Journal of Chromatography B, 960 (2014) 247–252

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of glimepiride and pioglitazone in human plasma by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic study Xiao-Jia Ni a,1 , Zhan-Zhang Wang b,1 , De-Wei Shang a , Ming Zhang a , Jin-Qing Hu b , Chang Qiu b , Yu-Guan Wen a,∗ a Clinical Laboratory of Phase I, Institution of National Drug Clinical Trials of Guangzhou Brain Hospital, Guangzhou Medical University, 36 MingXin Road, Guangzhou 510370, China b Clinical Pharmacy of Guangzhou Brain Hospital, Guangzhou Medical University, 36 MingXin Road, Guangzhou 510370, China

a r t i c l e

i n f o

Article history: Received 8 February 2014 Accepted 19 April 2014 Available online 28 April 2014 Keywords: LC–MS/MS Glimepiride Pioglitazone Pharmacokinetic

a b s t r a c t The rapid, sensitive, and selective liquid chromatography–electrospray ionization-tandem mass spectrometry method (LC–ESI-MS/MS) for the simultaneous estimation and pharmacokinetic investigation of glimepiride and pioglitazone in human plasma has been developed and fully validated. Glimepiride and pioglitazone, compounds which exert synergistic effects on blood glucose control, were investigated in human plasma using deuterium-labeled analogs as internal standards (IS). Liquid–liquid extraction was carried out on 0.2 mL of human plasma using ethyl acetate, and chromatographic separation was performed on an Agilent Eclipse plus C18 column (4.6 mm × 100 mm, 3.5 ␮m) using a mobile phase consisting of methanol–water–formic acid (95:5:0.1, v/v/v, plus 5 mM ammonium acetate) at a flow rate of 0.8 mL/min. To quantify glimepiride, pioglitazone and their IS, multiple reaction monitoring (MRM) transitions of m/z 491.2→ 352.2, m/z 496.2 → 357.2, m/z 357.2 → 134.2 and m/z 361.2 → 138.2 were performed in positive mode. The total run time was 3.0 min and the elution time was about 2.4 min. The method exhibited good separation of analytes, without interference from endogenous substances. The linear calibration curves were 0.2–250 ng/mL for glimepiride and 0.2–1250 ng/mL for pioglitazone; the lower limit of quantification (LLOQ) was 0.2 ng/mL for both analytes. Intra- and inter-day reproducibility was less than 10% for glimepiride and less than 5% for pioglitazone, with relative errors ranging from −8.00% to 2.80% at the three concentrations of analytes used for quality control (QC). The matrix effect was negligible and recoveries were similar for each analyte and its IS. Glimepiride and pioglitazone were found to be stable under the assay conditions and the method was successfully applied to the evaluation of pharmacokinetic studies of glimepiride and pioglitazone, following oral doses of 2 mg glimepiride tablets and 15 mg pioglitazone tablets to 16 healthy volunteers. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Pioglitazone hydrochloride ((±)-5-({4-[2-(5-ethylpyridin-2yl)ethoxy]phenyl}methyl)-1,3-thiazolidine-2,4-dione hydrochloride, Fig. 1) is a widely used thiazolidinedione (TZD) that selectively activates peroxisome proliferator-activated receptor-␥ (PPAR␥) and increases insulin-stimulated glucose uptake in peripheral tissues [1] as well as insulin sensitivity in hepatic and adipose

∗ Corresponding author. Tel.: +86 20 8126 8052; fax: +86 20 8189 1391. E-mail address: [email protected] (Y.-G. Wen). 1 Both authors contributed equally to the project and are considered co-first authors. http://dx.doi.org/10.1016/j.jchromb.2014.04.039 1570-0232/© 2014 Elsevier B.V. All rights reserved.

tissue [2–4]. Glimepiride (3-ethyl-4-methyl-N-{2-[4-({[(4methylcyclohexyl)carbamoyl]amino}sulfonyl)phenyl]ethyl}2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamide, Fig. 1) is a long-acting sulfonylurea that inhibits ATP-sensitive potassium channels and stimulates secretion of insulin from pancreatic ␤-cells into the blood [5]. Concomitant use of glimepiride and pioglitazone exerts a synergistic action and should achieve better control of blood glucose levels. Combination products containing the two oral antihyperglycemic agents, pioglitazone and glimepiride, have been launched and there is a growing demand from clinical trials of these combinations for a high throughput detection method that could quantify both compounds. Although quantitative determination of each single analyte by LC–MS/MS has been reported [6–11], analytical

248

X.-J. Ni et al. / J. Chromatogr. B 960 (2014) 247–252

Fig. 1. Chemical structures and mass spectra of (A) glimepiride (M + H)+ , (B) glimepiride-d5 (M + H)+ , (C) pioglitazone (M + H)+ , (D) pioglitazone-d4 (M + H)+ .

methods that allow simultaneous detection of both compounds in human plasma are limited. As far as we know, only one simultaneous determination assay have been reported in human plasma by LC–MS/MS [11] and other published methods are in biological fluid of rat plasma [12], dog plasma [13], equine plasma and equine urin [14], or in pharmaceutical dosage forms [13,15] using LC–MS/MS or HPLC method, namely, disadvantage of diffferent endogenous interferents might exist in biofluids, also have the disadvantage of long run times (up to 30 min), a high limit of quantification (∼50 ng/mL) and need a large plasma volume, making them unsatisfactory and impracticable as routine analytical methods for large numbers of clinical samples. Hess et al. [11] presented the simultaneous assay which allowed identification of 14 and quantification of 11 oral antidiabetics in human plasma for an unclear hypoglycaemia in case of forensic practice or negative autopsy. As the pKa values differed owing to their chemical structures, the price for the integration was the loss of efficiency and sensitivity. The cumbersome double extraction procedure combined with pH adjustment and high LLOQs (1 ng/mL for pioglitazone and 10 ng/mL for glimepiride) limitted the use in clinical trials. We have now developed and validated a rapid, reliable, and practicable LC–ESI-MS/MS method for the simultaneous determination of pioglitazone and glimepiride in a single run. Simultaneous detection showed significant advantages in terms of convenience, speed, cost, and throughput capacity and could easily be adapted for analysis of large numbers of samples from pharmacokinetic studies [16–20]. In this method, a simple and economical liquid–liquid extraction was used instead of more cumbersome solid-phase extraction. It was not found necessary to use gradient elution and the detection time was decreased to 3 min. Sensitivity and selectivity were enhanced by using an MRM detector coupled with an acidic mobile phase, rather than UV detection. LLOQs of 0.2 ng/mL were achieved for both analytes. Deuterium-labeled IS were used to reduce differences in retention time, peak shapes, stability, and matrix effects compared with the analytes. To the best of our knowledge, this simultaneous estimation of pioglitazone and glimepiride in human plasma could be successfully applied to a human pharmacokinetic study following oral administration of glimepiride and pioglitazone tablets and provides

a practical, sensitive, and simultaneous analytical method for use in clinical research. 2. Materials and methods 2.1. Chemicals and reagents Pioglitazone tablets (Actos® , 15 mg, lot: 147A) were purchased from Takeda Pharmaceutical Co., Ltd. (Japan) and glimepiride tablets (Amaryl® , 2 mg, lot: B2007) were bought from SanofiAventis Pharmaceutical Co., Ltd. (China). The purity of standard reference of glimepiride was 99.5% and that of pioglitazone was 99.1%, as determined by the National Institute for the Control of Pharmaceutical and Biological Products. The IS, pioglitazoned4 (97.0%) and glimepiride-d5 (99.6%), were supplied by Toronto Research Chemicals. HPLC grade methanol (lot: 3226794), ethyl acetate and methanol were purchased from Dikma (USA) and chromatography grade ammonium formate was obtained from Sigma (USA). Deionized water was produced using a Milli-Q academic reagent grade water purification system (Millipore, USA). 2.2. Apparatus An Agilent 1200 HPLC system coupled to an Agilent 6410 triple-quadrupole mass spectrometer (Agilent Technologies, Inc., USA) was used to detect the analytes and IS. A quaternary pump, autosampler and column oven degasser were included in the chromatographic analysis system. The mass spectrometer was equipped with an electrospray ionization source and drying gas of ultra-high purity (UHP) was used. We used Salvis Lab vacuum drying apparatus (Vacucenter VC20, CH-6343 Rotkreuz, Switzerland) to evaporate extracts from human plasma, an Agilent MassHunter Chemstation (B.01.03) to process raw data, and DAS 3.2.4 software to calculate the pharmacokinetic parameters. 2.3. LC–ESI-MS/MS Separations were carried out on a C18 column (Agilent Eclipseplus, 4.6 mm × 100 mm, 3.5 ␮m). The separation temperature was set at 35 ◦ C and a mixture of 95% methanol, 5% water, 5 mM

X.-J. Ni et al. / J. Chromatogr. B 960 (2014) 247–252

ammonium formate and 0.1% formic acid was used as the mobile phase, at a flow rate of 0.8 mL/min. Quantification was carried out in MRM ion mode. The parameters of the mass spectrometer were optimized and set as follows: HV capillary at 4000 V, nebulizer at 50 psi, and drying gas flow rate at 10 L/min at 350 ◦ C. The impact energy and split voltage for glimepiride and glimepiride-d5 were set at 6 units and 125 V, and those for pioglitazone and pioglitazoned4 were set at 28 units and 155 V, with a dwell time of 0.1 s. The run time was 3.0 min, and analytes eluted at about 2.4 min. 2.4. Preparation of stock solutions, calibration curves and QC samples Stock solutions of glimepiride, pioglitazone, and IS were prepared by dissolving accurately weighed samples in the mobile phase to obtain concentrations of 1 g/L for analytes and 0.1 g/L for IS. The stock solutions were then serially diluted with mobile phase to obtain reference solutions of 2, 5, 50, 500, 1000, 2000, and 2500 ng/mL for glimepiride; 2, 5, 200, 2000, 5000, 10,000, and 12,500 ng/mL for pioglitazone; and 500 ng/mL for both glimepirided5 and pioglitazone-d4. These solutions were also used to construct plasma calibration curves. All solutions were stored at 4 ◦ C and brought to room temperature before use. Calibration samples were prepared by spiking 0.2 mL of plasma with 10 ␮L of the pioglitazone and glimepiride solutions and 20 ␮L of the IS solutions to obtain final concentrations of 0.2, 0.5, 5, 50, 100, 200, and 250 ng/mL for glimepiride; 0.2, 0.5, 20, 200, 500, 1000, and 1250 ng/mL for pioglitazone; and 50 ng/mL for glimepiride-d5 and pioglitazone-d4. QC samples were prepared at concentrations of 0.5, 50, and 200 ng/mL for glimepiride; and 0.5, 200, and 1000 ng/mL for glimepiride, respectively. 2.5. Sample preparation Plasma samples were processed by liquid–liquid extraction. 0.2 mL of plasma and 20 ␮L of IS stock solution were pipetted into a 2.0 mL polypropylene tube and vortexed for 15 s. Ethyl acetate (1 mL) was added to the mixture and the whole vortexed for 50 s to extract compounds from the plasma. After centrifugation at 15,000 r/min for 3 min, the supernatant was transferred into a 1.5 mL V-bottom Eppendorf centrifuge tube and then vacuumdried at 42 ◦ C. The samples were redissolved using 100 ␮L of mobile phase, then the mixture vortexed for 15 s and centrifuged at 15,000 r/min for 5 min. For the analysis, 2 ␮L of the supernatant was injected into the LC–ESI-MS/MS system. 2.6. Method validation 2.6.1. Selectivity and lower limit of quantification Selectivity was determined by testing plasma samples from six healthy volunteers for the presence of interfering peaks in the retention window. No interfering peaks were observed and the selectivity was considered to be acceptable. The LLOQ was the concentration at which the precision and variance of accuracy were ≤20%, and the signal-to-noise ratio (S/N) was ≥5. Relative standard deviation (RSD, %) was used to assess precision. Relative error (RE, %) was used to estimate accuracy, calculated as ((measured conc. − nominal conc.)/nominal conc.) × 100%. 2.6.2. Calibration curves In the calculation of calibration curves, linear weighted leastsquares analysis and a weighting factor of 1/x2 were used. A correlation coefficient (r) > 0.99 was expected in all calibration curves. Residues were tested for by injecting a blank sample after three injections at the highest calibration concentration; the

249

measurement for the blank sample was required to be less than 20% of the LLOQ. 2.6.3. Precision and accuracy To determine intra-day precision and accuracy, five sets of spiked QC samples were prepared and analyzed on the same day. Another ten sets of QC samples were freshly prepared and analyzed over two consecutive days. Calibration curves were freshly prepared to calculate the QC samples. RSD and RE were used to estimate the precision and accuracy. RSD values of less than 15% and RE values in the range −15% to 15% were required to meet our precision and accuracy requirements. 2.6.4. Recovery and matrix effects The absolute extraction recoveries of analytes at three different QC concentrations and of IS at 50 ng/mL were measured by comparing peak areas of spiked samples of plasma, processed as described in Section 2.5 (plasma was obtained from five healthy volunteers, one batch with haemolysis, peak area labeled as B) to those of compounds diluted with mobile phase (peak area labeled as A). The matrix effects of human plasma were evaluated by comparing the peak area of compound mixed with plasma that remained after extraction (C) to peak area (A) at equivalent concentrations. The ratio (B/A × 100) is defined as the recovery and the ratio of C/A × 100 is defined as the matrix effect. An RSD value less than 15% is considered to be satisfactory. A matrix effect value of 100% indicates that the matrix components have a small impact on the quantification of the analyte. Similar recovery or matrix effect values between analyte and IS usually indicate similar impacts of the extraction procedure or matrix components on analytes and IS. 2.6.5. Stability The stability of reference solutions was tested by measuring the concentrations of analytes after 3 h at room temperature and 3 days at 4 ◦ C. The stability of the biological matrix was examined by calculating the concentration of analytes after short-term (24 h at 4 ◦ C) and long-term storage (50 days at −20 ◦ C). Freeze/thaw stability was determined by analysis of samples subjected to three freeze/thaw cycles (−20 ◦ C to 25 ◦ C). Stability under test conditions was investigated by measuring the concentrations of analytes after storage in an autosampler rack (24 h at 25 ◦ C). Generally, a sample was considered to be stable in the biological matrix if the concentration variance was less than 15% of the freshly prepared samples. 2.7. Pharmacokinetic study Sixteen healthy Chinese volunteers (male, 19–28 years, BMI 19–24) were included in a pharmacokinetic study. Health status of the applicants was evaluated by reviewing medical history and tests prior to enrollment, and only eligible candidates were chosen. Prior to enrollment, the investigator have provided written information sheets and a full explanation of the purpose, methods, and possible adverse effects of the clinical research which had been reviewed and approved by the ethics committee of Guangzhou Brain Hospital to every healthy subject who was old enough to read and understand. All participants have given written informed consent. After authorization by the ethics committee of the Guangzhou Brain Hospital, the pharmacokinetic study began. Each subject was fasted for 10 h before medication and received one pioglitazone tablet (15 mg) and one glimepiride tablet (2 mg) along with 250 mL water by oral administration. Blood samples were collected in heparin tubes before medication and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36 and 48 h after drug administration. All samples were immediately centrifuged at 3000 r/min for 10 min and then stored at −20 ◦ C before analysis.

250

X.-J. Ni et al. / J. Chromatogr. B 960 (2014) 247–252

Fig. 2. Representative chromatograms for human plasma (A); human plasma spiked with 0.2 ng/mL pioglitazone or glimepiride (LLOQ) and 50 ng/mL of the internal standards (B); human plasma from a volunteer after oral administration of one 15 mg glimepiride tablet and one 2 mg pioglitazone tablet (C).

3. Results and discussion 3.1. Selection of IS In LC–ESI-MS/MS detection, the purpose of an IS is primarily to correct detection errors [21]. Structural analogs have previously been reported as the IS in detection of glimepiride [10] and pioglitazone [7], but structural variations led to inconsistent extraction recovery. In the present study, deuterium-labeled analogs of glimepiride-d5 and pioglitazone-d4 were used as IS to eliminate any variation in retention time, recovery or matrix effect caused by structural differences between the analyte and IS. 3.2. Sample preparation Different sample pre-treatment methods have been evaluated during this study. Compared with solid phase extraction, liquid–liquid extraction was found to offer satisfactory efficiency together with low cost and good safety. We investigated and compared extraction solvents with different polarities. Ethyl acetate was found to give the highest recovery (75–80%), followed by ethyl acetate–dichloromethane (4:1, v/v; ∼70%) and n-hexane (∼50%). Ethyl acetate was thus chosen as the extraction solvent. 3.3. HPLC–ESI-MS/MS Analyte responses in positive and negative ESI were observed and compared; the positive ionization mode, with a strong and stable MS signal, was chosen to optimize the ionization polarity. The composition of the mobile phase has a significant impact on the separation and ionization of analyte and IS. Of the two frequently used organic modifiers, methanol and acetonitrile, methanol was preferred because of the higher signal response. The effect of the percentage of methanol (range 75–95%) was evaluated and 95%

was found to give the best signal response and exerted fast elution. Addition of 5 mM ammonium acetate to the aqueous medium gave good peak shapes and a higher detection response; and addition of 0.1% formic acid enhanced the mass-to-charge [M + H]+ precursor ion. After this evaluation, a mobile phase consisting of methanol–water (95:5, v/v, plus 5 mM ammonium acetate and 0.1% formic acid) was selected for the simultaneous determination of glimepiride and pioglitazone. To obtain a high S/N, the most intense product ion transitions (m/z 491.2 → 352.2 for glimepiride, m/z 496.2 → 357.2 for glimepiride-d5, m/z 357.2 → 134.2 for pioglitazone and m/z 361.2 → 138.2 for pioglitazone-d4) were chosen (Fig. 1). No interfering signals were observed at the retention times of glimepiride (2.4 min), glimepiride-d5 (2.4 min), pioglitazone (2.3 min), or pioglitazone-d5 (2.3 min) (Fig. 2).

3.4. Method validation 3.4.1. Selectivity and LLOQ In this assay, the LLOQ values reached 0.2 ng/mL in plasma (S/N > 5) for both glimepiride and pioglitazone, with a small injection volume of 2 ␮L. We have compared our method to previously reported LC–ESI-MS/MS methods for a single analyte, which showed LLOQs of 0.5–9 ng/mL for pioglitazone and 0.02–5 ng/mL for glimepiride, with injection volumes of at least 10 ␮L [6–8,10,22]. Although the use of higher injection volumes can achieve good LLOQs, the analytical column lifetime and sensitivity are reduced since the analyte signal does not increase proportionally with injection volume because of peak broadening [6]. Residual interference in complicated biological fluids may also be detrimental to detection. In the present study, an injection volume of 2 ␮L was found to provide satisfactory LLOQs. To balance sensitivity and high throughput detection, we chose a mobile phase containing 95% methanol plus 5 mM ammonium acetate and 0.1% formic acid

X.-J. Ni et al. / J. Chromatogr. B 960 (2014) 247–252

251

Table 1 The precision and accuracy of the determination of pioglitazone and glimepiride in human subjects. Measurement interval

Glimepiride

Nominal conc. (ng/mL) Intra-day (n = 5)

0.5 50 200

Inter-day (n = 15)

0.5 50 200

Pioglitazone

RSD (%)

RE (%)

0.48 ± 0.03 49.45 ± 1.15

9.40 4.27

−4.00 −1.10

196.87 ± 2.48

2.50

−1.57

0.47 ± 0.03 48.43 ± 1.46

5.59 3.02

−6.00 −3.14

195.68 ± 2.17

1.11

−2.16

Measured conc. (ng/mL) (mean ± SD)

to enhance the analyte response and a C18 column of 3.5 ␮m to separate the analyte peaks from interference. Representative chromatograms for human plasma, human plasma spiked with 0.2 ng/mL pioglitazone or glimepiride at their LLOQs together with 50 ng/mL of the IS are shown in Fig. 2 (rank 1 and 2), respectively. The precision of glimepiride at LLOQ was 9.34% and the accuracy was −10.00%. For pioglitazone at LLOQ, the precision was 6.43% and the accuracy was 5.00%. No endogenous interference peaks from the plasma matrix were observed at the retention times of analytes or internal standards. Typical chromatograms for a sample from a volunteer 48 h after oral administration of glimepiride and pioglitazone tablets are presented in Fig. 2 (rank 3). 3.4.2. Linearity The assay showed a good linear response over the range 0.2–250 ng/mL for glimepiride and 0.2–1250 ng/mL for pioglitazone in human plasma. The regression equations (n = 7) were y = 0.9420x − 0.0001 (r = 0.9991) for glimepiride and y = 0.8855x + 0.0003 (r = 0.9983) for pioglitazone, where y is the peak area ratio of analyte to IS and x is the nominal plasma concentration. The residues were 0.00% for glimepiride and 10.37% for pioglitazone. At calibration curve concentrations, the RSD and RE values ranged from 1.14% to 5.36% and −10.00% to 10.00% for glimepiride and from 1.15% to 5.17% and −10.00% to 8.00% for pioglitazone. 3.4.3. Precision and accuracy Precision and accuracy are summarized in Table 1. The intra- and inter-day precisions were within 10% for glimepiride and within 5% for pioglitazone at all plasma QC concentrations. Accuracy ranged from −2.16 to −6.00% for glimepiride and from −8.00% to 2.80% for pioglitazone. The RSD values for precision were less than 10% at the LLOQ and 6% at other QC concentrations for the two analytes, indicating that the assay is precise and accurate. 3.4.4. Recovery and matrix effects The absolute recovery data for glimepiride, pioglitazone and their IS are shown in Table 2. Recovery was found to be in the range 83.41–88.51% for pioglitazone and 80.80% for its IS, 77.11–78.53%

Nominal conc. (ng/mL)

RSD (%)

RE (%)

0.46 ± 0.02 198.57 ± 2.54

4.89 1.38

−8.00 −0.72

1027.95 ± 10.96

1.15

2.80

0.47 ± 0.02 198.57 ± 2.54

4.28 1.28

−6.00 −0.72

1027.95 ± 10.96

1.07

2.80

Measured conc. (ng/mL) (mean ± SD)

0.5 200 1000 0.5 200 1000

Table 2 The recoveries and matrix effects of pioglitazone and glimepiride in human plasma (mean ± SD, n = 5). Analytes

Nominal conc. (ng/mL)

Recoveries (%)

Matrix effects (%)

Glimepiride

0.5 50 200

77.75 ± 3.33 78.53 ± 3.25 77.11 ± 2.26

92.48 ± 4.56 87.49 ± 4.92 84.35 ± 3.35

Pioglitazone

0.5 200 1000

88.51 ± 5.78 86.63 ± 7.84 83.41 ± 3.33

85.06 ± 6.69 87.57 ± 11.03 91.18 ± 9.97

50 50

74.90 ± 2.47 80.80 ± 3.14

91.08 ± 4.34 89.67 ± 6.57

Glimepiride-d5 Pioglitazone-d4

for glimepiride and 74.90% for glimepiride-d5. After the extraction procedures, the recoveries were consistent and precise between analytes and their IS. The matrix effect was assessed by comparing the signals of analytes in plasma with those of analytes dissolved in mobile phase at the same concentration. The matrix effects were 92.48%, 87.45%, and 84.35% for glimepiride; and 85.06%, 87.57%, and 91.18% for pioglitazone at three QC concentrations. The matrix effects were 91.08% for glimepiride-d5 and 89.67% for pioglitazone-d4. Negligible matrix effects were observed, indicating consistent suppression of ionization. The high precision, accuracy, recovery and low matrix effects seen in this assay can be attributed, at least in part, to the similar structures and behavior of the deuterium-labeled IS. 3.4.5. Stability The stabilities of pioglitazone and glimepiride in solvent and human plasma were fully evaluated under all conditions that samples might be exposed to before detection. Stability results are summarized in Table 3. Solutions of glimepiride, pioglitazone and IS stored at 4 ◦ C for 3 days and at room temperature (25 ◦ C) for 3 h showed good stabilities ranging from 95.30% to 112.00%. There was no obvious degradation of glimepiride or pioglitazone in plasma after short-term storage, three freeze–thaw cycles, long-term storage, or storage in the autosample rack. The expected delays in testing during normal assay operation appeared to have no effect on the detection of analytes. The method was, therefore, shown to be suitable for routine testing.

Table 3 The stability of pioglitazone and glimepiride in human plasma under test conditions (mean ± SD, n = 5). Analytes

Nominal conc. (ng/mL)

Short-term (24 h, 25 ◦ C)

Freeze–thaw (3-cycles, −20 ◦ C to 25 ◦ C)

Long-term (50 d, −20 ◦ C)

Autosampler (24 h, 25 ◦ C)

Pioglitazone

0.5 200 1000

0.45 ± 0.01 195.80 ± 0.93 1018.87 ± 3.00

0.48 ± 0.04 200.78 ± 0.88 1028.09 ± 3.69

0.47 ± 0.01 201.30 ± 2.01 1044.06 ± 14.12

0.46 ± 0.02 196.34 ± 3.39 1011.35 ± 1.75

Glimepiride

0.5 50 200

0.47 ± 0.05 49.18 ± 0.44 195.54 ± 0.28

0.51 ± 0.01 50.08 ± 0.38 198.26 ± 1.24

0.49 ± 0.02 50.16 ± 0.31 199.41 ± 2.67

0.47 ± 0.03 48.94 ± 0.84 195.06 ± 3.70

252

X.-J. Ni et al. / J. Chromatogr. B 960 (2014) 247–252

Table 4 Pharmacokinetic parameters after oral administration of 15 mg pioglitazone and 2 mg glimepiride tablets to 16 volunteers (mean ± SD, n = 16). Parametersa

T1/2 (h)

Tmax (h)

Cmax (ng/mL)

AUC0–t (ng h/mL)

AUCinf (ng h/mL)

AUCt-inf (%)

Pioglitazone Glimepiride

7.11 ± 2.42 7.37 ± 2.24

1.66 ± 0.70 2.53 ± 0.62

641.21 ± 114.17 163.77 ± 45.73

5890.18 ± 1498.55 817.36 ± 287.74

6001.64 ± 1640.89 823.04 ± 290.87

99.36 ± 0.40 98.56 ± 2.01

a

T1/2 , elimination half-life; Cmax , maximum plasma concentration; Tmax , time to Cmax ; AUC, area under the plasma concentration–time curve.

developed a high throughput assay for both analytes using a simple liquid–liquid extraction method and MRM detection and, with an LLOQ of 0.2 ng/mL, have achieved superior sensitivity compared to previous methods. Our new assay method has been successfully applied to a pharmacokinetic study of pioglitazone and glimepiride tablets in humans. References

Fig. 3. Mean plasma concentration–time curves of glimepiride and pioglitazone in 16 volunteers after oral administration of one 15 mg pioglitazone tablet and one 2 mg glimepiride tablet.

3.5. Application of the method The method was applied to the measurement of plasma samples from 16 healthy volunteers after an oral dose of one 15 mg pioglitazone tablet and one 2 mg glimepiride tablet. The mean pioglitazone and glimepiride plasma concentrations versus time profiles are represented in Fig. 3. As shown in Table 4, glimepiride reached a peak concentration at 2.53 ± 0.62 h and pioglitazone at about 1.66 ± 0.70 h. Both compounds were undetectable 36 h after oral administration. High rate of AUCt-inf (98.56%∼99.36%) demonstrated that our method was sensitive enough to capture the terminal elimination phase. The pharmacokinetic values of Cmax and AUC0–t for both glimepiride and pioglitazone were also in agreement with published values [6,23,24]. The present assay method has thus been shown to be suitable for pharmacokinetic studies. 4. Conclusion In our study, a simultaneous LC–ESI-MS/MS quantification method for pioglitazone and glimepiride in human plasma was developed and fully validated for clinical trials. We have

[1] Y. Miyazaki, A. Mahankali, M. Matsuda, L. Glass, S. Mahankali, E. Ferrannini, K. Cusi, L.J. Mandarino, R.A. DeFronzo, Diabetes Care 24 (2001) 710. [2] Y. Miyazaki, A. Mahankali, E. Wajcberg, M. Bajaj, L.J. Mandarino, R.A. DeFronzo, Clin. Endocrinol. Metab. 89 (2004) 4312. [3] R. Kawamori, M. Matsuhisa, J. Kinoshita, K. Mochizuki, M. Niwa, T. Arisaka, M. Ikeda, M. Kubota, M. Wada, T. Kanda, M. Ikebuchi, R. Tohdo, Y. Yamasaki, Diabetes Res. Clin. Pract. 41 (1998) 35. [4] M. Kawaguchi-Suzuki, R.F. Frye, Front Pharmacol. 4 (2013) 147. [5] F.M. Ashcroft, P. Rorsman, Biophys. Mol. Biol. 54 (1989) 87. [6] Y.J. Xue, K.C. Turner, J.B. Meeker, J. Pursley, M. Arnold, S. Unger, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 795 (2003) 215. [7] Z.J. Lin, W. Ji, D. Desai-Krieger, L. Shum, J. Pharm. Biomed. Anal. 33 (2003) 101. [8] C. Pistos, M. Koutsopoulou, I. Panderi, Biomed. Chromatogr. 19 (2005) 394. [9] L. Chakradhar, R. Kallem, A. Karthik, B.T. Sundari, S. Ramesh, R. Mullangi, N.R. Srinivas, Biomed. Chromatogr. 22 (2008) 58. [10] I. Salem, J. Idrees, J.I. Al Tamimi, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 799 (2004) 103. [11] C. Hess, F. Musshoff, B. Madea, Anal. Bioanal. Chem. 400 (2011) 33. [12] P.B. Musmade, K.B. Talole, P.B. Deshpande, A. Karthik, S.M. Pathak, S. Pandey, N. Udupa, Arzneim. Forsch. 61 (2011) 23. [13] K. Arumugam, G. Subramanian, C.M. Rao, K. Bhat, A. Ranjithkumar, P. Musmade, M. Surulivelrajan, K. Karthikeyan, N. Udupa, J. Pharm. Sci 21 (2008) 421. [14] E.N. Ho, K.C. Yiu, T.S. Wan, B.D. Stewart, K.L. Watkins, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 811 (2004) 65. [15] D. Jain, S. Jain, D. Jain, M. Amin, J. Chromatogr. Sci. 46 (2008) 501. [16] V. Nitsche, H. Mascher, Arzneim. Forsch. 30 (1980) 1855. [17] H. Tada, A. Fujisaki, K. Itoh, T. Suzuki, Clin. Pharm. Ther. 28 (2003) 229. [18] S. Sved, D.L. Wilson, Biopharm. Drug Dispos. 1 (1980) 111. [19] X. Liu, X.J. Ni, D.W. Shang, M. Zhang, J.Q. Hu, C. Qiu, F.T. Luo, Y.G. Wen, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 941 (2013) 10. [20] A. Buchwald, K. Winkler, T. Epting, MC Pharmacol. Toxicol. 12 (2012) 2. [21] E. Stokvis, H. Rosing, J.H. Beijnen, Rapid Commun. Mass Spectrom. 19 (2005) 401. [22] J. Martin, W. Buchberger, J.L. Santos, E. Alonso, I. Aparicio, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 895–896 (2012) 94. [23] L. Tian, Y.L. Huang, L. Hua, J.J. Jiang, Y.S. Li, Chin. J. Clin. Pharmacol. Ther. 11 (2006) 868. [24] B. Jagadeesh, D.V. Bharathi, C. Pankaj, V.S. Narayana, V. Venkateswarulu, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 930 (2013) 136.

Simultaneous determination of glimepiride and pioglitazone in human plasma by liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic study.

The rapid, sensitive, and selective liquid chromatography-electrospray ionization-tandem mass spectrometry method (LC-ESI-MS/MS) for the simultaneous ...
1MB Sizes 0 Downloads 6 Views

Recommend Documents