- Email: [email protected]
Determination of cilnidipine, a new calcium antagonist, in human plasma using high performance liquid chromatography with tandem mass spectrometric detection
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Determination of cilnidipine, a new calcium antagonist, in human plasma using high performance liquid chromatography with tandem mass spectrometric detection Xianhua Zhang a , Suodi Zhai a,∗∗ , Rongsheng Zhao a , Jin Ouyang b , Xiaoguang Li a , Willy R.G. Baeyens c,∗ a b c
Peking University Third Hospital, Peking University TDMCT Center, Beijing 100083, PR China Department of Chemistry, Beijing Normal University, Beijing 100875, PR China Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
a r t i c l e
i n f o
a b s t r a c t
Article history:
A rapid, sensitive and reliable high performance liquid chromatographic method coupled
Received 5 July 2006
with tandem mass spectrometry (HPLC–MS/MS) has been developed and validated for
Received in revised form
the determination of cilnidipine, a relatively new calcium antagonist, in human plasma.
19 November 2006
The reversed-phase chromatographic system was interfaced with a TurboIonSpray (TIS)
Accepted 27 November 2006
source. Nimodipine was employed as the internal standard (IS). Sample extracts follow-
Published on line 6 December 2006
ing protein precipitation were injected into the HPLC–MS/MS system. The analyte and
Keywords:
and NH4 Ac (96:4, v/v). The ions were detected by a triple quadrupole mass spectromet-
Cilnidipine
ric detector in the negative mode. Quantification was performed using multiple reaction
Nimodipine
monitoring (MRM) of the transitions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 for cilnidip-
High performance liquid
ine and for the IS, respectively. The analysis time for each run was 3.0 min. The calibration
chromatographic method coupled
curve fitted well over the concentration range of 0.1–10 ng mL−1 , with the regression equa-
IS were eluted isocratically on a C18 column, with a mobile phase consisting of CH3 OH
with tandem mass spectrometry
tion Y = (0.103 ± 0.002)X + (0.014 ± 0.003) (n = 5), r = 0.9994. The intra-day and inter-day R.S.D.%
(HPLC–MS/MS)
were less than 12.51% at all concentration levels within the calibration range. The recover-
Human plasma
ies were between 92.71% and 97.64%. The long-term stability and freeze-thaw stability were satisfying at each level. The present method provides a modern, rapid and robust tool for pharmacokinetic studies of cilnidipine. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Cilnidipine (FRC-8653) is a newly synthesized dihydropyridine calcium antagonist that has a slow onset and long duration of action. It can regulate the catecholamine secretion closely linked to intracellular Ca2+ levels. Comparing with other calcium antagonists, it has a slow-onset, long-lasting
∗
anti-hypertensive effect and unique inhibitory actions on sympathetic neurotransmission [1]. It shifts the lower limits for autoregulation of the cerebral blood flow downward, which may remain intact even if excessive hypotension is induced by cilnidipine [2]. Hence, cilnidipine has high potentials in the therapy of hypertension. Due to its low therapeutic doses, the plasma concentration of cilnidipine is usually less than
Corresponding author. Tel.: +32 9 2648097; fax: +32 9 2648196. Corresponding author. Fax: +86 10 62017691 6673. E-mail addresses: [email protected] (S. Zhai), [email protected], [email protected] (W.R.G. Baeyens). 0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.11.072 ∗∗
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
1 ng mL−1 after oral administration of a single dose of 10 mg, which challenges the development of a modern analytical method. Several methods including UV and HPLC/UV have been reported for the determination of cilnidipine [3,4]. However, these methods are not sensitive enough to determine average plasma concentrations of this molecule. Furthermore, laborious and time-consuming procedures of sample preparation and analysis restrict the application of HPLC/UV. A fluorescence probe method was recently reported [5], but again failed to be applied to biofluids due to a sensitivity shortcoming and to the interference of a plasma substance. LC coupled with single MS was another choice for the determination of cilnidipine in rat plasma [6], but the sensitivity of single MS was not satisfying and at least 1 mL of plasma was needed. Moreover, the extraction and concentrating procedures of this method are laborious. Gas chromatography (GC) and GC–MS [7–10] are usually applied for the determination of many other calcium channel antagonists in biological fluids. However, these methods cannot be applied to cilnidipine due to its decomposition under GC conditions. Although LC–MS/MS was also used to determine calcium channel antagonists in current studies [11–15], it has not been applied to cilnidipine so far. On the other hand, in pharmacokinetic studies of cilnidipine, low concentration levels, little amounts of available samples and high throughput needs lead to modern analytical techniques that offer reduced analysis time. Therefore, the development of a rapid and robust analytical method was envisaged. The objective of the present investigation was to develop an HPLC–MS/MS method to determine plasma concentrations of cilnidipine. Its high selectivity has greatly simplified the process of method development and of sample preparation. Considering these points, an advanced reversed-phase LC–MS/MS technique combined with rapid sample preparation by simple precipitation seemed the best choice for the quantification of cilnidipine. In this work, nimodipine was employed as the IS. The proposed method provides a highly sensitive and selective way to determine cilnidipine in plasma with a detection limit of 0.02 ng mL−1 , using 0.2 mL of plasma, only. The method was validated and applied successfully to a pharmacokinetic study after single dose administration of 10 mg cilnidipine to healthy volunteers.
2.
Experimental
2.1.
Chemicals and reagents
2.2.
Instrumentation
LC–MS/MS analysis was performed using an Agilent 1100 HPLC system consisting of binary pumps, an autosampler, a vacuum degasser, and an API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source (Applied Biosystems, Foster City, CA), run by Analyst software (version 1.4).
2.3.
Standard solution and sample preparation
Stock solutions of cilnidipine and the IS were prepared in methanol. Working solutions were made by further dilution of the stock solutions with methanol. All the solutions were stored at 4 ◦ C. The samples were prepared by precipitating plasma with acetonitrile. More details about standard solution and sample preparation are described in supplementary note 1 (supplementary information, SI).
2.4.
Chromatographic conditions
Samples were separated on an Agilent C18 column (150 mm × 4.6 mm, 5 m) at ambient temperature. The isocratic mobile phase consisted of 96% of methanol and 4% (v/v) of aqueous ammonium acetate solution (0.1 mol L−1 , pH 7.0) at a flow-rate of 1.0 mL min−1 . The injection volume was 40 L. To avoid contamination of the mass spectrometer by excessive matrix molecules, the effluent from the column was split and 30% of it was directed into the mass spectrometer.
2.5.
Mass spectrometric conditions
Ionization was performed in the negative mode and MS/MS was operated at unit resolution in MRM mode. Collisioninduced dissociation was achieved by using nitrogen as the collision gas. The detailed MS/MS parameters are presented in supplementary note 2 in SI. Transition ions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 were selected for cilnidipine and nimodipine, respectively.
2.6.
Calibration and validation
Linear regression analysis was performed by plotting the relative peak area cilnidipine/nimodipine (Y) versus analyte concentration (X) in ng mL−1 , with a weighting factor 1/X. Precision, stability and matrix effects were evaluated at three concentration levels. Method details about validation are given in supplementary note 3 in SI.
2.7. Cilnidipine and nimodipine (IS) were provided by Jingxin Pharmaceutical Company (Zhejiang Province, China). Methanol (Fisher), ammonium acetate (Dikma) and acetonitrile (Dikma) were of HPLC grade. The mobile phase was filtrated with a 0.45 m film before use. Drug-free human plasma was purchased from the local blood bank (Beijing Red Cross Blood Center) and stored at −40 ◦ C. The water used was deionized and distilled.
143
Application to a human pharmacokinetic study
The method was applied to a human pharmacokinetic study. Nineteen healthy male volunteers, age between 20 and 28, were selected for this study. Informed consent forms were signed according to institutional guidelines. This study was approved by the Human Subjects Committee of Peking University Third Hospital. Experimental details were depicted in supplementary note 4 in SI.
144
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
3.
Results and discussion
3.1.
MS/MS detection
Both the positive and negative modes were investigated and 40 L of cilnidipine (0.5 ng mL−1 ) was injected into the LC–MS/MS system. The chromatograms were recorded and the sensitivity was compared in SI (Figure S1). The signal-to-noise ratio (S/N) calculated by the Analyst software was 221.5 for the negative mode and the value was only 14 for the positive one. That means the negative mode was much more sensitive. Hence, it was employed in the following experiments. In the negative mode, cilnidipine and the IS gave deprotonated precursor molecular ions [M–H]− . The major precursor ions were m/z = 491.2 for cilnidipine and m/z = 417.1 for the IS. Each of the precursor ions was subjected to collisioninduced dissociation in order to obtain the product ions. The MS/MS conditions were optimized and the full product spectra of both compounds were acquired. The pathways of main product fragments were also proposed (provided in Figures S2a and S2b in SI) based on their MS/MS spectra and chemical structures. The MS/MS spectra of both compounds showed similar fragmentation with base peak ions at m/z 122. Therefore, transition ions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 were used for cilnidipine and the IS, respectively.
3.2.
Optimization of chromatographic conditions
Several analytical columns, including a Restek Pinnacle C18 column (50 mm × 2.1 mm, 5 m), an Ultra C18 column (100 mm × 2.1 mm, 5 m) and an Agilent Eclipse C18 column (150 mm × 4.6 mm, 5 m) were evaluated to yield sharp and symmetrical peaks. The peak width extended on the former two columns and only the Agilent Eclipse C18 column (150 mm × 4.6 mm, 5 m) provided satisfying peak shapes and was hence employed in the following experiments. The mobile phase was likewise optimized. Initially, methanol and acetonitrile were compared as the mobile phase. The results indicated that the response was higher with methanol than with acetonitrile in mobile phase and hence the former was used in the following experiments. To increase the ionization efficiency and to adjust the retention time, a certain proportion of ammonium acetate was added to the mobile phase. The proportion of NH4 Ac was likewise optimized and eventually, 4% of NH4 Ac (0.1 mol L−1 , pH 7.0) was selected because it provided adequate retention and good peak shapes for cilnidipine and for the IS as well. The flow-rate was 1.0 mL min−1 . Under the optimal conditions, the retention
Fig. 1 – Chromatograms obtained from a plasma sample of a healthy volunteer (1.5 h after oral administration of 10 mg cilnidipine). Chromatograms: (a) cilnidipine; (b) IS.
times of cilnidipine and the IS were of 2.0 and 1.8 min, respectively. The total HPLC–MS/MS analysis time was 3 min for each run.
3.3.
Method validation and matrix effect
No analyte-interfering peaks were observed due to the high selectivity of MRM. Drug-free plasma was precipitated by acetonitrile and the results were recorded. Figure S3 in SI shows the representative HPLC chromatograms for a drugfree plasma sample, indicating that no endogenous peaks are present at the retention times (tR ) of cilnidipine or of the IS. The chromatograms of a plasma sample from a healthy volunteer after oral administration of 10 mg cilnidipine (1.5 h after administration) are depicted in Fig. 1. The weighted regression (1/X) calibration was linear over the concentration range of 0.1–10 ng mL−1 in human plasma. The average equation of linearity was Y = (0.103 ± 0.002)X + (0.014 ± 0.003) (n = 5), with each of the
Table 1 – Data of precision, accuracy, recovery and matrix effect in human plasma Nominal plasma concentration (ng mL−1 )
Precision Intra-day R.S.D. (%, n = 5)
0.5 2 8
12.51 3.23 2.03
Recovery (%, n = 3)
Ionization enhancement (%, n = 3)
97.64 ± 6.98 93.48 ± 9.04 92.71 ± 6.64
133.03 ± 19.20 122.87 ± 17.34 102.85 ± 12.84
Inter-day R.S.D. (%, n = 5) 8.15 8.40 5.80
Accuracy (%, n = 5)
101.88 ± 9.28 96.44 ± 9.06 95.86 ± 6.22
145
51.67 ± 6.24 62.19 ± 8.47 54.03 ± 4.68 a
Aw , peak areas of samples under weak light; As , peak areas of samples under strong light; A0 , peak areas of samples without being exposed to light.
86.16 ± 4.81 119.17 ± 2.72 88.89 ± 5.06
As a /A0 × 100 ± S.D. (n = 3) Aw a /A0 a × 100 ± S.D. (n = 3)
Photodegradation of cilnidipine in plasma (n = 3) Long-term stability (bias after 21 days, −40 ◦ C, n = 3)
−2.38 11.49 −4.99 −4.93 −9.50 −9.91 8.22 −1.08 −0.22 0.5 2 8
All samples were handled within 6 h after thawing. Therefore, QC samples were exposed to environment for 6 h to investigate the stability. The bias of the results ranged from −0.22% to 8.22% comparing with those of immediately analyzed samples (Table 2). It showed that the spiked samples were stable after being exposed to ambient temperature for at least 6 h. The freeze-thaw stability was also examined in these experiments. After two freeze-thaw cycles, the samples were analyzed and the bias comparing with nominal value is summarized in Table 2. The experimental concentrations at three levels (0.5, 2, 8 ng mL−1 ) were of 0.45 ± 0.03, 1.81 ± 0.15 and 7.29 ± 0.21 ng mL−1 (n = 3), respectively. The long-term stability was assessed by freezing the QC samples for 21 days (−40 ◦ C) and the bias of the results ranged from −2.38% to 11.49% comparing with those of immediately analyzed samples (see Table 2). The experimental results for three levels were of 0.41 ± 0.005, 1.94 ± 0.42, and 6.85 ± 1.02 ng mL−1 (n = 3), respectively. QC samples and also the corresponding working solutions were exposed to weak light (full wavelength light with intensity of 24.1 Lx) and strong light (full wavelength light with intensity of 11,700 Lx) to investigate the photostability. After 4 h under light, the above samples were analyzed and peak areas (A) were recorded. The data showed that after 4 h under strong light, almost all the drugs in working solutions degraded while only 20% did under weak light. A similar trend was observed in plasma, the degradation in plasma, however, being much slower than in methanol. In plasma, an average of 56% cilnidipine at three levels degraded after 4 h under strong
Freeze-thaw stability (bias after two cycles, n = 3)
Stability
Bias of plasma samples at room temperature (after 6 h, n = 3)
3.4.
Nominal plasma concentration (ng mL−1 )
correlation coefficients (r) greater than 0.9990. The use of the weighted regression resulted in less than 10% deviation between the nominal and experimental concentrations calculated from the equations. The detection limit was 0.02 ng mL−1 (S/N = 3). An assessment of intra-day and inter-day precisions was conducted by analyzing quality control (QC) samples at three levels. Data of precisions are presented in Table 1. The precision of the assay was less than 12.51% and the accuracy was between 95.86% and 101.88% of the nominal value at three levels, that is, 0.5, 2, 8 ng mL−1 . There is a continuing demand for high-throughput bioanalytical methods based on HPLC–MS/MS systems for the evaluation of new chemical entities. Matrix ionization suppression/enhancement is considered to be more likely a problem when using the protein precipitation method for sample preparation as compared to liquid–liquid extraction and solid-phase extraction methods. In spite of this concern, the protein precipitation method has been employed as the routine sample preparation procedure for HPLC–MS/MS assays in our laboratory, mainly due to its simplicity. A small absolute enhancement of ionization was indeed observed in this experiment (see Table 1) and it might be due to the presence of non-detectable endogenous compounds in the plasma sample affecting ionization of the analyte. However, it did not substantially affect the results and this conclusion was confirmed by the satisfying accuracy results. Furthermore, simplicity of sample preparation procedures brought high recovery and the averaged recoveries were approximately over 90% (see Table 1).
Table 2 – Data of stability of plasma samples at room temperature, freeze-thaw stability, long-term stability and photostability
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
146
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
cilnidipine after application of an oral dose of 10 mg to healthy volunteers. The method proves practical for pharmacokinetic studies and for in vivo detection of cilnidipine.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2006.11.072.
references Fig. 2 – Mean plasma concentration–time profile of 19 healthy male volunteers after a single oral administration of 10 mg cilnidipine.
light while the degradation was almost negligible under weak light (see Table 2). Therefore, in order to prevent photodegradation, all procedures were performed under weak light.
3.5.
Application to pharmacokinetic studies
The described assay was successfully applied to the quantification of cilnidipine in human plasma. Fig. 2 shows the mean plasma concentration–time profile of 19 subjects. Important pharmacokinetic parameters are as follows: Cmax = 11.08 ± 4.42 ng mL−1 , tmax = 1.30 ± 0.56 h, t1/2 = 3.99 ± 1.90 h.
4.
Conclusion
An HPLC–MS/MS method was developed and validated for the quantification of cilnidipine in human plasma. The method is rapid, selective and highly sensitive with a detection limit of 0.02 ng mL−1 . With this method, only 200 L of plasma is needed for analysis. The method offering a wide range of linearity allows quantification over the range 0.1–10 ng mL−1 . The analysis time was decreased comparing to conventional HPLC methods and for each run, only 3 min was needed. The method was successfully applied to evaluate the pharmacokinetics of
[1] M. Tominaga, Y. Ohya, A. Tsukashima, K. Kobayashi, Y. Takata, T. Koga, Y. Yamashita, I. Abe, M. Fujishima, Cardiovasc. Drugs Ther. 11 (1997) 43. [2] Y. Murai, H. Uneyama, H. Ishibashi, K. Takahama, N. Akaike, Brain Res. 854 (2000) 6. [3] W.Y. Shen, Y.X. Du, C. Huang, Y.G. Xu, J. China Pharmaceut. Univ. 33 (2002) 544. [4] L.Y. He, G.Y. Hu, Y.B. Zhou, J.H. Liu, West China J. Pharmaceut. Sci. 19 (2004) 70. [5] S.Z. Tan, J.H. Jiang, G.Y. Shen, G.L. Shen, R.Q. Yu, Anal. Chim. Acta 547 (2005) 215. [6] K. Hatada, M. Kimura, I. Ono, M. Ozaki, J. Chromatogr. 583 (1992) 116. [7] J.D.Y. Dru, J.Y.K. Hsieh, B.K. Matuszewski, M.R. Dobrinska, J. Chromatogr. B 666 (1995) 259. [8] M.E. Ortiz, L.J.N. Vergara, C. Camargo, J.A. Squella, Pharm. Res. 21 (2004) 428. [9] M.P. Marques, N.A. Santos, E.B. Coelho, P.S. Bonato, V.L. Lanchote, J. Chromatogr. B: Biomed. Sci. Appl. 762 (2001) 87. [10] B. Marciniec, M. Ogrodowczyk, Acta Pol. Pharm. 60 (2003) 151. [11] A.B. Baranda, C.A. Mueller, R.M. Alonso, R.M. Jimenez, W. Weinmann, Ther. Drug Monit. 27 (2005) 44. [12] L.H. Miglioranc, R.E. Barrientos-Astigarraga, B.S. Schug, H.H. Blume, A.S. Pereira, G. De Nucci, J. Chromatogr. B 814 (2005) 217. [13] F. Qiu, X. Chen, X. Li, D. Zhong, J. Chromatogr. B 802 (2004) 291. [14] J. Fiori, R. Gotti, C. Bertucci, V. Cavrini, J. Pharm. Biomed. Anal. 41 (2006) 176. [15] N.V. Ramakrishna, K.N. Vishwottam, S. Puran, S. Manoj, M. Santosh, M. Koteshwara, J. Mass Spectrom. 39 (2004) 824.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Determination of cilnidipine, a new calcium antagonist, in human plasma using high performance liquid chromatography with tandem mass spectrometric detection Xianhua Zhang a , Suodi Zhai a,∗∗ , Rongsheng Zhao a , Jin Ouyang b , Xiaoguang Li a , Willy R.G. Baeyens c,∗ a b c
Peking University Third Hospital, Peking University TDMCT Center, Beijing 100083, PR China Department of Chemistry, Beijing Normal University, Beijing 100875, PR China Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
a r t i c l e
i n f o
a b s t r a c t
Article history:
A rapid, sensitive and reliable high performance liquid chromatographic method coupled
Received 5 July 2006
with tandem mass spectrometry (HPLC–MS/MS) has been developed and validated for
Received in revised form
the determination of cilnidipine, a relatively new calcium antagonist, in human plasma.
19 November 2006
The reversed-phase chromatographic system was interfaced with a TurboIonSpray (TIS)
Accepted 27 November 2006
source. Nimodipine was employed as the internal standard (IS). Sample extracts follow-
Published on line 6 December 2006
ing protein precipitation were injected into the HPLC–MS/MS system. The analyte and
Keywords:
and NH4 Ac (96:4, v/v). The ions were detected by a triple quadrupole mass spectromet-
Cilnidipine
ric detector in the negative mode. Quantification was performed using multiple reaction
Nimodipine
monitoring (MRM) of the transitions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 for cilnidip-
High performance liquid
ine and for the IS, respectively. The analysis time for each run was 3.0 min. The calibration
chromatographic method coupled
curve fitted well over the concentration range of 0.1–10 ng mL−1 , with the regression equa-
IS were eluted isocratically on a C18 column, with a mobile phase consisting of CH3 OH
with tandem mass spectrometry
tion Y = (0.103 ± 0.002)X + (0.014 ± 0.003) (n = 5), r = 0.9994. The intra-day and inter-day R.S.D.%
(HPLC–MS/MS)
were less than 12.51% at all concentration levels within the calibration range. The recover-
Human plasma
ies were between 92.71% and 97.64%. The long-term stability and freeze-thaw stability were satisfying at each level. The present method provides a modern, rapid and robust tool for pharmacokinetic studies of cilnidipine. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Cilnidipine (FRC-8653) is a newly synthesized dihydropyridine calcium antagonist that has a slow onset and long duration of action. It can regulate the catecholamine secretion closely linked to intracellular Ca2+ levels. Comparing with other calcium antagonists, it has a slow-onset, long-lasting
∗
anti-hypertensive effect and unique inhibitory actions on sympathetic neurotransmission [1]. It shifts the lower limits for autoregulation of the cerebral blood flow downward, which may remain intact even if excessive hypotension is induced by cilnidipine [2]. Hence, cilnidipine has high potentials in the therapy of hypertension. Due to its low therapeutic doses, the plasma concentration of cilnidipine is usually less than
Corresponding author. Tel.: +32 9 2648097; fax: +32 9 2648196. Corresponding author. Fax: +86 10 62017691 6673. E-mail addresses: [email protected] (S. Zhai), [email protected], [email protected] (W.R.G. Baeyens). 0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.11.072 ∗∗
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
1 ng mL−1 after oral administration of a single dose of 10 mg, which challenges the development of a modern analytical method. Several methods including UV and HPLC/UV have been reported for the determination of cilnidipine [3,4]. However, these methods are not sensitive enough to determine average plasma concentrations of this molecule. Furthermore, laborious and time-consuming procedures of sample preparation and analysis restrict the application of HPLC/UV. A fluorescence probe method was recently reported [5], but again failed to be applied to biofluids due to a sensitivity shortcoming and to the interference of a plasma substance. LC coupled with single MS was another choice for the determination of cilnidipine in rat plasma [6], but the sensitivity of single MS was not satisfying and at least 1 mL of plasma was needed. Moreover, the extraction and concentrating procedures of this method are laborious. Gas chromatography (GC) and GC–MS [7–10] are usually applied for the determination of many other calcium channel antagonists in biological fluids. However, these methods cannot be applied to cilnidipine due to its decomposition under GC conditions. Although LC–MS/MS was also used to determine calcium channel antagonists in current studies [11–15], it has not been applied to cilnidipine so far. On the other hand, in pharmacokinetic studies of cilnidipine, low concentration levels, little amounts of available samples and high throughput needs lead to modern analytical techniques that offer reduced analysis time. Therefore, the development of a rapid and robust analytical method was envisaged. The objective of the present investigation was to develop an HPLC–MS/MS method to determine plasma concentrations of cilnidipine. Its high selectivity has greatly simplified the process of method development and of sample preparation. Considering these points, an advanced reversed-phase LC–MS/MS technique combined with rapid sample preparation by simple precipitation seemed the best choice for the quantification of cilnidipine. In this work, nimodipine was employed as the IS. The proposed method provides a highly sensitive and selective way to determine cilnidipine in plasma with a detection limit of 0.02 ng mL−1 , using 0.2 mL of plasma, only. The method was validated and applied successfully to a pharmacokinetic study after single dose administration of 10 mg cilnidipine to healthy volunteers.
2.
Experimental
2.1.
Chemicals and reagents
2.2.
Instrumentation
LC–MS/MS analysis was performed using an Agilent 1100 HPLC system consisting of binary pumps, an autosampler, a vacuum degasser, and an API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source (Applied Biosystems, Foster City, CA), run by Analyst software (version 1.4).
2.3.
Standard solution and sample preparation
Stock solutions of cilnidipine and the IS were prepared in methanol. Working solutions were made by further dilution of the stock solutions with methanol. All the solutions were stored at 4 ◦ C. The samples were prepared by precipitating plasma with acetonitrile. More details about standard solution and sample preparation are described in supplementary note 1 (supplementary information, SI).
2.4.
Chromatographic conditions
Samples were separated on an Agilent C18 column (150 mm × 4.6 mm, 5 m) at ambient temperature. The isocratic mobile phase consisted of 96% of methanol and 4% (v/v) of aqueous ammonium acetate solution (0.1 mol L−1 , pH 7.0) at a flow-rate of 1.0 mL min−1 . The injection volume was 40 L. To avoid contamination of the mass spectrometer by excessive matrix molecules, the effluent from the column was split and 30% of it was directed into the mass spectrometer.
2.5.
Mass spectrometric conditions
Ionization was performed in the negative mode and MS/MS was operated at unit resolution in MRM mode. Collisioninduced dissociation was achieved by using nitrogen as the collision gas. The detailed MS/MS parameters are presented in supplementary note 2 in SI. Transition ions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 were selected for cilnidipine and nimodipine, respectively.
2.6.
Calibration and validation
Linear regression analysis was performed by plotting the relative peak area cilnidipine/nimodipine (Y) versus analyte concentration (X) in ng mL−1 , with a weighting factor 1/X. Precision, stability and matrix effects were evaluated at three concentration levels. Method details about validation are given in supplementary note 3 in SI.
2.7. Cilnidipine and nimodipine (IS) were provided by Jingxin Pharmaceutical Company (Zhejiang Province, China). Methanol (Fisher), ammonium acetate (Dikma) and acetonitrile (Dikma) were of HPLC grade. The mobile phase was filtrated with a 0.45 m film before use. Drug-free human plasma was purchased from the local blood bank (Beijing Red Cross Blood Center) and stored at −40 ◦ C. The water used was deionized and distilled.
143
Application to a human pharmacokinetic study
The method was applied to a human pharmacokinetic study. Nineteen healthy male volunteers, age between 20 and 28, were selected for this study. Informed consent forms were signed according to institutional guidelines. This study was approved by the Human Subjects Committee of Peking University Third Hospital. Experimental details were depicted in supplementary note 4 in SI.
144
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
3.
Results and discussion
3.1.
MS/MS detection
Both the positive and negative modes were investigated and 40 L of cilnidipine (0.5 ng mL−1 ) was injected into the LC–MS/MS system. The chromatograms were recorded and the sensitivity was compared in SI (Figure S1). The signal-to-noise ratio (S/N) calculated by the Analyst software was 221.5 for the negative mode and the value was only 14 for the positive one. That means the negative mode was much more sensitive. Hence, it was employed in the following experiments. In the negative mode, cilnidipine and the IS gave deprotonated precursor molecular ions [M–H]− . The major precursor ions were m/z = 491.2 for cilnidipine and m/z = 417.1 for the IS. Each of the precursor ions was subjected to collisioninduced dissociation in order to obtain the product ions. The MS/MS conditions were optimized and the full product spectra of both compounds were acquired. The pathways of main product fragments were also proposed (provided in Figures S2a and S2b in SI) based on their MS/MS spectra and chemical structures. The MS/MS spectra of both compounds showed similar fragmentation with base peak ions at m/z 122. Therefore, transition ions m/z 491.2 → 122.1 and m/z 417.1 → 122.1 were used for cilnidipine and the IS, respectively.
3.2.
Optimization of chromatographic conditions
Several analytical columns, including a Restek Pinnacle C18 column (50 mm × 2.1 mm, 5 m), an Ultra C18 column (100 mm × 2.1 mm, 5 m) and an Agilent Eclipse C18 column (150 mm × 4.6 mm, 5 m) were evaluated to yield sharp and symmetrical peaks. The peak width extended on the former two columns and only the Agilent Eclipse C18 column (150 mm × 4.6 mm, 5 m) provided satisfying peak shapes and was hence employed in the following experiments. The mobile phase was likewise optimized. Initially, methanol and acetonitrile were compared as the mobile phase. The results indicated that the response was higher with methanol than with acetonitrile in mobile phase and hence the former was used in the following experiments. To increase the ionization efficiency and to adjust the retention time, a certain proportion of ammonium acetate was added to the mobile phase. The proportion of NH4 Ac was likewise optimized and eventually, 4% of NH4 Ac (0.1 mol L−1 , pH 7.0) was selected because it provided adequate retention and good peak shapes for cilnidipine and for the IS as well. The flow-rate was 1.0 mL min−1 . Under the optimal conditions, the retention
Fig. 1 – Chromatograms obtained from a plasma sample of a healthy volunteer (1.5 h after oral administration of 10 mg cilnidipine). Chromatograms: (a) cilnidipine; (b) IS.
times of cilnidipine and the IS were of 2.0 and 1.8 min, respectively. The total HPLC–MS/MS analysis time was 3 min for each run.
3.3.
Method validation and matrix effect
No analyte-interfering peaks were observed due to the high selectivity of MRM. Drug-free plasma was precipitated by acetonitrile and the results were recorded. Figure S3 in SI shows the representative HPLC chromatograms for a drugfree plasma sample, indicating that no endogenous peaks are present at the retention times (tR ) of cilnidipine or of the IS. The chromatograms of a plasma sample from a healthy volunteer after oral administration of 10 mg cilnidipine (1.5 h after administration) are depicted in Fig. 1. The weighted regression (1/X) calibration was linear over the concentration range of 0.1–10 ng mL−1 in human plasma. The average equation of linearity was Y = (0.103 ± 0.002)X + (0.014 ± 0.003) (n = 5), with each of the
Table 1 – Data of precision, accuracy, recovery and matrix effect in human plasma Nominal plasma concentration (ng mL−1 )
Precision Intra-day R.S.D. (%, n = 5)
0.5 2 8
12.51 3.23 2.03
Recovery (%, n = 3)
Ionization enhancement (%, n = 3)
97.64 ± 6.98 93.48 ± 9.04 92.71 ± 6.64
133.03 ± 19.20 122.87 ± 17.34 102.85 ± 12.84
Inter-day R.S.D. (%, n = 5) 8.15 8.40 5.80
Accuracy (%, n = 5)
101.88 ± 9.28 96.44 ± 9.06 95.86 ± 6.22
145
51.67 ± 6.24 62.19 ± 8.47 54.03 ± 4.68 a
Aw , peak areas of samples under weak light; As , peak areas of samples under strong light; A0 , peak areas of samples without being exposed to light.
86.16 ± 4.81 119.17 ± 2.72 88.89 ± 5.06
As a /A0 × 100 ± S.D. (n = 3) Aw a /A0 a × 100 ± S.D. (n = 3)
Photodegradation of cilnidipine in plasma (n = 3) Long-term stability (bias after 21 days, −40 ◦ C, n = 3)
−2.38 11.49 −4.99 −4.93 −9.50 −9.91 8.22 −1.08 −0.22 0.5 2 8
All samples were handled within 6 h after thawing. Therefore, QC samples were exposed to environment for 6 h to investigate the stability. The bias of the results ranged from −0.22% to 8.22% comparing with those of immediately analyzed samples (Table 2). It showed that the spiked samples were stable after being exposed to ambient temperature for at least 6 h. The freeze-thaw stability was also examined in these experiments. After two freeze-thaw cycles, the samples were analyzed and the bias comparing with nominal value is summarized in Table 2. The experimental concentrations at three levels (0.5, 2, 8 ng mL−1 ) were of 0.45 ± 0.03, 1.81 ± 0.15 and 7.29 ± 0.21 ng mL−1 (n = 3), respectively. The long-term stability was assessed by freezing the QC samples for 21 days (−40 ◦ C) and the bias of the results ranged from −2.38% to 11.49% comparing with those of immediately analyzed samples (see Table 2). The experimental results for three levels were of 0.41 ± 0.005, 1.94 ± 0.42, and 6.85 ± 1.02 ng mL−1 (n = 3), respectively. QC samples and also the corresponding working solutions were exposed to weak light (full wavelength light with intensity of 24.1 Lx) and strong light (full wavelength light with intensity of 11,700 Lx) to investigate the photostability. After 4 h under light, the above samples were analyzed and peak areas (A) were recorded. The data showed that after 4 h under strong light, almost all the drugs in working solutions degraded while only 20% did under weak light. A similar trend was observed in plasma, the degradation in plasma, however, being much slower than in methanol. In plasma, an average of 56% cilnidipine at three levels degraded after 4 h under strong
Freeze-thaw stability (bias after two cycles, n = 3)
Stability
Bias of plasma samples at room temperature (after 6 h, n = 3)
3.4.
Nominal plasma concentration (ng mL−1 )
correlation coefficients (r) greater than 0.9990. The use of the weighted regression resulted in less than 10% deviation between the nominal and experimental concentrations calculated from the equations. The detection limit was 0.02 ng mL−1 (S/N = 3). An assessment of intra-day and inter-day precisions was conducted by analyzing quality control (QC) samples at three levels. Data of precisions are presented in Table 1. The precision of the assay was less than 12.51% and the accuracy was between 95.86% and 101.88% of the nominal value at three levels, that is, 0.5, 2, 8 ng mL−1 . There is a continuing demand for high-throughput bioanalytical methods based on HPLC–MS/MS systems for the evaluation of new chemical entities. Matrix ionization suppression/enhancement is considered to be more likely a problem when using the protein precipitation method for sample preparation as compared to liquid–liquid extraction and solid-phase extraction methods. In spite of this concern, the protein precipitation method has been employed as the routine sample preparation procedure for HPLC–MS/MS assays in our laboratory, mainly due to its simplicity. A small absolute enhancement of ionization was indeed observed in this experiment (see Table 1) and it might be due to the presence of non-detectable endogenous compounds in the plasma sample affecting ionization of the analyte. However, it did not substantially affect the results and this conclusion was confirmed by the satisfying accuracy results. Furthermore, simplicity of sample preparation procedures brought high recovery and the averaged recoveries were approximately over 90% (see Table 1).
Table 2 – Data of stability of plasma samples at room temperature, freeze-thaw stability, long-term stability and photostability
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
146
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 142–146
cilnidipine after application of an oral dose of 10 mg to healthy volunteers. The method proves practical for pharmacokinetic studies and for in vivo detection of cilnidipine.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.aca.2006.11.072.
references Fig. 2 – Mean plasma concentration–time profile of 19 healthy male volunteers after a single oral administration of 10 mg cilnidipine.
light while the degradation was almost negligible under weak light (see Table 2). Therefore, in order to prevent photodegradation, all procedures were performed under weak light.
3.5.
Application to pharmacokinetic studies
The described assay was successfully applied to the quantification of cilnidipine in human plasma. Fig. 2 shows the mean plasma concentration–time profile of 19 subjects. Important pharmacokinetic parameters are as follows: Cmax = 11.08 ± 4.42 ng mL−1 , tmax = 1.30 ± 0.56 h, t1/2 = 3.99 ± 1.90 h.
4.
Conclusion
An HPLC–MS/MS method was developed and validated for the quantification of cilnidipine in human plasma. The method is rapid, selective and highly sensitive with a detection limit of 0.02 ng mL−1 . With this method, only 200 L of plasma is needed for analysis. The method offering a wide range of linearity allows quantification over the range 0.1–10 ng mL−1 . The analysis time was decreased comparing to conventional HPLC methods and for each run, only 3 min was needed. The method was successfully applied to evaluate the pharmacokinetics of
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