CHIRALITY (2015)
Short Communication Determination of Trantinterol Enantiomers in Human Plasma by HighPerformance Liquid Chromatography – Tandem Mass Spectrometry Using Vancomycin Chiral Stationary Phase and Solid Phase Extraction and Stereoselective Pharmacokinetic Application FENG QIN,1 YANJUAN WANG,1 LIJUAN WANG,1 LONGSHAN ZHAO,1 LI PAN,2 MAOSHENG CHENG,2 AND FAMEI LI1* Department of Analytical Chemistry, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China 2 Department of Pharmaceutical Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China 1
ABSTRACT A sensitive and enantioselective vancomycin chiral stationary phase highperformance liquid chromatography–tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile–methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from 6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human. Chirality 00:00–00, 2015. © 2015 Wiley Periodicals, Inc. KEY WORDS: trantinterol; enantiomers; enantioselective HPLC-MS/MS; extraction; human plasma; stereoselective pharmacokinetics Trantinterol,2-(4-amino-3-chloro-5-trifluoromethyl-phenyl)2-tert-butylamino-ethanol, is a novel phenylethanolamine β2 adrenoceptor agonist currently undergoing phase III clinical trials in China. It has exhibited both a potent trachea-relaxing activity and high β2 selectivity with low cardiac side effect.1 Chemically, trantinterol is a chiral molecule, and the structure is given in Figure 1A. In contrast to its (+)-enantiomer and racemate, ( )-trantinterol has more powerful and selective activity, which may be beneficial for asthma treatment.2 The pharmacokinetics of trantinterol were stereoselective3 and a bidirectional chiral inversion of trantinterol enantiomers4 was observed in rats. To answer whether the enantiomers of trantinterol have different pharmacokinetic profiles and chiral inversion in human, it is necessary to develop and validate a sensitive and enantioselective method for determination of the enantiomers in human plasma. Several chiral methods have been reported for the separation of ( )- and (+)-trantinterol. But there is no report on the quantitative determination of trantinterol enantiomers in human plasma. Yang et al. developed capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) using a Chiralpak AS-H chiral column for enantioseparation of trantinterol.5,6 But the sensitivity of those methods with a UV detector was not adequate for bioanalysis. A method of precolumn derivatization utilizing diacetyl-l-tartaric anhydride as chiral derivatization reagents with a mass spectroscopy (MS) detector for the separation © 2015 Wiley Periodicals, Inc.
solid
phase
of trantinterol enantiomers in rat plasma was reported.3 But the necessary derivatization was time-consuming, complicated, and might involve undesirable side reactions, formation of decomposition products, and racemization.7 Recently, we published an HPLC-MS/MS method based on the chiral stationary phase (CSP) to determine ( )-trantinterol in rat plasma, which had a lower limit of quantification (LLOQ) of 0.270 ng/mL.8 Here we present a sensitive, reproducible, and enantioselective method using vancomycin CSP and solid phase extraction for measuring trantinterol enantiomers in human plasma carefully optimized based on the previous work.8 The method was fully validated and applied to the study of stereoselective pharmacokinetics of trantinterol enantiomers in human after oral administration of trantinterol racemate. MATERIALS AND METHODS Chemicals and Reagents Racemic, ( )- and (+)-trantinterol were synthesized at the Department of Pharmaceutical Chemistry, Shenyang Pharmaceutical University *Correspondence to: Famei Li, Department of Analytical Chemistry, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103#, Shenyang 110016, PR China. E-mail: [email protected] Received for publication 26 August 2014; Accepted 13 January 2015 DOI: 10.1002/chir.22432 Published online in Wiley Online Library (wileyonlinelibrary.com).
QIN ET AL.
Sample Preparation
Fig. 1. Chemical structures of trantinterol (A) and verapamil (IS) (B). The chiral center is labeled (*).
A 100-μL aliquot of IS working solution (96.0 ng/mL) was pipetted into a 10-mL clean glass tube and evaporated to dryness. The residue was vortex-mixed with 1000 μL plasma and 400 μL of 1 mol/L NaOH for 30 s. Then the mixture was loaded onto a Waters Oasis 10 mg HLB solid-phase extraction (SPE) cartridge with a flow rate of about 0.5–1 mL/min and all processing was performed on an SPE vacuum manifold. The SPE cartridge was preconditioned with 2 mL of methanol and 2 mL of water successively. Care was taken that the cartridges did not run dry. The SPE cartridge was washed with 1 mL of water, and the analytes were eluted with 2 mL of methanol. The eluate was evaporated under a nitrogen stream at 40°C. The residue was reconstituted in 100 μL of acetonitrile-methanol (60:40, v/v), and an aliquot of 20 μL was injected into HPLC-MS/MS system for analysis.
Method Validation (Shenyang, China) with a purity higher than 99.4%. Verapamil (internal standard [IS], Fig. 1B) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and methanol of HPLC grade were purchased from Tedia (Fairfield, OH). HPLC-grade glacial acetic acid was from Dikma (Richmond Hill, NY). Ammonia (28%, w/v, HPLC-grade) was from Sigma–Aldrich (St. Louis, MO). Other chemicals were all of analytical grade.
Apparatus and Operating Conditions The analysis was carried out on an ACQUITY UPLC system (Waters, Milford, MA) with cooling autosampler and column oven. A Chirobiotic V (150 × 4.6 mm, 5 μm, Astec, Whippany, NJ) column was employed for the separation at 20°C. The autosampler temperature was kept at a temperature of 4°C and the injection volume was 20 μL. New polar ionic mode was employed with an isocratic mobile phase of acetonitrile– methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. The flow was split using a T-piece such that only 300 μL/min was directed to the ESI interface. A triple quadrupole tandem mass spectrometer (Micromass Quattro micro API mass spectrometer, Waters) with an electrospray ionization (ESI) interface was employed for analyte detection. The ESI source was operated in positive ionization mode with optimal operation parameters as follows: capillary 1.0 kV, cone 15 V, source temperature 110°C and desolvation temperature 500°C. Nitrogen was used as the desolvation and cone gas with a flow rate of 700 and 30 L/h, respectively. Argon was used as the collision gas at a pressure of 1.30 × 3 10 mbar. The collision energy was 15 V for trantinterol and 20 V for IS. The analyses were carried out in multiple reaction monitoring (MRM) mode of the transition m/z 311→238 for trantinterol and m/z 455→165 for IS, with a scan time of 0.2 s per transition. All data were collected in centroid mode and processed using MassLynx NT 4.1 software with a QuanLynx program (Waters).
Preparation of Standards and Quality Control Samples Working standards of each enantiomer in a concentration range of 0.606–303 ng/mL were prepared by dilution of 1.01 μg/mL stock solution with methanol. A 96.0 ng/mL IS working solution was obtained by diluting the stock solution of verapamil with methanol. All the solutions were stored at 4°C and brought to room temperature before use. Calibration standards were prepared daily by spiking 1000 μL of the blank plasma to the residue of 100 μL of the appropriate standard solutions giving concentrations of 0.0606, 0.121, 0.404, 1.21, 4.04, 10.1, and 30.3 ng/mL. Quality control (QC) working solutions were prepared separately using another stock solution. QC samples, which were used in the validation and during the pharmacokinetic study, were prepared at the beginning of the experiment by independent dilution at three levels of plasma concentration: 0.107, 1.79, and 21.5 ng/mL for ( )-trantinterol and 0.118, 1.97, and 23.6 ng/mL for (+)-trantinterol and stored at 70°C after preparation. The standards and quality controls were extracted on each analysis day along with the unknown samples. Chirality DOI 10.1002/chir
The method was validated for selectivity, linearity, accuracy, precision, extract recovery, and stability according to the FDA guideline9 for validation of bioanalytical methods. Validation runs were conducted on 3 consecutive days. Each validation run consisted of calibration standards and six replicates of QC plasma samples at three concentrations. The peak area ratios of ( )- or (+)-trantinterol/IS of QC samples were interpolated from the calibration curve on the same day to give the concentrations of ( )- or (+)-trantinterol. The results from QC plasma samples in three runs were used to evaluate the precision and accuracy of the method developed. The selectivity of this method was investigated by preparing and analyzing six individual human blank plasma samples with corresponding plasma samples spiked with ( )- and (+)-trantinterol and plasma samples after oral administration of trantinterol racemate. The linearity was determined using seven standard plasma samples in the 0.0606–30.3 ng/mL range by plotting the peak area ratio of ( )- or (+)trantinterol to I.S versus the nominal concentration of ( )- or (+)trantinterol in plasma. The calibration curves were constructed by 2 weighted (1/x ) least squares linear regression. The precision and accuracy were determined by repeated analysis of six replicates at LLOQ and each QC level (low, mid, and high levels) on 3 different days. The precision was defined as the RSD (%) (relative standard deviation) and the accuracy was expressed as relative error (RE). The extraction recovery of ( )- and (+)-trantinterol at three QC concentrations was determined by comparing the peak area of extracted samples spiked with a known amount of the analytes with that of spiked postextraction at corresponding concentrations. To evaluate the matrix effect, ( )- and (+)-trantinterol at three concentration levels were added to the extract of 1 mL of blank plasma, dried, and reconstituted with 100 μL of acetonitrile-methanol (60:40, v/v). The corresponding peak areas (A) were compared with those of the standard solutions containing equivalent amounts of the two compounds dried directly and reconstituted with the same solvent (B). The ratio (A/B×100)% was used to evaluate the matrix effect. The extraction recovery and matrix effect of IS were also evaluated using the same procedure. Stability experiments were performed to evaluate the stability of ( )and (+)-trantinterol in plasma samples under different temperature and timing conditions. Three aliquots of QC samples (low and high levels) were stored at 70°C for 14 days and at ambient temperature for 4 h to determine long-term and short-term stability respectively. Postpreparative stability was assessed by analyzing the extracted QC samples kept in the autosampler at 4°C for 12 h. All stability testing QC samples was determined by using the calibration curve of freshly prepared standards. The concentrations obtained were compared with the nominal values.
Application to Stereoselective Pharmacokinetic Study The pharmacokinetic study was approved by the local Ethics Committee and carried out in the hospital. Two volunteers gave their signed informed consent to participate in the study according to the principles of the Declaration of Helsinki and 15 mg racemate trantinterol hydrochloride was administered to each healthy male volunteer after 12 h fasting.
DETERMINATION OF TRANTINTEROL ENANTIOMERS IN HUMAN PLASMA
Blood samples were collected before and 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 12.0, 24.0, and 36.0 h postdosing. Plasma was separated by centrifugation at 3000g for 10 min and stored at 70°C until analyzed.
RESULTS AND DISCUSSION Chiral HPLC Optimization
In our previous study8 we developed a method for the determination of ( )-trantinterol in rat plasma by using chiral stationary phase. In this work, chiral HPLC operation parameters were carefully optimized for the determination of trantinterol enantiomers based on the previous study. Vancomycin chiral stationary phase has been widely used for enantiomeric separation in bioanalysis.10–15 The mobile phase in polar ionic mode is compatible with MS because of its high volatility and beneficial ionization effect. Therefore, a new polar ionic mode was chosen for the separation of trantinterol enantiomers. The chromatographic conditions were modified to obtain high resolution. The separation and ionization of trantinterol enantiomers were affected by the composition of mobile phase. The mobile phase system of acetonitrile and methanol containing ammonia and acetic acid were the same as that used in the determination in rat plasma. The composition of mobile phase has a substantial influence on the resolution. No baseline separation was observed in the absence of acetonitrile when the mobile phase consisted of methanol-ammonia-acetic acid (100:0.01:0.02, v/v/v). Acetonitrile is a solvent suitable for LC-MS analysis and the combination of methanol and acetonitrile has been reported for chiral separation.16–18 The baseline separation of the trantinterol enantiomers was obtained by using mobile phases containing 40–80% acetonitrile (Fig. 2). In view of the enantioresolution, 60% acetonitrile was finally chosen. The ratio and concentration of acetic acid and ammonia in mobile phase has some influence on the resolution. The best chiral resolution was observed at the ratio of 2:1 of acid to base and 0.01% ammonia. Sample Preparation
Sample preparation is an important part of an analytical method. Protein precipitation (PPT), liquid–liquid extraction (LLE), and solid phase extraction (SPE) are the most widely employed biological sample preparation techniques. Protein precipitation is easy and rapid to perform, but often introduces significant matrix effects due to its inability to remove many residual matrix components such as phospholipids.19 In the literature8,20,21 LLE was selected. In our study a 1-mL plasma sample was used for good sensitivity and this resulted
in low recovery (
Short Communication Determination of Trantinterol Enantiomers in Human Plasma by HighPerformance Liquid Chromatography – Tandem Mass Spectrometry Using Vancomycin Chiral Stationary Phase and Solid Phase Extraction and Stereoselective Pharmacokinetic Application FENG QIN,1 YANJUAN WANG,1 LIJUAN WANG,1 LONGSHAN ZHAO,1 LI PAN,2 MAOSHENG CHENG,2 AND FAMEI LI1* Department of Analytical Chemistry, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China 2 Department of Pharmaceutical Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China 1
ABSTRACT A sensitive and enantioselective vancomycin chiral stationary phase highperformance liquid chromatography–tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile–methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from 6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human. Chirality 00:00–00, 2015. © 2015 Wiley Periodicals, Inc. KEY WORDS: trantinterol; enantiomers; enantioselective HPLC-MS/MS; extraction; human plasma; stereoselective pharmacokinetics Trantinterol,2-(4-amino-3-chloro-5-trifluoromethyl-phenyl)2-tert-butylamino-ethanol, is a novel phenylethanolamine β2 adrenoceptor agonist currently undergoing phase III clinical trials in China. It has exhibited both a potent trachea-relaxing activity and high β2 selectivity with low cardiac side effect.1 Chemically, trantinterol is a chiral molecule, and the structure is given in Figure 1A. In contrast to its (+)-enantiomer and racemate, ( )-trantinterol has more powerful and selective activity, which may be beneficial for asthma treatment.2 The pharmacokinetics of trantinterol were stereoselective3 and a bidirectional chiral inversion of trantinterol enantiomers4 was observed in rats. To answer whether the enantiomers of trantinterol have different pharmacokinetic profiles and chiral inversion in human, it is necessary to develop and validate a sensitive and enantioselective method for determination of the enantiomers in human plasma. Several chiral methods have been reported for the separation of ( )- and (+)-trantinterol. But there is no report on the quantitative determination of trantinterol enantiomers in human plasma. Yang et al. developed capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) using a Chiralpak AS-H chiral column for enantioseparation of trantinterol.5,6 But the sensitivity of those methods with a UV detector was not adequate for bioanalysis. A method of precolumn derivatization utilizing diacetyl-l-tartaric anhydride as chiral derivatization reagents with a mass spectroscopy (MS) detector for the separation © 2015 Wiley Periodicals, Inc.
solid
phase
of trantinterol enantiomers in rat plasma was reported.3 But the necessary derivatization was time-consuming, complicated, and might involve undesirable side reactions, formation of decomposition products, and racemization.7 Recently, we published an HPLC-MS/MS method based on the chiral stationary phase (CSP) to determine ( )-trantinterol in rat plasma, which had a lower limit of quantification (LLOQ) of 0.270 ng/mL.8 Here we present a sensitive, reproducible, and enantioselective method using vancomycin CSP and solid phase extraction for measuring trantinterol enantiomers in human plasma carefully optimized based on the previous work.8 The method was fully validated and applied to the study of stereoselective pharmacokinetics of trantinterol enantiomers in human after oral administration of trantinterol racemate. MATERIALS AND METHODS Chemicals and Reagents Racemic, ( )- and (+)-trantinterol were synthesized at the Department of Pharmaceutical Chemistry, Shenyang Pharmaceutical University *Correspondence to: Famei Li, Department of Analytical Chemistry, School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103#, Shenyang 110016, PR China. E-mail: [email protected] Received for publication 26 August 2014; Accepted 13 January 2015 DOI: 10.1002/chir.22432 Published online in Wiley Online Library (wileyonlinelibrary.com).
QIN ET AL.
Sample Preparation
Fig. 1. Chemical structures of trantinterol (A) and verapamil (IS) (B). The chiral center is labeled (*).
A 100-μL aliquot of IS working solution (96.0 ng/mL) was pipetted into a 10-mL clean glass tube and evaporated to dryness. The residue was vortex-mixed with 1000 μL plasma and 400 μL of 1 mol/L NaOH for 30 s. Then the mixture was loaded onto a Waters Oasis 10 mg HLB solid-phase extraction (SPE) cartridge with a flow rate of about 0.5–1 mL/min and all processing was performed on an SPE vacuum manifold. The SPE cartridge was preconditioned with 2 mL of methanol and 2 mL of water successively. Care was taken that the cartridges did not run dry. The SPE cartridge was washed with 1 mL of water, and the analytes were eluted with 2 mL of methanol. The eluate was evaporated under a nitrogen stream at 40°C. The residue was reconstituted in 100 μL of acetonitrile-methanol (60:40, v/v), and an aliquot of 20 μL was injected into HPLC-MS/MS system for analysis.
Method Validation (Shenyang, China) with a purity higher than 99.4%. Verapamil (internal standard [IS], Fig. 1B) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and methanol of HPLC grade were purchased from Tedia (Fairfield, OH). HPLC-grade glacial acetic acid was from Dikma (Richmond Hill, NY). Ammonia (28%, w/v, HPLC-grade) was from Sigma–Aldrich (St. Louis, MO). Other chemicals were all of analytical grade.
Apparatus and Operating Conditions The analysis was carried out on an ACQUITY UPLC system (Waters, Milford, MA) with cooling autosampler and column oven. A Chirobiotic V (150 × 4.6 mm, 5 μm, Astec, Whippany, NJ) column was employed for the separation at 20°C. The autosampler temperature was kept at a temperature of 4°C and the injection volume was 20 μL. New polar ionic mode was employed with an isocratic mobile phase of acetonitrile– methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. The flow was split using a T-piece such that only 300 μL/min was directed to the ESI interface. A triple quadrupole tandem mass spectrometer (Micromass Quattro micro API mass spectrometer, Waters) with an electrospray ionization (ESI) interface was employed for analyte detection. The ESI source was operated in positive ionization mode with optimal operation parameters as follows: capillary 1.0 kV, cone 15 V, source temperature 110°C and desolvation temperature 500°C. Nitrogen was used as the desolvation and cone gas with a flow rate of 700 and 30 L/h, respectively. Argon was used as the collision gas at a pressure of 1.30 × 3 10 mbar. The collision energy was 15 V for trantinterol and 20 V for IS. The analyses were carried out in multiple reaction monitoring (MRM) mode of the transition m/z 311→238 for trantinterol and m/z 455→165 for IS, with a scan time of 0.2 s per transition. All data were collected in centroid mode and processed using MassLynx NT 4.1 software with a QuanLynx program (Waters).
Preparation of Standards and Quality Control Samples Working standards of each enantiomer in a concentration range of 0.606–303 ng/mL were prepared by dilution of 1.01 μg/mL stock solution with methanol. A 96.0 ng/mL IS working solution was obtained by diluting the stock solution of verapamil with methanol. All the solutions were stored at 4°C and brought to room temperature before use. Calibration standards were prepared daily by spiking 1000 μL of the blank plasma to the residue of 100 μL of the appropriate standard solutions giving concentrations of 0.0606, 0.121, 0.404, 1.21, 4.04, 10.1, and 30.3 ng/mL. Quality control (QC) working solutions were prepared separately using another stock solution. QC samples, which were used in the validation and during the pharmacokinetic study, were prepared at the beginning of the experiment by independent dilution at three levels of plasma concentration: 0.107, 1.79, and 21.5 ng/mL for ( )-trantinterol and 0.118, 1.97, and 23.6 ng/mL for (+)-trantinterol and stored at 70°C after preparation. The standards and quality controls were extracted on each analysis day along with the unknown samples. Chirality DOI 10.1002/chir
The method was validated for selectivity, linearity, accuracy, precision, extract recovery, and stability according to the FDA guideline9 for validation of bioanalytical methods. Validation runs were conducted on 3 consecutive days. Each validation run consisted of calibration standards and six replicates of QC plasma samples at three concentrations. The peak area ratios of ( )- or (+)-trantinterol/IS of QC samples were interpolated from the calibration curve on the same day to give the concentrations of ( )- or (+)-trantinterol. The results from QC plasma samples in three runs were used to evaluate the precision and accuracy of the method developed. The selectivity of this method was investigated by preparing and analyzing six individual human blank plasma samples with corresponding plasma samples spiked with ( )- and (+)-trantinterol and plasma samples after oral administration of trantinterol racemate. The linearity was determined using seven standard plasma samples in the 0.0606–30.3 ng/mL range by plotting the peak area ratio of ( )- or (+)trantinterol to I.S versus the nominal concentration of ( )- or (+)trantinterol in plasma. The calibration curves were constructed by 2 weighted (1/x ) least squares linear regression. The precision and accuracy were determined by repeated analysis of six replicates at LLOQ and each QC level (low, mid, and high levels) on 3 different days. The precision was defined as the RSD (%) (relative standard deviation) and the accuracy was expressed as relative error (RE). The extraction recovery of ( )- and (+)-trantinterol at three QC concentrations was determined by comparing the peak area of extracted samples spiked with a known amount of the analytes with that of spiked postextraction at corresponding concentrations. To evaluate the matrix effect, ( )- and (+)-trantinterol at three concentration levels were added to the extract of 1 mL of blank plasma, dried, and reconstituted with 100 μL of acetonitrile-methanol (60:40, v/v). The corresponding peak areas (A) were compared with those of the standard solutions containing equivalent amounts of the two compounds dried directly and reconstituted with the same solvent (B). The ratio (A/B×100)% was used to evaluate the matrix effect. The extraction recovery and matrix effect of IS were also evaluated using the same procedure. Stability experiments were performed to evaluate the stability of ( )and (+)-trantinterol in plasma samples under different temperature and timing conditions. Three aliquots of QC samples (low and high levels) were stored at 70°C for 14 days and at ambient temperature for 4 h to determine long-term and short-term stability respectively. Postpreparative stability was assessed by analyzing the extracted QC samples kept in the autosampler at 4°C for 12 h. All stability testing QC samples was determined by using the calibration curve of freshly prepared standards. The concentrations obtained were compared with the nominal values.
Application to Stereoselective Pharmacokinetic Study The pharmacokinetic study was approved by the local Ethics Committee and carried out in the hospital. Two volunteers gave their signed informed consent to participate in the study according to the principles of the Declaration of Helsinki and 15 mg racemate trantinterol hydrochloride was administered to each healthy male volunteer after 12 h fasting.
DETERMINATION OF TRANTINTEROL ENANTIOMERS IN HUMAN PLASMA
Blood samples were collected before and 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 12.0, 24.0, and 36.0 h postdosing. Plasma was separated by centrifugation at 3000g for 10 min and stored at 70°C until analyzed.
RESULTS AND DISCUSSION Chiral HPLC Optimization
In our previous study8 we developed a method for the determination of ( )-trantinterol in rat plasma by using chiral stationary phase. In this work, chiral HPLC operation parameters were carefully optimized for the determination of trantinterol enantiomers based on the previous study. Vancomycin chiral stationary phase has been widely used for enantiomeric separation in bioanalysis.10–15 The mobile phase in polar ionic mode is compatible with MS because of its high volatility and beneficial ionization effect. Therefore, a new polar ionic mode was chosen for the separation of trantinterol enantiomers. The chromatographic conditions were modified to obtain high resolution. The separation and ionization of trantinterol enantiomers were affected by the composition of mobile phase. The mobile phase system of acetonitrile and methanol containing ammonia and acetic acid were the same as that used in the determination in rat plasma. The composition of mobile phase has a substantial influence on the resolution. No baseline separation was observed in the absence of acetonitrile when the mobile phase consisted of methanol-ammonia-acetic acid (100:0.01:0.02, v/v/v). Acetonitrile is a solvent suitable for LC-MS analysis and the combination of methanol and acetonitrile has been reported for chiral separation.16–18 The baseline separation of the trantinterol enantiomers was obtained by using mobile phases containing 40–80% acetonitrile (Fig. 2). In view of the enantioresolution, 60% acetonitrile was finally chosen. The ratio and concentration of acetic acid and ammonia in mobile phase has some influence on the resolution. The best chiral resolution was observed at the ratio of 2:1 of acid to base and 0.01% ammonia. Sample Preparation
Sample preparation is an important part of an analytical method. Protein precipitation (PPT), liquid–liquid extraction (LLE), and solid phase extraction (SPE) are the most widely employed biological sample preparation techniques. Protein precipitation is easy and rapid to perform, but often introduces significant matrix effects due to its inability to remove many residual matrix components such as phospholipids.19 In the literature8,20,21 LLE was selected. In our study a 1-mL plasma sample was used for good sensitivity and this resulted
in low recovery (
