Journal of Chromatographic Science Advance Access published July 16, 2015 Journal of Chromatographic Science, 2015, 1–9 doi: 10.1093/chromsci/bmv097 Article
Article
Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC–MS-MS: Application to Pharmacokinetic Interaction Study Yan-rong Ma1,2, Zhi Rao1, A-xi Shi1,2, Ya-feng Wang1,2,3, Jing Huang1,2, Miao Han1,2, Xin-dong Wang1,2, Yong-wen Jin1,2, Guo-qiang Zhang1, Yan Zhou1, Fan Zhang1, Hong-yan Qin1, and Xin-an Wu1,* 1
Department of Pharmacy, The First Hospital of Lanzhou University, Lanzhou 730000, China, 2School of Pharmacy, Lanzhou University, Lanzhou 730000, China, and 3Department of Pharmacy, Qing Hai Provincial Hospital, Xining 810000, China *Author to whom correspondence should be addressed. Email: [email protected] Received 11 December 2014; Revised 9 June 2015
Abstract A simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed and validated for the simultaneous quantitation of metformin (MTF), metoprolol (MET), α-hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma using paracetamol as an internal standard (IS), respectively. The sample preparation involved a proteinprecipitation method with methanol after the addition of IS. The separation was performed on an Agilent HC-C18 column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, using methanol–water containing 0.1% formic acid (39:61, v/v) as mobile phase, and total run time was 8.5 min. MS–MS detection was accomplished in multiple reaction monitoring mode with positive electrospray ionization. The monitored transitions were m/z 130.1 → 60.2 for MTF, m/z 268.2 → 116.1 for MET, m/z 284.2 → 116.1 for HMT, m/z 254.2 → 116.1 for DMT and m/z 152.3 → 110.1 for IS. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 19.53–40,000 ng/mL for MTF, 3.42–7,000 ng/mL for MET, 2.05–4,200 ng/mL for HMT and 1.95–4,000 ng/mL for DMT, respectively. The analytical method was successfully applied to drug interaction study of MTF and MET after oral administration of MTF and MET. Results suggested that the coadministration of MTF and MET results in a significant drug interaction in rat.
Introduction Metformin (MTF), a biguanide derivative, is one of the first-line drugs for the treatment of type 2 diabetes mellitus (T2DM), and it is often used alone or in combination with other drugs to control blood glucose level (1). Although the mechanism that is responsible for blood glucose improvement is not well understood, MTF, to some extent, decrease hepatic glucose production and improve insulin sensitivity by increasing the uptake and utilization of peripheral glucose (2, 3). The main adverse effects of MTF involve gastrointestinal intolerance
and lethal lactic acidosis (4, 5). In vivo, MTF is not metabolized by hepatic cytochrome P450 and it is excreted into urine via active secretion (∼78.9–99.9%) by renal tubular (6). Metoprolol (MET), a selective β-adrenoceptor antagonist, is used in clinical practice in the racemic form for the treatment of cardiovascular disorders, such as hypertension, arrhythmia and heart failure (7, 8). MET is metabolized through O-demethylation (65% of dose), α-hydroxylation (10% of dose) and N-dealkylation, which form the correspondent metabolites O-desmethylmetoprolol (DMT), α-hydroxymetoprolol
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
1
2 (HMT) and N-dealkylmetoprolol (9–11). The α-hydroxylation is catalyzed almost entirely, and O-demethylation partially, via CYP2D6 (12, 13). About 85% of the administered drug is excreted in the urine as metabolites, along with small amounts of unchanged parent drug (0.990 and the deviations of the calculated concentrations were within ±15% of the nominal concentrations. The lower limit of quality quantification (LLOQ) of the assay was defined as the lowest concentration that could be quantified (LLOQ, relative standard deviation (RSD) < 20% and S/N ≥ 10). Precision and accuracy The accuracy and precision of the established method were evaluated by QC samples at low, medium and high concentrations. Three validation batches, each containing six replicates of LQC, MQC and HQC, were assayed to assess the precision and accuracy of the method
Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC–MS-MS on three consecutive validation days. The percentage deviation of the mean from true values, expressed as relative error (RE), and the RSD serve as the measure of accuracy and precision, respectively. Extraction recovery and matrix effect The extraction recovery was evaluated by comparing peak areas of analytes and IS obtained from extracted spiked samples with those of the postextracted spiked samples at corresponding concentrations. The matrix effect was accessed by comparing the peak areas of blank plasmas spiked with analytes and IS after extraction with those for the standard solutions at the same concentration. Stability The stability of analytes in rat plasma was assessed by analyzing QC samples at three concentration levels through three freeze-thaw cycles (−20°C to room temperature as a cycle), on the bench at room temperature for 24 h (short-term stability), at −20°C for 21 days (long-term stability) and 24 h after prepared at 4°C.
Pharmacokinetic interaction between MTF and MET in rats All studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Wistar rats aged 8 weeks with body weigh ∼180–220 g were obtained from the Experimental Animal Center of Lanzhou University (Lanzhou, China). Rats were housed in plastic cages and maintained at 25°C under 12–12 h alternating light–dark cycle with free access to food and water. Male rats were randomly divided into three groups (n = 8/group). Rats in Group 1 were orally administered MTF (100 mg/kg, a clinically equivalent dose) (18, 19); rats in Group 2 were given MET (40 mg/ kg, a clinically equivalent dose) and rats in Group 3 were given MTF (100 mg/kg) plus MET (40 mg/kg). Blood samples (each ∼250 µL) were collected via the femoral artery into heparinized tubes before and 2, 5, 10, 20, 40, 60, 90, 120, 240, 360, 480 and 720 min after oral administration, respectively. Rats were immediately supplemented with the equal volume (250 µL) of heparin–saline solution after each blood collection. The blood samples were immediately centrifuged at 10,000 g for 10 min. The plasma layer was transferred to clean tubes and stored at −20°C. Blank plasma was obtained from the rat without any drug administration and was used to investigate the method development and validation. Plasma concentration–time profile for each rat was analyzed with Drug and Statistics 2.0 program (DAS, Medical College of Wannan, China) software by noncompartmental analysis. The pharmacokinetic parameters included the area under the plasma concentration–time curve (AUC), the time-averaged total body clearance (Cl), the terminal half-life (t1/2) and apparent volume of distribution at steady state (Vd). The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were directly read from the experimental data. Statistical analysis was computed using SPSS 13.0 software. Data were expressed as mean ± SD. Statistically significant differences of data from two sets were compared using one-way analysis of variance. In all statistical analyses, P < 0.05 or P < 0.01 was considered to indicate a statistically significant result.
Results Method development Under the ESI condition chosen, all of the compounds exhibited higher sensitivity in the positive mode than the negative mode, and the
3
most abundant ions are [M + H]+ for them. Therefore, [M + H]+ from MTF, MET, HMT, DMT and IS were selected as the precursor ions and subsequently fragmented in MS/MS mode to obtain the product ion spectra (Figure 1). The purpose is that the background noise could be reduced and the compounds confirmation could be more accurately, and the risk of false positive due to the interference of matrix was greatly reduced. The choice of mobile phase should be concerned based on the consideration of ionization efficiency before the analytes enter the MS/MS system in order to obtain nice resolution and high sensitivity. As for the choice of strong elution mobile phase, methanol and acetonitrile were considered as two candidates. The result showed that there was no obvious difference in the responses of analytes between methanol– water and acetonitrile–water. Therefore, methanol was chosen in the study for its lower price than acetonitrile. To obtain the maximum sensitivity, we investigated the effects of pH with various mobile phases on the ionization efficiency. Both analytes and IS were found to have the highest response and the best peak shapes in the mobile phase containing 0.1% formic acid. The LC mobile phase was optimized with varying percentages of organic solvent and different modifiers in water to obtain high sensitivity. Analytes and IS were separated on an Agilent HC-C18 column with a mobile phase consisting of methanol and water containing 0.1% formic acid (39:61, v/v). Moreover, width peak, S/N and system stability during an analytical batch (50 h) were considered to find the optimal injection volume (10 µL). An efficient cleanup for bio-samples to remove protein and potential interferences prior to LC–MS-MS analysis was an important point in the method development. The effective and simple protein precipitation was employed in our work. Methanol was chosen as the protein-precipitation solvent because it exhibited better effect than acetonitrile and perchloric acid.
Method validation Selectivity Figure 2 shows the typical chromatograms of blank plasma, blank plasma spiked with analytes at LLOQ and plasma sample collected at 10 min after administration of MET (40 mg/kg) and MTF (100 mg/kg). The retention times of MET, HMT, DMT, MTF and IS were 6.9, 3.6, 3.8, 3.1 and 4.4 min, respectively. The total chromatographic run time was 8.5 min. Figure 2c (1) shows that the interference was completely separated from MET, and retention time of the interference was 4.0 min. However, there were no obvious endogenous interferences under the described chromatographic conditions. Calibration curve and lower limit of quantification The calibration curves were created by plotting the peak area ratios of the various analytes to IS versus nominal concentration of the analyte standards. The four calibration curves were linear over the concentration range of 3.42–7,000 ng/mL for MET, 2.05–4,200 ng/mL for HMT, 1.95–4,000 ng/mL for DMT and 19.53–40,000 ng/mL for MTF, with a correlation coefficient (R 2) ≥ 0.9980 for both analytes. The lowest concentration with the RSD < 20% and S/N ≥ 10 was taken as LLOQ, which was found to be 0.86, 2.05, 1.95 and 2.79 ng/mL for MET, HMT, DMT and MTF, respectively. Precision and accuracy Intraday and interday precision for all of the QC samples did not exceed 15% RSD, and accuracy values did not exceed 15% deviation from the true value for the analytes at any tested concentrations. Precision and accuracy determinations are listed in Table I. These results
4
Ma et al.
Figure 1. Chemical structures and product ion mass spectra of MET (a), HMT (b), DMT (c), MTF (d) and IS (e).
indicated that the present method has a satisfactory accuracy, precision and reproducibility. Extraction recovery and matrix effect Table II lists the extraction recovery and matrix effect data of MTF, MET, HMT and DMT at LQC, MQC and HQC in plasma. The data of extraction efficiency measured for MTF, MET, HMT, DMT and IS in rat plasma were consistent, precise and reproducible. The mean extraction recoveries of MET, HMT, DMT and MTF were ranged from 87.1 to 101.1%, 90.2 to 105.6%, 89.8 to 97.3% and 89.2 to 97.1% for MTF, respectively, at each QC level. The mean extraction recovery of the IS was 93.3 ± 5.2%. The matrix effects of MTF, MET, HMT and DMT at each QC level were ranged from 90.8 to 103.2%, and matrix effect of IS was 98.2 ± 5.4%. These results showed that ion suppression or enhancement from plasma matrix was negligible in the present condition. Stability The stability of the analytes in rat plasma was investigated for three freeze-thaw cycles, for 24 h at room temperature, for 21 days at −20°C in the freezer and for 24 h in the postprepared solutions at 4°C. Stability data in Table III show good stability for all the analytes over four storage conditions.
in Table IV. The plasma concentrations of MTF in Group 3 were significantly lower than those in Group 1 at 20, 40, 60, 90, 120 and 240 min after oral administration. As shown in Table IV, the parameters Cmax and AUC0 → t of MTF in Group 3 were significantly decreased compared with that in Group 1, whereas the Cl and Vd of MTF in Group 3 were significantly increased by 43 and 72%, respectively, when compared with that in Group 1. On the other hand, there were no significant differences in ka of MTF between Group 1 and Group 3. The mean plasma concentration–time curves of MET and two major metabolites, HMT and DMT, are shown in Figure 4, and the main relevant pharmacokinetic parameters are listed in Table V. Plasma MET, HMT and DMT concentration in Group 3 were significantly higher than those in Group 2 at 5, 10 and 20 min after oral administration, respectively. In addition, parameters ka of MET, HMT and DMT were significantly increased in Group 3 compared with that in Group 2, respectively, indicating that MTF could promote the absorption of MET. By contrast, the plasma concentration of MET in Group 3 was decreased significantly compared with that in Group 2 at 60 and 90 min. Meanwhile, the plasma concentrations of HMT were markedly elevated in Group 3 compared with that in Group 2.
Discussion Pharmacokinetic interaction between MTF and MET study The mean plasma concentration–time curves of MTF are shown in Figure 3, and the main relevant pharmacokinetic parameters are listed
Several methods such as HPLC and LC–MS-MS have been reported for the determination of MTF (20–23) or MET and/or its metabolites (10, 24–27) in biological fluids. Recently, a LC–MS-MS method for simultaneous determination of MET, HMT and DMT in human
5
Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC–MS-MS
Figure 2. Typical MRM chromatograms of MET (1), HMT (2), DMT (3), MTF (4) and IS (5) in rat plasma: (a) blank rat plasma; (b) blank rat plasma spiked with MET (0.86 ng/mL), HMT (2.05 ng/mL), DMT (1.95 ng/mL), MTF (2.79 ng/mL) and IS (300 ng/mL); (c) a plasma sample obtained at 10 min after oral administration of MTF (100 mg/kg) and MET (40 mg/kg) in rats.
Table I. Precision and Accuracy From QC Samples in Rat Plasma Analytes
MET
HMT
DMT
HTF
Spiked concentration (ng/mL)
6.84 109.38 3,500.00 4.10 65.63 2,100.00 3.91 62.50 2,000.00 39.06 625.00 20,000.00
Intraday (n = 6)
Interday (n = 6 × 5)
Measured concentration (ng/mL)
Precision (%, RSD)
Accuracy (%, RE)
Measured concentration (ng/mL)
Precision (%, RSD)
Accuracy (%, RE)
6.80 ± 0.51 115.34 ± 6.31 3,445.43 ± 111.12 3.83 ± 0.54 58.76 ± 5.91 2,043.10 ± 56.11 3.88 ± 0.42 64.13 ± 4.89 1,977.71 ± 89.24 35.65 ± 4.22 612.88 ± 34.90 19,451.15 ± 122.18
7.5 5.8 3.2 14.1 10.1 2.7 10.8 7.6 4.5 11.8 5.7 0.6
−0.6 5.4 −1.6 −6.6 −10.5 −2.7 −0.8 2.6 −1.1 −8.7 −1.9 −2.7
7.11 ± 0.47 110.45 ± 8.12 3.389.13 ± 134.51 3.89 ± 0.37 68.34 ± 9.32 1,895.33 ± 89.21 3.55 ± 0.51 65.27 ± 5.31 2,108.21 ± 42.21 35.71 ± 4.32 597.21 ± 45.23 20,124.1 ± 98.7
6.6 7.3 4.0 9.5 13.6 4.7 14.4 8.1 2.0 12.1 7.6 0.5
3.9 −1.0 −3.2 −5.1 4.1 −9.7 −9.2 4.4 5.4 −8.5 −4.4 0.6
plasma has been reported, which was rapid and sensitive (28). However, this method possesses many disadvantages such as low extraction recovery of IS (
Article
Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC–MS-MS: Application to Pharmacokinetic Interaction Study Yan-rong Ma1,2, Zhi Rao1, A-xi Shi1,2, Ya-feng Wang1,2,3, Jing Huang1,2, Miao Han1,2, Xin-dong Wang1,2, Yong-wen Jin1,2, Guo-qiang Zhang1, Yan Zhou1, Fan Zhang1, Hong-yan Qin1, and Xin-an Wu1,* 1
Department of Pharmacy, The First Hospital of Lanzhou University, Lanzhou 730000, China, 2School of Pharmacy, Lanzhou University, Lanzhou 730000, China, and 3Department of Pharmacy, Qing Hai Provincial Hospital, Xining 810000, China *Author to whom correspondence should be addressed. Email: [email protected] Received 11 December 2014; Revised 9 June 2015
Abstract A simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed and validated for the simultaneous quantitation of metformin (MTF), metoprolol (MET), α-hydroxymetoprolol (HMT) and O-desmethylmetoprolol (DMT) in rat plasma using paracetamol as an internal standard (IS), respectively. The sample preparation involved a proteinprecipitation method with methanol after the addition of IS. The separation was performed on an Agilent HC-C18 column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min, using methanol–water containing 0.1% formic acid (39:61, v/v) as mobile phase, and total run time was 8.5 min. MS–MS detection was accomplished in multiple reaction monitoring mode with positive electrospray ionization. The monitored transitions were m/z 130.1 → 60.2 for MTF, m/z 268.2 → 116.1 for MET, m/z 284.2 → 116.1 for HMT, m/z 254.2 → 116.1 for DMT and m/z 152.3 → 110.1 for IS. The method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, matrix effect and recovery over a concentration range of 19.53–40,000 ng/mL for MTF, 3.42–7,000 ng/mL for MET, 2.05–4,200 ng/mL for HMT and 1.95–4,000 ng/mL for DMT, respectively. The analytical method was successfully applied to drug interaction study of MTF and MET after oral administration of MTF and MET. Results suggested that the coadministration of MTF and MET results in a significant drug interaction in rat.
Introduction Metformin (MTF), a biguanide derivative, is one of the first-line drugs for the treatment of type 2 diabetes mellitus (T2DM), and it is often used alone or in combination with other drugs to control blood glucose level (1). Although the mechanism that is responsible for blood glucose improvement is not well understood, MTF, to some extent, decrease hepatic glucose production and improve insulin sensitivity by increasing the uptake and utilization of peripheral glucose (2, 3). The main adverse effects of MTF involve gastrointestinal intolerance
and lethal lactic acidosis (4, 5). In vivo, MTF is not metabolized by hepatic cytochrome P450 and it is excreted into urine via active secretion (∼78.9–99.9%) by renal tubular (6). Metoprolol (MET), a selective β-adrenoceptor antagonist, is used in clinical practice in the racemic form for the treatment of cardiovascular disorders, such as hypertension, arrhythmia and heart failure (7, 8). MET is metabolized through O-demethylation (65% of dose), α-hydroxylation (10% of dose) and N-dealkylation, which form the correspondent metabolites O-desmethylmetoprolol (DMT), α-hydroxymetoprolol
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
1
2 (HMT) and N-dealkylmetoprolol (9–11). The α-hydroxylation is catalyzed almost entirely, and O-demethylation partially, via CYP2D6 (12, 13). About 85% of the administered drug is excreted in the urine as metabolites, along with small amounts of unchanged parent drug (0.990 and the deviations of the calculated concentrations were within ±15% of the nominal concentrations. The lower limit of quality quantification (LLOQ) of the assay was defined as the lowest concentration that could be quantified (LLOQ, relative standard deviation (RSD) < 20% and S/N ≥ 10). Precision and accuracy The accuracy and precision of the established method were evaluated by QC samples at low, medium and high concentrations. Three validation batches, each containing six replicates of LQC, MQC and HQC, were assayed to assess the precision and accuracy of the method
Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC–MS-MS on three consecutive validation days. The percentage deviation of the mean from true values, expressed as relative error (RE), and the RSD serve as the measure of accuracy and precision, respectively. Extraction recovery and matrix effect The extraction recovery was evaluated by comparing peak areas of analytes and IS obtained from extracted spiked samples with those of the postextracted spiked samples at corresponding concentrations. The matrix effect was accessed by comparing the peak areas of blank plasmas spiked with analytes and IS after extraction with those for the standard solutions at the same concentration. Stability The stability of analytes in rat plasma was assessed by analyzing QC samples at three concentration levels through three freeze-thaw cycles (−20°C to room temperature as a cycle), on the bench at room temperature for 24 h (short-term stability), at −20°C for 21 days (long-term stability) and 24 h after prepared at 4°C.
Pharmacokinetic interaction between MTF and MET in rats All studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Wistar rats aged 8 weeks with body weigh ∼180–220 g were obtained from the Experimental Animal Center of Lanzhou University (Lanzhou, China). Rats were housed in plastic cages and maintained at 25°C under 12–12 h alternating light–dark cycle with free access to food and water. Male rats were randomly divided into three groups (n = 8/group). Rats in Group 1 were orally administered MTF (100 mg/kg, a clinically equivalent dose) (18, 19); rats in Group 2 were given MET (40 mg/ kg, a clinically equivalent dose) and rats in Group 3 were given MTF (100 mg/kg) plus MET (40 mg/kg). Blood samples (each ∼250 µL) were collected via the femoral artery into heparinized tubes before and 2, 5, 10, 20, 40, 60, 90, 120, 240, 360, 480 and 720 min after oral administration, respectively. Rats were immediately supplemented with the equal volume (250 µL) of heparin–saline solution after each blood collection. The blood samples were immediately centrifuged at 10,000 g for 10 min. The plasma layer was transferred to clean tubes and stored at −20°C. Blank plasma was obtained from the rat without any drug administration and was used to investigate the method development and validation. Plasma concentration–time profile for each rat was analyzed with Drug and Statistics 2.0 program (DAS, Medical College of Wannan, China) software by noncompartmental analysis. The pharmacokinetic parameters included the area under the plasma concentration–time curve (AUC), the time-averaged total body clearance (Cl), the terminal half-life (t1/2) and apparent volume of distribution at steady state (Vd). The peak plasma concentration (Cmax) and time to reach Cmax (Tmax) were directly read from the experimental data. Statistical analysis was computed using SPSS 13.0 software. Data were expressed as mean ± SD. Statistically significant differences of data from two sets were compared using one-way analysis of variance. In all statistical analyses, P < 0.05 or P < 0.01 was considered to indicate a statistically significant result.
Results Method development Under the ESI condition chosen, all of the compounds exhibited higher sensitivity in the positive mode than the negative mode, and the
3
most abundant ions are [M + H]+ for them. Therefore, [M + H]+ from MTF, MET, HMT, DMT and IS were selected as the precursor ions and subsequently fragmented in MS/MS mode to obtain the product ion spectra (Figure 1). The purpose is that the background noise could be reduced and the compounds confirmation could be more accurately, and the risk of false positive due to the interference of matrix was greatly reduced. The choice of mobile phase should be concerned based on the consideration of ionization efficiency before the analytes enter the MS/MS system in order to obtain nice resolution and high sensitivity. As for the choice of strong elution mobile phase, methanol and acetonitrile were considered as two candidates. The result showed that there was no obvious difference in the responses of analytes between methanol– water and acetonitrile–water. Therefore, methanol was chosen in the study for its lower price than acetonitrile. To obtain the maximum sensitivity, we investigated the effects of pH with various mobile phases on the ionization efficiency. Both analytes and IS were found to have the highest response and the best peak shapes in the mobile phase containing 0.1% formic acid. The LC mobile phase was optimized with varying percentages of organic solvent and different modifiers in water to obtain high sensitivity. Analytes and IS were separated on an Agilent HC-C18 column with a mobile phase consisting of methanol and water containing 0.1% formic acid (39:61, v/v). Moreover, width peak, S/N and system stability during an analytical batch (50 h) were considered to find the optimal injection volume (10 µL). An efficient cleanup for bio-samples to remove protein and potential interferences prior to LC–MS-MS analysis was an important point in the method development. The effective and simple protein precipitation was employed in our work. Methanol was chosen as the protein-precipitation solvent because it exhibited better effect than acetonitrile and perchloric acid.
Method validation Selectivity Figure 2 shows the typical chromatograms of blank plasma, blank plasma spiked with analytes at LLOQ and plasma sample collected at 10 min after administration of MET (40 mg/kg) and MTF (100 mg/kg). The retention times of MET, HMT, DMT, MTF and IS were 6.9, 3.6, 3.8, 3.1 and 4.4 min, respectively. The total chromatographic run time was 8.5 min. Figure 2c (1) shows that the interference was completely separated from MET, and retention time of the interference was 4.0 min. However, there were no obvious endogenous interferences under the described chromatographic conditions. Calibration curve and lower limit of quantification The calibration curves were created by plotting the peak area ratios of the various analytes to IS versus nominal concentration of the analyte standards. The four calibration curves were linear over the concentration range of 3.42–7,000 ng/mL for MET, 2.05–4,200 ng/mL for HMT, 1.95–4,000 ng/mL for DMT and 19.53–40,000 ng/mL for MTF, with a correlation coefficient (R 2) ≥ 0.9980 for both analytes. The lowest concentration with the RSD < 20% and S/N ≥ 10 was taken as LLOQ, which was found to be 0.86, 2.05, 1.95 and 2.79 ng/mL for MET, HMT, DMT and MTF, respectively. Precision and accuracy Intraday and interday precision for all of the QC samples did not exceed 15% RSD, and accuracy values did not exceed 15% deviation from the true value for the analytes at any tested concentrations. Precision and accuracy determinations are listed in Table I. These results
4
Ma et al.
Figure 1. Chemical structures and product ion mass spectra of MET (a), HMT (b), DMT (c), MTF (d) and IS (e).
indicated that the present method has a satisfactory accuracy, precision and reproducibility. Extraction recovery and matrix effect Table II lists the extraction recovery and matrix effect data of MTF, MET, HMT and DMT at LQC, MQC and HQC in plasma. The data of extraction efficiency measured for MTF, MET, HMT, DMT and IS in rat plasma were consistent, precise and reproducible. The mean extraction recoveries of MET, HMT, DMT and MTF were ranged from 87.1 to 101.1%, 90.2 to 105.6%, 89.8 to 97.3% and 89.2 to 97.1% for MTF, respectively, at each QC level. The mean extraction recovery of the IS was 93.3 ± 5.2%. The matrix effects of MTF, MET, HMT and DMT at each QC level were ranged from 90.8 to 103.2%, and matrix effect of IS was 98.2 ± 5.4%. These results showed that ion suppression or enhancement from plasma matrix was negligible in the present condition. Stability The stability of the analytes in rat plasma was investigated for three freeze-thaw cycles, for 24 h at room temperature, for 21 days at −20°C in the freezer and for 24 h in the postprepared solutions at 4°C. Stability data in Table III show good stability for all the analytes over four storage conditions.
in Table IV. The plasma concentrations of MTF in Group 3 were significantly lower than those in Group 1 at 20, 40, 60, 90, 120 and 240 min after oral administration. As shown in Table IV, the parameters Cmax and AUC0 → t of MTF in Group 3 were significantly decreased compared with that in Group 1, whereas the Cl and Vd of MTF in Group 3 were significantly increased by 43 and 72%, respectively, when compared with that in Group 1. On the other hand, there were no significant differences in ka of MTF between Group 1 and Group 3. The mean plasma concentration–time curves of MET and two major metabolites, HMT and DMT, are shown in Figure 4, and the main relevant pharmacokinetic parameters are listed in Table V. Plasma MET, HMT and DMT concentration in Group 3 were significantly higher than those in Group 2 at 5, 10 and 20 min after oral administration, respectively. In addition, parameters ka of MET, HMT and DMT were significantly increased in Group 3 compared with that in Group 2, respectively, indicating that MTF could promote the absorption of MET. By contrast, the plasma concentration of MET in Group 3 was decreased significantly compared with that in Group 2 at 60 and 90 min. Meanwhile, the plasma concentrations of HMT were markedly elevated in Group 3 compared with that in Group 2.
Discussion Pharmacokinetic interaction between MTF and MET study The mean plasma concentration–time curves of MTF are shown in Figure 3, and the main relevant pharmacokinetic parameters are listed
Several methods such as HPLC and LC–MS-MS have been reported for the determination of MTF (20–23) or MET and/or its metabolites (10, 24–27) in biological fluids. Recently, a LC–MS-MS method for simultaneous determination of MET, HMT and DMT in human
5
Simultaneous Determination of Metformin, Metoprolol and its Metabolites in Rat Plasma by LC–MS-MS
Figure 2. Typical MRM chromatograms of MET (1), HMT (2), DMT (3), MTF (4) and IS (5) in rat plasma: (a) blank rat plasma; (b) blank rat plasma spiked with MET (0.86 ng/mL), HMT (2.05 ng/mL), DMT (1.95 ng/mL), MTF (2.79 ng/mL) and IS (300 ng/mL); (c) a plasma sample obtained at 10 min after oral administration of MTF (100 mg/kg) and MET (40 mg/kg) in rats.
Table I. Precision and Accuracy From QC Samples in Rat Plasma Analytes
MET
HMT
DMT
HTF
Spiked concentration (ng/mL)
6.84 109.38 3,500.00 4.10 65.63 2,100.00 3.91 62.50 2,000.00 39.06 625.00 20,000.00
Intraday (n = 6)
Interday (n = 6 × 5)
Measured concentration (ng/mL)
Precision (%, RSD)
Accuracy (%, RE)
Measured concentration (ng/mL)
Precision (%, RSD)
Accuracy (%, RE)
6.80 ± 0.51 115.34 ± 6.31 3,445.43 ± 111.12 3.83 ± 0.54 58.76 ± 5.91 2,043.10 ± 56.11 3.88 ± 0.42 64.13 ± 4.89 1,977.71 ± 89.24 35.65 ± 4.22 612.88 ± 34.90 19,451.15 ± 122.18
7.5 5.8 3.2 14.1 10.1 2.7 10.8 7.6 4.5 11.8 5.7 0.6
−0.6 5.4 −1.6 −6.6 −10.5 −2.7 −0.8 2.6 −1.1 −8.7 −1.9 −2.7
7.11 ± 0.47 110.45 ± 8.12 3.389.13 ± 134.51 3.89 ± 0.37 68.34 ± 9.32 1,895.33 ± 89.21 3.55 ± 0.51 65.27 ± 5.31 2,108.21 ± 42.21 35.71 ± 4.32 597.21 ± 45.23 20,124.1 ± 98.7
6.6 7.3 4.0 9.5 13.6 4.7 14.4 8.1 2.0 12.1 7.6 0.5
3.9 −1.0 −3.2 −5.1 4.1 −9.7 −9.2 4.4 5.4 −8.5 −4.4 0.6
plasma has been reported, which was rapid and sensitive (28). However, this method possesses many disadvantages such as low extraction recovery of IS (
