Arch Pharm Res Vol 30, No 9, 1174-1178, 2007
http://apr.psk.or.kr
Determination of Eperisone in Human Plasma by Liquid Chromatography-ESI-Tandem Mass Spectrometry Min Kyo Jeoung, Eun Sook Jeong, Nam Hee Kim, Chang-Soo Kim, Youn-Bok Chung, Yong-Moon Lee, Su-Youn Ahn, Hwang-Eui Cho, Yong Hwa Lee, Jin Tae Hong, and Dong-Cheul Moon College of Pharmacy, CBITRC, Chungbuk National University, Cheongju 361-763, Korea
(Received April 10, 2007)
A sensitive and selective method for the determination of 4’-ethyl-3-methyl-3-piperidinopropiophenone hydrochloride (eperisone hydrochloride) in human plasma was developed and validated. The procedure employed an internal standard and a solvent extraction step followed by chromatography on a Xterra C18 minibore column. Detection was by electrospray ionization tandem mass spectrometry with multiple reaction monitoring. The mass transitions of eperisone and tolperisone (IS) were m/z 260 → 98 and m/z 246 → 98, respectively. The method has a limit of detection of 0.1 pg/mL for eperisone based on the three times signal-tonoise value with a linear range from 0.01 to 10.0 ng/mL for the analyte. Extraction recovery was on average 98.6±7.2% (SD) for eperisone. The Intra- and inter-day assay accuracy ranged from 93 to 114% and precision (RSD) was better than 8.5%. The method was successfully employed to analyze plasma samples and evaluate pharmacokinetics of eperisone in healthy male volunteers. Key words: Eperisone, Tolperisone, Tandem mass spectrometry, Human plasma, Pharmacokinetics
INTRODUCTION Eperisone hydrochloride (4’-ethyl-2-methyl-3-piperidinopropiopenone hydrochloride), a central muscle relaxant (Kuroiwa et al., 1981; Iwase et al., 1992), is widely used for the treatment of spastic paralysis and is administered orally at a dose of 50-100 mg (Kuroiwa et al., 1981). Eperisone has a short muscle relaxant activity on account of its extensive first-pass metabolism after oral administration (Fujita et al., 1981; Mihara et al., 2001). It has a very low concentration in plasma because of its low bioavailability. Therefore, a very sensitive analytical technique employing a simple and rapid clean-up step is required to quantify the drug level in plasma. A few methods have been reported to estimate eperisone hydrochloride in biological fluids. For GC/MS method (Takenaka et al., 1992), the limit of quantification (LOQ) is 0.2 ng/mL, which is not sensitive enough for pharmacokinetic studies. A LC-ESI/MS method using Correspondence to: Dong-Cheul Moon, College of Pharmacy, CBITRC, Chungbuk National University, Gaeshin-dong 12, Heungduk-gu, Cheongju 361-763, Korea Tel: 82-43-261-2819, Fax: 82-43-275-6131 E-mail:
[email protected]
single-quadrupole MS (Li et al., 2004) used [M+H]+ ions for detecting both eperisone and internal standard, and it required 1 mL of plasma sample for clean-up process. There has been a great deal of interest in using LC/MS/ MS for the analysis of drugs in biological fluids because of its excellent specificity, speed, and sensitivity (Ackermann et al., 2002). This paper describes the development and validation of a rapid, sensitive, and specific LC-MS/MS method for the determination of eperisone in human plasma using tolperisone as an internal standard. Owing to the specificity of MS/MS detection, sample processing is minimal and the analysis run time is considerably shorter. The assay method has been applied to pharmacokinetic studies of eperisone tablets in human volunteers.
MATERIALS AND METHODS Materials and reagents
Eperisone hydrochloride and tolperisone hydrochloride (internal standard) were provided by the Goo-Joo Pharm. Co. (Seoul, Korea). Purity of both standards was higher than 99.0%. HPLC-grade methanol was purchased from Fisher Scientific Co. (Fairlawn, NJ, U.S.A.), and analytical
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reagent-grade glacial acetic acid and diethyl ether were obtained from Sigma (St. Louis, MO, U.S.A.). All other chemicals were of the highest quality available and were used without further purification. Water was passed through Millipore Milli-RO4 and Milli-Q water purification systems (Bedford, MA, U.S.A.). The mobile phase was filtered through a 0.2-mm membrane filter (Phenomenex, CA, U.S.A.) and degassed ultrasonically prior to use. The micro-tubes (2.0 mL) used for the sample mixture were purchased from Axygen (CA, U.S.A.). Human plasma was obtained with consent from healthy male volunteers in the written form after the approval of the Korean Food and Drug Administration (KFDA) for the study protocol.
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multiple reaction monitoring (MRM) mode employing collision energy of 25 eV with dwell time of 300 ms for the mass transitions, m/z 260 → 98 for eperisone and m/z 246 → 98 for tolperisone, respectively. Quadrupoles, Q1 and Q3 were set to unit resolution. Automated data acquisition and analysis were performed using the Analyst software (version 1.4.1).
Sample preparation
Stock solutions of eperisone (100 µg/mL) and tolperisone (10 µg/mL) were freshly prepared in methanol and stored at 4oC. Standard solutions were prepared by serial dilution of the stock solutions with methanol to required concentrations. Quality control (QC) samples were prepared by spiking the stock solutions with drug-free human plasma to make the final concentrations at 0 (blank), 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 5.0, and 10 ng/mL of eperisone hydrochloride in plasma, respectively.
To a 500-µL aliquot of plasma samples, which was placed into a 2-mL micro-tube with a teflon-lined cap, were added 10-µL of internal standard (tolperisone, 50 ng/ mL) and 200 µL of a saturated K2CO3. The sample mixture was stirred for 5 min using a vortex mixer. After the addition of 1.2 mL of diethyl ether to the mixture, it was vortex-mixed additionally for 5 min, then centrifuged at 9000 × g for 3 min. The resulting supernatant solution (1 mL) was transferred to another tube followed by addition of deionized water (500 µL). It was vortex mixed (1 min) and centrifuged at 9000 × g for 3 min. The separated organic phase was evaporated to dryness using a speed-vacuum concentrator (Hanil Co. Ltd., Seoul, Korea). The residue was reconstituted in 100 µL of a freshly prepared mobile phase from which 10-µL aliquots were injected on to the LC/MS/MS system.
Chromatography
Method validation
Preparation of standard solutions and quality control (QC) samples
The HPLC system consisted of an Agilent 1100 series with a degasser, binary pump and autosampler (Agilent Technologies, Palo Alto, CA, U.S.A.). Separation was performed on a X-terraTM C18 column (5 mm, 150×2.1 mm i.d.; Waters Co., U.S.A.) with the isocratic elution of 1% acetic acid-methanol (30:70, v/v) at a flow rate of 0.2 mL/ min. The column temperature was maintained at room temperature. The HPLC eluent was introduced directly into the positive electrospray ionization source at a flow rate of 0.2 mL/min.
LC/ESI-MS/MS analysis
The plasma concentration of eperisone was quantified using liquid chromatography-mass spectrometry with a Sciex API 3000 triple quadrupole mass spectrometer (Applied Biosystems, MDS Sciex, Concord, Canada) equipped with a TurboIonSpray interface to generate the positive ions [M+H]+. The ion spray interface was operated in positive ion mode at 5.0 kV with a turbo gas temperature at 360oC. The operating conditions were investigated by a flow injection of a mixture of all the analytes and optimized as follows: nebulizing gas flow, 1.31 L/min (setting 11); auxiliary gas flow, 6.1 L/min; curtain gas flow, 0.95 L/min (setting 7); orifice voltage, 53 V; ring voltage 400 V; and collision gas (nitrogen) pressure, 3.58×105 Torr (setting 7). Quantitation was performed by
The precision and accuracy of the method were determined by replicate analyses for batches of the QC sample (0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 5.0 and 10 ng/mL eperisone hydrochloride) on a day and five separate days. The precision was calculated by the intra- and inter-day percent relative standard deviation (RSD, %). The matrix effect and recoveries of eperisone were assessed by analyzing three sets of standards at eight concentrations (0.01, 0.02, 0.05, 0.1, 0.5, 1, 5 and 10 ng/mL) according to the approach of Matuszewski (Matuszewski et al., 2003). The matrix effect for eperisone was assessed by comparing mean peak areas of the analyte at eight concentrations spiked after extraction into plasma extracts from seven different plasmas (set 2) to mean peak areas for neat solutions of the analyte in mobile phase (set 1). The recovery of eperisone was determined by comparing mean peak areas of the analytes spiked before extraction into the same seven different plasma as set 2 (set 3) with those of the analyte spiked after extraction into the seven different blank plasma at eight concentrations (set 2).
Pharmacokinetic study
The pharmacokinetic studies were carried out on eight healthy-male volunteers aged from The pharmacokinetic studies were carried out on eight healthy-male volunteers aged from 20 to 25 years. Two tablets (100 mg eperisone
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hydrpchloride) of a test drug (Goo-Joo Pharm. Co.) were administered to each volunteer with 200 mL of tap water after an overnight fast. After each administration, blood samples were drawn into venoject heparin-containing tubes immediately before and up to 12 h after administration. After centrifugation at 9000 × g for 10 min at 4oC, the plasma samples were deep frozen at -70oC prior to the assay. A non-compartmental pharmacokinetic analysis was performed on the plasma concentrations using PCNONLIN software. Levels less than the quantification limit were considered zero. The maximum plasma concentrations (Cmax) and the corresponding time to those (Tmax) were obtained through direct observations of the plasma concentration-time curves. The area under the plasma concentration-time curves from time zero to the time of the last quantifiable concentration (AUC0-last) was calculated using the trapezoidal rule whereas AUC0-∞ was calculated according to AUC0-∞ = AUC0-last + Clast/k, where k is the slope of the terminal phase of the plasma concentration curve using the log-transformed concentrations and Clast is the final concentration higher than the quantification limit. The plasma half-life (t1/2) was calculated using the formula, t 1/2 = 0.693/k.
RESULTS AND DISCUSSION Mass spectra
The LC/ESI-MS/MS conditions were optimized to obtain sensitivity and signal stability during infusion of the analytes in the continuous flow of mobile phase to electrospray ion source operated in positive ion mode at a flow rate of 10 µL/min. The predominant peaks in the primary ESI spectra of eperisone and tolperisone correspond to the MH+ ions at m/z 260 and m/z 246, respectively. Both product ions for eperisone and tolperisone scanned in Q3 after a collision with nitrogen in Q2 had a m/z of 98 (C6H12N+). The Q3 ions shown in Fig. 1 were formed by the C-C cleavage adjacent to C-N bond as is typical for 2alkyl-3-piperidinopropiophenones.
Assay characteristics
An advantage of using a specific and selective method such as LC/ESI-MS/MS is that sample processing can be minimized. The sample pretreatment was accomplished with 500 µL of plasma sample. Diethyl ether was used to give deproteination and extract the analytes from plasma under alkaline condition using saturated potassium carbonate instead of sodium carbonate or bicarbonate, which can reduce work-up volumes because the potassium salt has better solubility in aqueous phase. A structurally similar, commercially available compound, tolperisone was selected as an internal standard. The precision and accuracy were acceptable for eperisone using this internal
Fig. 1.
Product ion mass spectra of eperisone (A) and tolperisone (B)
standard. The internal standard substantially improved the precision and reliability of the quantitation of eperisone. The matrix effect for the measurement of eperisone was practically absent from the result in Table I. Extraction recoveries were 91.2-110.1% at eight different concentrations between 0.01-10 ng/mL of eperisone. The retention times for eperisone and tolperisone (I.S) were 1.57 and 1.52 min, respectively and the total run time was less than 3 min. Regeneration of the column using a gradient elution step was not necessary because Matrix effect and recovery for eperisone in human plasma (n=7) Eperisone. Spiked Metrix effect (%) Recovery (%) (ng/mL) [mean ± S.D.] [mean ± S.D.] 0.01 95.9 ± 8.3 110.1 ± 9.2 0.02 101.7 ± 3.8 106.2 ± 6.3 0.05 97.8 ± 6.3 103.3 ± 7.4 0.10 96.4 ± 0.8 95.3 ± 8.8 0.50 93.3 ± 6.0 96.0 ± 7.0 100. 102.9 ± 5.3 92.1 ± 6.8 500. 96.4 ± 4.6 94.2 ± 5.6 10000. 103.6 ± 3.0 91.2 ± 6.8 Mean 98.5 ± 3.8 98.6 ± 7.2 Table I.
Determination of Eperisone
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Fig. 2. Representative chromatograms of eperisone (left) and tolperisone (right) in human plasma. (A) Blank plasma. (B) Plasma spiked with 0.01 ng/mL of eperisone and 5 ng/mL of tolperisone (I.S). (C) Plasma sample 2.5 h after the oral administration of two tablets of eperisone to human volunteers.
there were no late-eluting peaks observed. The background was very low and the reconstituted samples did not cause any type of blockage in the tubes or needle sprayer due to non-precipitated proteins. As can be seen in Fig. 2(A), the MRM chromatograms of blank human plasma had no interfering peaks at the retention times of the compounds of interest. Fig. 2(B) show representative chromatograms for the plasma samples spiked with 0.01 ng/mL eperisone (left) and 5 ng/mL tolperisone (right), respectively. Fig. 2(C) show chromatograms of plasma samples 2.5 h after oral administration of eperisone (100 mg) to human volunteers. The drug concentration was equivalent to 0.9 ng/mL of eperisone.
Validation
The calibration curve for eperisone was linear over the concentration range, 0.01-10.0 ng/mL in human plasma with a correlation coefficient (r) of 0.9998 ± 0.0002. The mean equation of the regression line was, y = (0.4956 ± 0.029)x + (0.0079 ± 0.001). The limit of detection (LOD) was 0.1 pg/mL, as defined by the concentration of analyte giving a signal-to-noise (S/N) ratio of 3. And the limit of quantification (LOQ) was 0.01 ng/mL, as defined by the lowest concentration in the linear range that can be detected with a variation within 10%. The relative standard deviation (RSD) of seven replicate determinations of the eight different concentration, between 0.01 and 10 ng/mL, was in the range of 2.28-8.51%. The intra- (n=5) and inter-day (n=5) precision and
accuracy are summarized in Table II. The inter-day assay variations were determined by duplicates of QC sample on five separate days. In both cases, the accuracy ranged from 93 to 114% at the concentrations investigated, and the RSDs were less then 8.5% (Table II).
Application to a pharmacokinetics study
The method was used to determine the plasma pharmacokinetics of eperisone in the healthy human volunteers. Fig. 3 shows the plasma concentration versus time profile after the oral administration of 100 mg eperisone hydrochloride to eight volunteers. Table III lists the mean pharTable
II.
plasma
Precision and accuracy for the assay of eperisone in
Intra-assay (n=5) Inter-assay (n=5) Added to plasma Measured RSD Accuracy Measured RSD Accuracy concentration (ng/mL) concentration (%) (%) (ng/mL) (ng/mL) (%) (%) 00.01 00.02 00.05 00.10 00.50 01.00 05.00 10.00
0.01 0.02 0.06 0.10 0.49 1.02 5.05 9.95
6.6 8.5 5.9 2.3 3.0 4.0 5.5 6.0
093.4 097.6 111.7 098.8 097.8 102.4 101.0 099.5
00.01 00.02 00.06 00.11 00.48 01.00 04.91 10.12
8.2 5.1 6.8 4.0 3.0 4.2 5.4 7.5
114.0 099.3 110.3 104.7 095.6 099.8 098.2 101.2
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pharmacokinetic studies of the drug in human volunteers.
ACKNOWLEDGEMENTS This work was supported by the Regional Research Centers Program of the Ministry of Education & Human Resources Development in Korea and partly by 2006 General subject support by Chungbuk National University, Republic of Korea.
REFERENCES Mean plasma concentration-time profile of eperisone after the oral administration at 100 mg to the human volunteers. Each point represents the mean ± S.E. (n=8). Fig. 3.
Pharmacokinetic parameters of eperisone in human volunteers after a single oral dose of two tablets (100 mg) Table
III.
Parameters
Mean ± S.E
Cmax (ng/mL) Tmax (h) AUC0-12h (ng h/mL) AUC0- (ng h/mL) t1/2 (h)
1.25 ± 0.59 1.28 ± 0.64 3.85 ± 1.89 4.21 ± 2.13 3.16 ± 0.41
macokinetic parameters. The mean terminal half-life (t1/2) was 3.16 h; the maximum plasma concentration (Cmax) was 1.25 ng/mL; the time to maximum concentration (Tmax) was 1.38 h; and the area under the plasma concentration-time curves (the AUC0-12h and AUC0-∞) were 3.85 ± 1.89 and 4.21 ± 2.13 ng/mL/h, respectively.
CONCLUSION An LC/ESI-MS/MS method was developed and validated for the determination of eperisone in human plasma. This method is more specific, and sensitive for eperisone than the LC/MS method, requires a minimum sample pretreatment and has a shorter analysis time. This method showed acceptable precision and accuracy and recovery characteristics and was applied successfully for
Ackermann, B. L., Berna, M. J., and Murphy, A.T., Recent advances in use of LC/MS/MS for quantitative high throughput bioanalytical support of drug discovery. Curr. Top. Med. Chem., 2, 53-66 (2002). Fujita, T., Takamatsu, T., Hisamoto, T., Tsutsumi, J., Kinoshita, K., and Kanai, T., Studies on the metabolic fate of 4’-ethtyl-2methyl-3-piperidinopropiopenone hydrochloride(1):absorption, disposition and excretion in rat and guinea pigs. Pharmacometrics, 21, 835-846 (1981). Iwase, S., Mano, T., Saito, M., and Ishida, G., Effect of a centrally-acting muscle relaxant eperisone hydrochloride, on muscle symmmpathetic nerve activity in humans. Funct. Neurol., 7, 459-470 (1992). Kuroiwa, Y., Sobue, I., Tazak, Y. i, Nakanishi, T., Ohtomo, E., and Itahara, K., Effects of E-0646 on cases of spasticity - A double blind comparision using tolperisone hydrochloride. Clin. Eval., 9, 391-419 (1981). Li, D., Xin, W., Shengqiang, Z., Jianpin, S., and Yindi, Z., Rapid and sensitive liquid chromatography-electrospray ionizationmass specrtometry method for the determination of eperisone in human plasma: Method and Clinical Applications. J. Chromatogr Sci., 42, 254-258 (2004). Matuszewski, B. K., Constanzer M. L., and Chavez-Eng, C. M., Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem., 75, 3019–3030 (2003). Mihara, K., Masumura, M., Yoshioka, E., Hanada, K., Nakasa, H., Ohmori, S., Kitada, M., and Ogata, H., Intestinal first-pass metabolism of eperisone in the rat. Pharm. Res., 18, 1131-37 (2001). Takenaka, F., Hasui, M., and Ohkawa, T., Determination of eperisone in human plasma by gas chromatography - mass spectrometry. J. Chromatogr., 584, 261-266 (1992).
