. Preparation of standard solutions, calibration standards and quality controls Stock solutions of KRN1493 were prepared in duplicate in methanol at a concentration of 1 mg/mL, one for calibration curve samples and the other for quality control (QC) samples. The stock solution of internal standard (IS) was prepared as above to obtain a concentration of 1 mg/mL. Fresh working solutions were prepared by diluting the appropriate stock solution to a concentration of 10 g/mL for KRN1493 and 100 ng/mL for the IS. Calibration standards were prepared by diluting the corresponding working solutions with drug-free human plasma. The final concentrations of KRN1493 in plasma were 0.1, 0.25, 0.5, 1, 2, 5, 10 and 25 ng/mL. Six pools of quality control (QC) samples were prepared by spiking blank plasma with KRN1493 at concentrations of 0.1 (LLOQ),
In all, 250 L of plasma was spiked with 25 L of IS (100 ng/mL) and 500 L of 0.3 M aqueous ammonia. The mixture was vortexed for 30 s, centrifuged for 5 min (at 13,000 rpm) and applied to a Bond Elut C18 96-well plate (100 mg/1 mL) (Varian, Palo Alto, California). The plate was then washed with 500 L of 5 mM aqueous ammonia and 500 L of 5 mM aqueous ammonia/methanol (8:2, v/v) and finally eluted with 1 mL of acetonitrile. The elute was evaporated to dryness under a stream of nitrogen at room temperature. The residues were dissolved in 200 L of acetonitrile and vortexed for 1 min. Finally, 10 L of the sample was injected for a LC–MS/MS analysis. 2.6. Method validation Validation of this method was performed in compliance with the FDA guidelines for biological method validation [6], including validating the selectivity, calibration curve performance, accuracy and precision, LLOQ, stability of the analytes at various test conditions, recovery and the matrix effect. Selectivity was assessed by comparing the chromatograms of drug-free human plasma from 9 different lots with the corresponding spiked plasma to test for endogenous interferences. The calibration curves were constructed using 8 non-zero standards ranging from 0.1 to 25 ng/mL. The linearity of the relationship between the peak area ratio and the concentration was demonstrated by the correlation coefficient (R) obtained for the linear regression. The relative standard deviations were calculated for all calibration curve slopes. The intra- and inter-day assays of the method were evaluated by assessing 6 replicate QC samples at three concentration levels including LLOQ, MQC and ULOQ. The relative standard deviation (RSD%) and relative error (RE%) were calculated. The stability of the KRN1493 stock solution and the working solution was calculated by comparing the average peak ratio of KRN1493 to the IS for solutions stored at 4 ◦ C for 3 months with those of freshly prepared solutions at the same concentrations. The post-extraction stability was investigated by comparing the concentration of the extracted QC samples after being kept in the autosampler for 24 h with the initial concentrations. The freeze–thaw stability was determined by comparing the values for QC samples subjected to 3 freeze/thaw cycles (from −30 ◦ C to 25 ◦ C) with those of freshly prepared QC samples. Long-term storage stability was investigated by assessing QC samples stored at −30 ◦ C for 112 days, including the LQC, HQC and 1000 ng/mL QC samples that were diluted to 10 ng/mL with drug-free plasma before extraction. The extraction recovery of KRN1493 was expressed as the ratio of A (the average area ratio of KRN1493 to the IS from the extracted samples) to B (the average area ratio of KRN1493 to the IS from the samples spiked with analyte post-extraction) at the same concentration. To determine any matrix effects in the method, blank plasma samples from 6 different lots were used to prepare the QC samples at 3 concentrations, and the matrix effect was evaluated using the average peak areas in spiked samples post-extraction to those of the corresponding working solutions at same concentrations (n = 6). 2.7. Data treatment Data acquisition was performed using Analyst software (version 1.4). The descriptive statistics (mean, SD, RSD% and RE%) were
F. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 237–241
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Fig. 2. Representative MRM chromatograms of KRN 1493 and IS from (A) a blank plasma sample without any analyte and internal standard added; (B) a real sample collected at 120 h after oral administration of 120 mg cinacalcet hydrochloride (0.26 ng/mL); (C) the LLOQ sample (0.1 ng/mL).
calculated using Watson LIMS (version 7.3). The calibration curve was created by plotting the peak area ratio of the analyte to the internal standard versus the plasma concentration. A weighted 1/y2 linear regression of the type y = ax + b was used, where x represented the concentration and y represented the peak area ratio. The concentrations of the QC samples and the unknown samples were calculated by interpolation using the calibration curve.
3. Results and discussion 3.1. Chromatographic analysis Pharmacokinetic studies involve the analysis of a large number of biological samples, so a high-throughput method is needed to perform this type of analysis. The extraction method described in this paper, using an automatic SPE device, is efficient, less laborious, rapid and reproducible. The analytes were extracted more efficiently under basic conditions. Though the extraction recovery was not particularly high, the lower limit of quantitation of the method was satisfactory for the clinical study. To avoid interference from other compounds (exogenous or endogenous) that may co-elute with the analytes, the instrument’s MRM scan mode was selected to ensure high specificity. The ionization of the analytes was achieved using an ESI ion source operated in positive mode. The protonated ion of each analyte was selected as the precursor ion, and the most abundant or specific fragment ion was chosen as the product ion in the MRM acquisition. To obtain the highest and most stable signal response for the MRM transition, the mass spectrometric parameters were optimized, including the ion spray voltage, source temperature, nebulizer gas, collision gas and collision energy. Furthermore, good separation from interference of matrix was obtained by optimizing the mobile phase system, including the choice and the ratios of the solvents.
3.2. Method validation 3.2.1. Selectivity Representative MRM chromatograms (Fig. 2A) of blank plasma from 9 different lots, plasma sample spiked with drugs and a real human plasma sample (Fig. 2B) were obtained under the selected analytical conditions. The results demonstrate that no significant interference from endogenous substances was found at the retention times of the analyte and the IS. 3.2.2. Sensitivity The lower limit of quantitation (LLOQ) was defined as the concentration of the lowest non-zero calibration standard, which met the acceptance criteria for accuracy and precision of 17% (RSD%) and 1.8% (RE%). The LLOQ was set at 0.1 ng/mL, and a typical chromatogram of a plasma sample containing 0.1 ng/mL KRN1493 is shown in Fig. 2C. 3.2.3. Linearity, precision and accuracy The calibration curves were validated over the concentration range of 0.1–25 ng/mL in human plasma. The correlation coefficients for all of the curves were greater than 0.99, and the deviations of the concentrations back-calculated from their nominal values were within ±15%. The intra- and inter-day precision and accuracy of the method were determined from the analysis of 6 QC samples at three different concentrations, and the results are summarized in Table 1. The method was reliable and reproducible, evidenced by an RSD% below 20% for LLOQ and below 15% for MQC and ULOQ. 3.2.4. Stability The KRN1493 stock solutions and working solutions were stable at 4 ◦ C for 3 months. The stability of the analyte in human plasma was investigated under a variety of storage and processing conditions, and it was found to be stable under the following conditions: at 10 ◦ C for 24 h post-extraction; after 3 cycles freeze/thaw (from
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Table 1 Intra- and inter-day accuracy and precision of quality control samples. Inter-day (ng/mL)
Mean SD Precision (RSD%) Accuracy (RE%) n
Intra-day (ng/mL)
LLOQ (0.1000)
MQC (2.000)
ULOQ 25.00)
LLOQ 0.1000)
MQC (2.000)
ULOQ (25.00)
0.0876 0.01 14.7 −12.4 6
1.966 0.12 6.4 −1.7 6
23.35 0.92 3.9 −6.6 6
0.0946 0.01 15.1 −5.4 18
1.932 0.111 5.5 −3.4 18
24.90 1.57 6.3 −0.4 18
Table 2 Stability results of QC samples at different conditions (n = 3). Stability tests
Initial conc. ± SD (ng/mL)
Found conc. ± SD (ng/mL)
Precision (RSD%)
Accuracy (RE%)
Stock solution (4 ◦ C for 3 months) Work solution (4 ◦ C for 3 months) Post-extraction (10 ◦ C for 24 h)
2.78a 0.34b 0.2148 ± 0.01 20.09 ± 0.19 0.1964 ± 0.02 20.15 ± 0.75 0.1995 ± 0.01 20.47 ± 0.95 1026 ± 28
2.83a 0.35b 0.1948 ± 0.02 17.80 ± 0.63 0.1775 ± 0.01 19.09 ± 0.16 0.2009 ± 0.02 22.15 ± 0.23 1113 ± 25
2.8 1.6 9.3 3.5 5.0 0.8 9.1 1.0 2.3
2.0 −2.6 −9.3 −11.4 −9.6 −5.3 0.7 8.2 8.4
Freeze thaw (from −30 ◦ C to 25 ◦ C, 3 cycles) Long term (−30 ◦ C for 112 days) a b
Peak area ratio of KRN1493 to IS (25 ng/mL of KRN1493). Peak area ratio of KRN1493 to IS (0.1 ng/mL of KRN1493).
−30 ◦ C to 25 ◦ C); and at −30 ◦ C for 112 days. The stability results are summarized in Table 2. 3.2.5. Recovery and matrix effects An automatic SPE method was used in the extraction of the analyte and the IS. The extraction recoveries of KRN1493 were 46.5%, 65.7% and 56.8% for the low, medium and high concentration QC samples (n = 5), and the extraction recovery of the IS was 55.6%. No apparent matrix effect was found in the determination of KRN1493. The values were 103.2%, 108.3% and 106.7% for the 3 QC samples (LQC, MQC and HQC). 3.2.6. Carryover test The MRM chromatogram of a blank sample (with added IS and without KRN1493) analyzed following five consecutive analyses of ULOQ samples showed no apparent carryover using the present method. 3.3. Application of the method in a pharmacokinetic study This HPLC/MS/MS method was used to evaluate a pharmacokinetic study of KRN1493 in human plasma. This was a phase I, single center, open-label study with 3 parallel groups and a single period used to assess the pharmacokinetics of KRN1493 in healthy Chinese subjects. Thirty healthy Chinese volunteers were enrolled and randomized into 3 groups (n = 10). The subjects in each group received a single oral dose of 25, 50 or 100 mg cinacalcet hydrochloride. Plasma samples were collected pre-dose and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, 72, 96 and 120 h post-dose and assayed by the validated method. The mean plasma concentration–time curves of KRN1493 are shown in Fig. 3. The pharmacokinetic parameters of KRN1493 in healthy Chinese subjects after a single oral administration of 50 mg of cinacalcet hydrochloride are compared with those acquired in western subjects [9] at the same dose level in Table 3. The Tmax of KRN1493 was approximately 4.5–5 h in both studies; however, faster elimination was observed in Chinese subjects. The mean AUC0→t , AUC0→∞ and Cmax in Chinese subjects were higher than those in western subjects, which may be explained by the differences in body weight between the Chinese and western subjects.
Fig. 3. Mean plasma concentration–time curves of KRN1493 after single oral administration of 25 mg, 50 mg and 100 mg cinacalcet hydrochloride (n = 10). Table 3 Comparison of pharmacokinetic parameters of KRN1493 between Chinese and western volunteers at 50 mg dose of cinacalcet hydrochloride (mean ± SD).
Number (n) Race Weight (kg) Age (y) AUC0→t (ng h mL−1 ) AUC0→∞ (ng h mL−1 ) Cmax (ng/mL) Tmax (h) t1/2 (h)
Chinese volunteers
Western volunteers [9]
10 Chinese 83.3 ± 8.21 50.8 ± 2.64 187 ± 70.7 205 ± 83.05 17 ± 6.33 4.55 ± 1.62 37.1 ± 14.67
6 White and black 61.4 ± 9.75 22.6 ± 1.9 156 ± 105 181 ± 131 11.9 ± 6.62 5 ± 3.8 49.2 ± 35.4
AUC0→t , area under the concentration–time curve (AUC) from 0 to time t; AUC0→∞ , AUC from 0 to infinity; Cmax , maximum plasma concentration; Tmax , time to Cmax ; t1/2 , terminal half-life.
4. Conclusions A rapid, sensitive and selective HPLC–MS-MS method has been developed and validated for the determination of KRN1493 in human plasma. The method, using an automatic SPE method for the extraction of KRN1493 and the IS from plasma, is sensitive, specific
F. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 237–241
and accurate. The present method was successfully applied in pharmacokinetic studies of KRN1493 in healthy Chinese volunteers.
[5]
Acknowledgment The authors thank Kirin Kunpeng (China) Biopharmaceutical Co. Ltd. for supplying the cinacalcet hydrochloride.
[6]
References [7] [1] M. Rodriguez, E. Nemeth, D. Martin, The calcium-sensing receptor: a key factor in the pathogenesis of secondary hyperparathyroidism, Am. J. Physiol. Renal. Physiol. 288 (2005) F253–F264. [2] I. Martinez, R. Saracho, J. Montenegro, F. Llach, The importance of dietary calcium and phosphorous in the secondary hyperparathyroidism of patients with early renal failure, Am. J. Kidney Dis. 29 (1997) 496–502. [3] E.W. Young, J.M. Albert, S. Satayathum, D.A. Goodkin, R.L. Pisoni, T. Akiba, T. Akizawa, K. Kurokawa, J. Bommer, L. Piera, F.K. Port, Predictors and consequences of altered mineral metabolism: the Dialysis Outcomes and Practice Patterns Study, Kidney Int. 67 (2005) 1179–1187. [4] M. Fukagawa, S. Yumita, T. Akizawa, E. Uchida, Y. Tsukamoto, M. Iwasaki, S. Koshikawa, KRN1493 study group, cinacalcet (KRN1493) effectively decreases the serum intact PTH level with favorable control of the serum phosphorus
[8]
[9]
[10]
241
and calcium levels in Japanese dialysis patients, Nephrol. Dial. Transplant. 23 (2008) 328–335. ˜ Torres, D. Fouque, S.H. Jacobson, F. Pétavy, B. Dehmel, E. Zitt, M. Rix, P. Urena M. Ryba, Effectiveness of cinacalcet in patients with recurrent/persistent secondary hyperparathyroidism following parathyroidectomy: results of the ECHO study, Nephrol. Dial. Transplant. 26 (2011) 1956–1961. N. Ohashi, T. Uematsu, S. Nagashima, M. Kanamaru, A. Togawa, A. Hishida, E. Uchida, T. Akizawa, S. Koshikawa, The calcimimetic agent KRN 1493 lowers plasma parathyroid hormone and ionized calcium concentrations in patients with chronic renal failure on haemodialysis both on the day of haemodialysis and on the day without haemodialysis, Br. J. Clin. Pharmacol. 57 (2004) 726–734. D. Padhi, R.Z. Harris, M. Salfi, J.T. Sullivan, No effect of renal function or dialysis on pharmacokinetics of cinacalcet (Sensipar/Mimpara), Clin. Pharmacokinet. 44 (2005) 509–516. D. Nakashima, H. Takama, Y. Ogasawara, T. Kawakami, T. Nishitoba, S. Hoshi, E. Uchida, H. Tanaka, Effect of cinacalcet hydrochloride, a new calcimimetic agent, on the pharmacokinetics of dextromethorphan: in vitro and clinical studies, J. Clin. Pharmacol. 47 (2007) 1311–1319. D. Padhi, R.Z. Harris, M. Salfi, R.J. Noveck, J.T. Sullivan, Pharmacokinetics and pharmacodynamics of cinacalcet in hepatic impairment: phase I, open-label, parallel-group, single-dose, single-centre study, Clin. Drug Investig. 28 (2008) 635–643. Food and Drug Administration (FDA), Guidance for Industry in Bioanalytical Method Validation, Rockville, MD, USA, 2001.
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Determination of cinacalcet hydrochloride in human plasma by liquid chromatography–tandem mass spectrometry Fen Yang 1 , Hongyun Wang 1 , Qian Zhao, Hongzhong Liu, Pei Hu, Ji Jiang ∗ Clinical Pharmacology Research Center, Peking Union Medical College Hospital and Chinese Academy of Medical Sciences, Beijing 100730, China
a r t i c l e
i n f o
Article history: Received 20 May 2011 Received in revised form 18 October 2011 Accepted 19 October 2011 Available online 25 October 2011 Keywords: Cinacalcet hydrochloride Calcimimetic agent SPE LC–MS/MS MRM
a b s t r a c t A sensitive and selective high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed to determine the cinacalcet hydrochloride in human plasma. The analyte was extracted from plasma samples using a 96-well plate automatic solid-phase extraction (SPE) device and chromatographed on an Inertsil SIL-150 (2.1 mm × 50 mm, i.d. 5 m) column using acetonitrile–water–formic acid (90:10:1) as the mobile phase with an isocratic flow rate of 0.35 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.1–25 ng/mL. The indicators of inter- and intra-day precision (RSD%) were all within 15.1%, and the accuracy (RE%) was within ±15%. The lower limit of quantitation (LLOQ) was 0.1 ng/mL. The average extraction recovery was 51.7%, and the detection was not affected by the matrix. The method was successfully applied to a pharmacokinetic study of cinacalcet hydrochloride in healthy Chinese volunteers. © 2011 Published by Elsevier B.V.
1. Introduction Patients with chronic kidney disease (CKD) often exhibit a combination of increased serum parathyroid hormone (PTH) levels, disordered calcium and phosphorus, and parathyroid hyperplasia commonly referred to as secondary hyperparathyroidism (SHPT) [1]. SHPT develops early and worsens as kidney function decreases [2]. HPT-associated disturbances in PTH and mineral metabolism in patients receiving dialysis contribute to an increased risk of mortality as well as vascular calcification and skeletal abnormalities collectively known as CKD-mineral and bone disorder. Traditional treatment options, including dietary phosphorus restriction, phosphorus-binding agents, calcium supplementation and 1,25-dihydroxy vitamin-D sterols, have proven inadequate in successfully controlling all aspects of SHPT [3]. Cinacalcet hydrochloride (hereinafter referred to as cinacalcet or KRN1493) is a calcimimetic agent that activates the calciumsensing receptor on the surface of parathyroid cells and that inhibits parathyroid hormone (PTH) secretion [4]. It was approved for the treatment of SHPT in patients with chronic kidney disease
(CKD) receiving dialysis and for the treatment of hypercalcaemia in patients with parathyroid carcinoma [5]. To our knowledge, there have been several reports [6–9] concerning the determination of KRN1493 in human plasma samples using the LC–MS/MS method. In these methods, KRN1493 was extracted using solid phase extraction (SPE) techniques [6,8] and 500 L of plasma sample was required [7]. A lower limit of quantitation of 0.1 ng/mL was used in all the above methods, which was sufficient for pharmacokinetic studies of cinacalcet hydrochloride. The present study describes an assay for the determination of KRN1493 using LC–MS/MS in MRM mode. This method requires 250 L of plasma sample, and the analyte was extracted using a 96-well plate automatic solid-phase extraction (SPE) device. The running time for each sample was only 2.2 min. In brief, the present method has advantages over the old methods that include the small plasma volume required, high-throughput sample preparation and a short running time. Furthermore, this method was validated according to FDA guidelines [10], and was successfully applied to pharmacokinetic studies of KRN1493 in healthy Chinese volunteers. 2. Materials and methods
∗ Corresponding author. Tel.: +86 10 8806 8357; fax: +86 10 8806 8864. E-mail address: [email protected] (J. Jiang). 1 These authors contributed equally to this work and should be considered as co-first authors. 0731-7085/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.jpba.2011.10.022
2.1. Chemical reagents KRN1493 (Fig. 1A) was supplied by Kirin Kunpeng (China) Biopharmaceutical Co., Ltd. Fendiline (internal standard, (IS), Fig. 1B)
238
F. Yang et al. / Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 237–241
0.2 (LQC), 2 (MQC), 20 (HQC), 25 (ULOQ) and 1000 ng/mL. All stock solutions and working solutions were stored at 4 ◦ C, and all quality control samples were stored at −30 ◦ C. Calibration standards were freshly prepared for each experiment. 2.5. Samples preparation
Fig. 1. Chemical structure of KRN1493 (A, MW 357) and internal standard fendiline (B, MW 315).
was purchased from Sigma–Aldrich (USA). Methanol and acetonitrile (HPLC grade) were purchased from Honeywell Burdick & Jackson (Muskegon, ML, USA). Formic acid and aqueous ammonia (both of analytical grades) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). Drug-free human plasma was supplied by the Peking Union Medical College Hospital blood bank. 2.2. HPLC The chromatographic separation was carried out on a Waters Alliance 2690 liquid chromatography system (Waters, USA). An Inertsil SIL-150 column (2.1 mm × 50 mm, i.d. 5 m, GL Sciences, Japan) and a guard column, an Inertsil SIL-150 (3.0 mm × 50 mm, i.d. 5 m, GL Sciences, Japan) were used for all the separations. The mobile phase consisted of acetonitrile–water–formic acid (90:10:1, v/v/v). The samples were delivered at a flow rate of 0.35 mL/min, and the injection volume was 10 L. 2.3. MS A mass spectrometric analysis was performed using an API 3000 triple quadruple mass spectrometer from Applied Biosystems Sciex (Toronto, Canada) equipped with an electro-spray ionization (ESI) source operating in positive mode. Detection was achieved in multiple reaction monitoring (MRM) mode with a dwell time of 200 ms for each transition. The MRM transitions of KRN1493 and the IS were 358.1 → 155.1 and 316.0 → 212.1, respectively. The mass spectrometric conditions were optimized as follows: source temperature, 300 ◦ C; curtain gas, 6 units; nebulizer gas, 5 units; auxiliary gas (7000 units); collision gas, 8 units; ion spray voltage, 5000 V; declustering potential, 38 V for KRN1493 and 66 V for the IS; collision energy, 35 eV for KRN1493 and 30 eV for the IS. Ultrahighpurity nitrogen gas was used as the collision gas. Data acquisition and processing were performed using Analyst software (version 1.4). 2.4. Preparation of standard solutions, calibration standards and quality controls Stock solutions of KRN1493 were prepared in duplicate in methanol at a concentration of 1 mg/mL, one for calibration curve samples and the other for quality control (QC) samples. The stock solution of internal standard (IS) was prepared as above to obtain a concentration of 1 mg/mL. Fresh working solutions were prepared by diluting the appropriate stock solution to a concentration of 10 g/mL for KRN1493 and 100 ng/mL for the IS. Calibration standards were prepared by diluting the corresponding working solutions with drug-free human plasma. The final concentrations of KRN1493 in plasma were 0.1, 0.25, 0.5, 1, 2, 5, 10 and 25 ng/mL. Six pools of quality control (QC) samples were prepared by spiking blank plasma with KRN1493 at concentrations of 0.1 (LLOQ),
In all, 250 L of plasma was spiked with 25 L of IS (100 ng/mL) and 500 L of 0.3 M aqueous ammonia. The mixture was vortexed for 30 s, centrifuged for 5 min (at 13,000 rpm) and applied to a Bond Elut C18 96-well plate (100 mg/1 mL) (Varian, Palo Alto, California). The plate was then washed with 500 L of 5 mM aqueous ammonia and 500 L of 5 mM aqueous ammonia/methanol (8:2, v/v) and finally eluted with 1 mL of acetonitrile. The elute was evaporated to dryness under a stream of nitrogen at room temperature. The residues were dissolved in 200 L of acetonitrile and vortexed for 1 min. Finally, 10 L of the sample was injected for a LC–MS/MS analysis. 2.6. Method validation Validation of this method was performed in compliance with the FDA guidelines for biological method validation [6], including validating the selectivity, calibration curve performance, accuracy and precision, LLOQ, stability of the analytes at various test conditions, recovery and the matrix effect. Selectivity was assessed by comparing the chromatograms of drug-free human plasma from 9 different lots with the corresponding spiked plasma to test for endogenous interferences. The calibration curves were constructed using 8 non-zero standards ranging from 0.1 to 25 ng/mL. The linearity of the relationship between the peak area ratio and the concentration was demonstrated by the correlation coefficient (R) obtained for the linear regression. The relative standard deviations were calculated for all calibration curve slopes. The intra- and inter-day assays of the method were evaluated by assessing 6 replicate QC samples at three concentration levels including LLOQ, MQC and ULOQ. The relative standard deviation (RSD%) and relative error (RE%) were calculated. The stability of the KRN1493 stock solution and the working solution was calculated by comparing the average peak ratio of KRN1493 to the IS for solutions stored at 4 ◦ C for 3 months with those of freshly prepared solutions at the same concentrations. The post-extraction stability was investigated by comparing the concentration of the extracted QC samples after being kept in the autosampler for 24 h with the initial concentrations. The freeze–thaw stability was determined by comparing the values for QC samples subjected to 3 freeze/thaw cycles (from −30 ◦ C to 25 ◦ C) with those of freshly prepared QC samples. Long-term storage stability was investigated by assessing QC samples stored at −30 ◦ C for 112 days, including the LQC, HQC and 1000 ng/mL QC samples that were diluted to 10 ng/mL with drug-free plasma before extraction. The extraction recovery of KRN1493 was expressed as the ratio of A (the average area ratio of KRN1493 to the IS from the extracted samples) to B (the average area ratio of KRN1493 to the IS from the samples spiked with analyte post-extraction) at the same concentration. To determine any matrix effects in the method, blank plasma samples from 6 different lots were used to prepare the QC samples at 3 concentrations, and the matrix effect was evaluated using the average peak areas in spiked samples post-extraction to those of the corresponding working solutions at same concentrations (n = 6). 2.7. Data treatment Data acquisition was performed using Analyst software (version 1.4). The descriptive statistics (mean, SD, RSD% and RE%) were
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Fig. 2. Representative MRM chromatograms of KRN 1493 and IS from (A) a blank plasma sample without any analyte and internal standard added; (B) a real sample collected at 120 h after oral administration of 120 mg cinacalcet hydrochloride (0.26 ng/mL); (C) the LLOQ sample (0.1 ng/mL).
calculated using Watson LIMS (version 7.3). The calibration curve was created by plotting the peak area ratio of the analyte to the internal standard versus the plasma concentration. A weighted 1/y2 linear regression of the type y = ax + b was used, where x represented the concentration and y represented the peak area ratio. The concentrations of the QC samples and the unknown samples were calculated by interpolation using the calibration curve.
3. Results and discussion 3.1. Chromatographic analysis Pharmacokinetic studies involve the analysis of a large number of biological samples, so a high-throughput method is needed to perform this type of analysis. The extraction method described in this paper, using an automatic SPE device, is efficient, less laborious, rapid and reproducible. The analytes were extracted more efficiently under basic conditions. Though the extraction recovery was not particularly high, the lower limit of quantitation of the method was satisfactory for the clinical study. To avoid interference from other compounds (exogenous or endogenous) that may co-elute with the analytes, the instrument’s MRM scan mode was selected to ensure high specificity. The ionization of the analytes was achieved using an ESI ion source operated in positive mode. The protonated ion of each analyte was selected as the precursor ion, and the most abundant or specific fragment ion was chosen as the product ion in the MRM acquisition. To obtain the highest and most stable signal response for the MRM transition, the mass spectrometric parameters were optimized, including the ion spray voltage, source temperature, nebulizer gas, collision gas and collision energy. Furthermore, good separation from interference of matrix was obtained by optimizing the mobile phase system, including the choice and the ratios of the solvents.
3.2. Method validation 3.2.1. Selectivity Representative MRM chromatograms (Fig. 2A) of blank plasma from 9 different lots, plasma sample spiked with drugs and a real human plasma sample (Fig. 2B) were obtained under the selected analytical conditions. The results demonstrate that no significant interference from endogenous substances was found at the retention times of the analyte and the IS. 3.2.2. Sensitivity The lower limit of quantitation (LLOQ) was defined as the concentration of the lowest non-zero calibration standard, which met the acceptance criteria for accuracy and precision of 17% (RSD%) and 1.8% (RE%). The LLOQ was set at 0.1 ng/mL, and a typical chromatogram of a plasma sample containing 0.1 ng/mL KRN1493 is shown in Fig. 2C. 3.2.3. Linearity, precision and accuracy The calibration curves were validated over the concentration range of 0.1–25 ng/mL in human plasma. The correlation coefficients for all of the curves were greater than 0.99, and the deviations of the concentrations back-calculated from their nominal values were within ±15%. The intra- and inter-day precision and accuracy of the method were determined from the analysis of 6 QC samples at three different concentrations, and the results are summarized in Table 1. The method was reliable and reproducible, evidenced by an RSD% below 20% for LLOQ and below 15% for MQC and ULOQ. 3.2.4. Stability The KRN1493 stock solutions and working solutions were stable at 4 ◦ C for 3 months. The stability of the analyte in human plasma was investigated under a variety of storage and processing conditions, and it was found to be stable under the following conditions: at 10 ◦ C for 24 h post-extraction; after 3 cycles freeze/thaw (from
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Table 1 Intra- and inter-day accuracy and precision of quality control samples. Inter-day (ng/mL)
Mean SD Precision (RSD%) Accuracy (RE%) n
Intra-day (ng/mL)
LLOQ (0.1000)
MQC (2.000)
ULOQ 25.00)
LLOQ 0.1000)
MQC (2.000)
ULOQ (25.00)
0.0876 0.01 14.7 −12.4 6
1.966 0.12 6.4 −1.7 6
23.35 0.92 3.9 −6.6 6
0.0946 0.01 15.1 −5.4 18
1.932 0.111 5.5 −3.4 18
24.90 1.57 6.3 −0.4 18
Table 2 Stability results of QC samples at different conditions (n = 3). Stability tests
Initial conc. ± SD (ng/mL)
Found conc. ± SD (ng/mL)
Precision (RSD%)
Accuracy (RE%)
Stock solution (4 ◦ C for 3 months) Work solution (4 ◦ C for 3 months) Post-extraction (10 ◦ C for 24 h)
2.78a 0.34b 0.2148 ± 0.01 20.09 ± 0.19 0.1964 ± 0.02 20.15 ± 0.75 0.1995 ± 0.01 20.47 ± 0.95 1026 ± 28
2.83a 0.35b 0.1948 ± 0.02 17.80 ± 0.63 0.1775 ± 0.01 19.09 ± 0.16 0.2009 ± 0.02 22.15 ± 0.23 1113 ± 25
2.8 1.6 9.3 3.5 5.0 0.8 9.1 1.0 2.3
2.0 −2.6 −9.3 −11.4 −9.6 −5.3 0.7 8.2 8.4
Freeze thaw (from −30 ◦ C to 25 ◦ C, 3 cycles) Long term (−30 ◦ C for 112 days) a b
Peak area ratio of KRN1493 to IS (25 ng/mL of KRN1493). Peak area ratio of KRN1493 to IS (0.1 ng/mL of KRN1493).
−30 ◦ C to 25 ◦ C); and at −30 ◦ C for 112 days. The stability results are summarized in Table 2. 3.2.5. Recovery and matrix effects An automatic SPE method was used in the extraction of the analyte and the IS. The extraction recoveries of KRN1493 were 46.5%, 65.7% and 56.8% for the low, medium and high concentration QC samples (n = 5), and the extraction recovery of the IS was 55.6%. No apparent matrix effect was found in the determination of KRN1493. The values were 103.2%, 108.3% and 106.7% for the 3 QC samples (LQC, MQC and HQC). 3.2.6. Carryover test The MRM chromatogram of a blank sample (with added IS and without KRN1493) analyzed following five consecutive analyses of ULOQ samples showed no apparent carryover using the present method. 3.3. Application of the method in a pharmacokinetic study This HPLC/MS/MS method was used to evaluate a pharmacokinetic study of KRN1493 in human plasma. This was a phase I, single center, open-label study with 3 parallel groups and a single period used to assess the pharmacokinetics of KRN1493 in healthy Chinese subjects. Thirty healthy Chinese volunteers were enrolled and randomized into 3 groups (n = 10). The subjects in each group received a single oral dose of 25, 50 or 100 mg cinacalcet hydrochloride. Plasma samples were collected pre-dose and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, 72, 96 and 120 h post-dose and assayed by the validated method. The mean plasma concentration–time curves of KRN1493 are shown in Fig. 3. The pharmacokinetic parameters of KRN1493 in healthy Chinese subjects after a single oral administration of 50 mg of cinacalcet hydrochloride are compared with those acquired in western subjects [9] at the same dose level in Table 3. The Tmax of KRN1493 was approximately 4.5–5 h in both studies; however, faster elimination was observed in Chinese subjects. The mean AUC0→t , AUC0→∞ and Cmax in Chinese subjects were higher than those in western subjects, which may be explained by the differences in body weight between the Chinese and western subjects.
Fig. 3. Mean plasma concentration–time curves of KRN1493 after single oral administration of 25 mg, 50 mg and 100 mg cinacalcet hydrochloride (n = 10). Table 3 Comparison of pharmacokinetic parameters of KRN1493 between Chinese and western volunteers at 50 mg dose of cinacalcet hydrochloride (mean ± SD).
Number (n) Race Weight (kg) Age (y) AUC0→t (ng h mL−1 ) AUC0→∞ (ng h mL−1 ) Cmax (ng/mL) Tmax (h) t1/2 (h)
Chinese volunteers
Western volunteers [9]
10 Chinese 83.3 ± 8.21 50.8 ± 2.64 187 ± 70.7 205 ± 83.05 17 ± 6.33 4.55 ± 1.62 37.1 ± 14.67
6 White and black 61.4 ± 9.75 22.6 ± 1.9 156 ± 105 181 ± 131 11.9 ± 6.62 5 ± 3.8 49.2 ± 35.4
AUC0→t , area under the concentration–time curve (AUC) from 0 to time t; AUC0→∞ , AUC from 0 to infinity; Cmax , maximum plasma concentration; Tmax , time to Cmax ; t1/2 , terminal half-life.
4. Conclusions A rapid, sensitive and selective HPLC–MS-MS method has been developed and validated for the determination of KRN1493 in human plasma. The method, using an automatic SPE method for the extraction of KRN1493 and the IS from plasma, is sensitive, specific
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and accurate. The present method was successfully applied in pharmacokinetic studies of KRN1493 in healthy Chinese volunteers.
[5]
Acknowledgment The authors thank Kirin Kunpeng (China) Biopharmaceutical Co. Ltd. for supplying the cinacalcet hydrochloride.
[6]
References [7] [1] M. Rodriguez, E. Nemeth, D. Martin, The calcium-sensing receptor: a key factor in the pathogenesis of secondary hyperparathyroidism, Am. J. Physiol. Renal. Physiol. 288 (2005) F253–F264. [2] I. Martinez, R. Saracho, J. Montenegro, F. Llach, The importance of dietary calcium and phosphorous in the secondary hyperparathyroidism of patients with early renal failure, Am. J. Kidney Dis. 29 (1997) 496–502. [3] E.W. Young, J.M. Albert, S. Satayathum, D.A. Goodkin, R.L. Pisoni, T. Akiba, T. Akizawa, K. Kurokawa, J. Bommer, L. Piera, F.K. Port, Predictors and consequences of altered mineral metabolism: the Dialysis Outcomes and Practice Patterns Study, Kidney Int. 67 (2005) 1179–1187. [4] M. Fukagawa, S. Yumita, T. Akizawa, E. Uchida, Y. Tsukamoto, M. Iwasaki, S. Koshikawa, KRN1493 study group, cinacalcet (KRN1493) effectively decreases the serum intact PTH level with favorable control of the serum phosphorus
[8]
[9]
[10]
241
and calcium levels in Japanese dialysis patients, Nephrol. Dial. Transplant. 23 (2008) 328–335. ˜ Torres, D. Fouque, S.H. Jacobson, F. Pétavy, B. Dehmel, E. Zitt, M. Rix, P. Urena M. Ryba, Effectiveness of cinacalcet in patients with recurrent/persistent secondary hyperparathyroidism following parathyroidectomy: results of the ECHO study, Nephrol. Dial. Transplant. 26 (2011) 1956–1961. N. Ohashi, T. Uematsu, S. Nagashima, M. Kanamaru, A. Togawa, A. Hishida, E. Uchida, T. Akizawa, S. Koshikawa, The calcimimetic agent KRN 1493 lowers plasma parathyroid hormone and ionized calcium concentrations in patients with chronic renal failure on haemodialysis both on the day of haemodialysis and on the day without haemodialysis, Br. J. Clin. Pharmacol. 57 (2004) 726–734. D. Padhi, R.Z. Harris, M. Salfi, J.T. Sullivan, No effect of renal function or dialysis on pharmacokinetics of cinacalcet (Sensipar/Mimpara), Clin. Pharmacokinet. 44 (2005) 509–516. D. Nakashima, H. Takama, Y. Ogasawara, T. Kawakami, T. Nishitoba, S. Hoshi, E. Uchida, H. Tanaka, Effect of cinacalcet hydrochloride, a new calcimimetic agent, on the pharmacokinetics of dextromethorphan: in vitro and clinical studies, J. Clin. Pharmacol. 47 (2007) 1311–1319. D. Padhi, R.Z. Harris, M. Salfi, R.J. Noveck, J.T. Sullivan, Pharmacokinetics and pharmacodynamics of cinacalcet in hepatic impairment: phase I, open-label, parallel-group, single-dose, single-centre study, Clin. Drug Investig. 28 (2008) 635–643. Food and Drug Administration (FDA), Guidance for Industry in Bioanalytical Method Validation, Rockville, MD, USA, 2001.