Jianjun Zou

Measurement of fexofenadine concentration in micro-sample human plasma by a rapid and sensitive LC-MS/MS employing protein precipitation: application to a clinical pharmacokinetic study.

A simple, rapid and sensitive liquid chromatography/positive ion electro-spray tandem mass spectrometry method (LC-MS/MS) was developed and validated for the quantification of fexofenadine with 100 microL human plasma employing glipizide as internal standard (IS). Protein precipitation was used in the sample preparation procedure. Chromatographic separation was achieved on a reversed-phase C(18 )column (5 microm, 100 x 2.1 mm) with methanol : buffer (containing 10 mmol/L ammonium acetate and 0.1% formic acid; 70 : 30, v/v) as mobile phase. The total chromatographic runtime was approximately 3.0 min with retention time for fexofenadine and IS at approximately 1.9 and 2.1 min, respectively. Detection of fexofenadine and IS was achieved by LC-MS/MS in positive ion mode using 502.1 --> 466.2 and 446.0 --> 321.1 transitions, respectively. The method was proved to be accurate and precise at linearity range of 1-600 ng/mL with a correlation coefficient (r) of > or =0.9976. The validated method was applied to a pharmacokinetic study in human volunteers following oral administration of 60 or 120 mg fexofenadine formulations, successfully.

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C(18) column with a mobile phase of methanol-water (70:30, v/v). HPLC-ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M-H(2)O-H](-), m/z 331.1 for andrographolide and [M-H](-), m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0-150.0ng/mL. The chromatographic separation was achieved in less than 6.5min. The lower limits of quantification (LLOQ) was 1.0ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.

Influence of CYP2C19 loss-of-function variants on the antiplatelet effects and cardiovascular events in clopidogrel-treated Chinese patients undergoing percutaneous coronary intervention

Background and objectivesA large number of clinical studies have well demonstrated that the loss-of-function variant allele CYP2C19*2 is associated with attenuated response to clopidogrel and increased risk of developing stent thrombosis (ST) in white or black patients with stenting. However, similar association studies on the effect of the CYP2C19*2 and *3 variants on maximum platelet aggregation (MPA) and the risk of cardiovascular events are currently unavailable for the Chinese patients. This work was aimed at assessing the impact of the CYP2C19 *2 and *3 variants on the antiplatelet effects and adverse cardiovascular events in clopidogrel-treated Chinese patients undergoing percutaneous coronary intervention (PCI).MethodsThe study population consisted of 617 patients undergoing PCI. Genotypes were determined using MALDI/TOF-MS. MPA was measured by light transmittance aggregometry. The clinical end-point was the 1-year incidence of adverse cardiovascular events, including ST.ResultsCarriers of CYP2C19 heterozygous (*1/*2, or *1/*3; n = 278) and mutant homozygous (*2/*2, *2/*3, or *3/*3, n = 80) genotypes had significantly higher MPA values than noncarriers (*1/*1; n = 259; P = 0.036 and 0.007 respectively). Moreover, the presence of the CYP2C19*2 or *3 mutant allele was significantly associated with an increased risk of developing ST, with the higher risk of ST being seen in patients homozygous for the CYP2C19*2 or *3 variant allele than in noncarriers (OR, 13.58; 95% CI, 1.49–123.31; P = 0.012). Furthermore, multivariate analysis revealed an independent association of the presence of CYP2C19*2 and/or *3 variant alleles with greater MPA values (P = 0.001) and increased risk of ST (OR, 11.67; 95% CI, 1.21–78.83; P = 0.022). However, there was no significant influence of CYP2C19*2 and *3 on the risk of developing other adverse cardiovascular events.ConclusionsCarriage of the loss-of-function genetic variants CYP2C19*2 and *3 is significantly associated with attenuated platelet response to clopidogrel and an increased risk of ST in Chinese patients treated with stenting.

Determination of betamethasone and betamethasone 17-monopropionate in human plasma by liquid chromatography-positive/negative electrospray ionization tandem mass spectrometry.

This study presents a high-performance liquid chromatography-positive/negative electrospray ionization tandem mass spectrometric (LC-ESI(+/-)-MS-MS) method for the determination of betamethasone (BOH) and betamethasone 17-monopropionate (B17P) in human plasma using beclomethasone dipropionate as the internal standard (I.S.). Both compounds were extracted from human plasma with ether-cyclohexane (4:1, v/v) and were separated by HPLC on a Hanbon Lichrospher C(18) column with a mobile phase of methanol-water (85:15, v/v) at a flow rate of 0.7ml/min. Calibration curves were linear over the range of 0.10-50ng/ml for BOH and 0.050-50ng/ml for B17P. The inter-run relative standard deviations were less than 14.4% for BOH and 12.3% for B17P. The intra-run relative standard deviations were less than 9.3% for BOH and 7.9% for B17P. The mean plasma extraction recovery for BOH and B17P were in the ranges of 82.7-85.9% and 83.6-85.3%, respectively. The method was successfully applied to study the pharmacokinetics of a new formulation of betamethasone phosphate/betamethasone dipropionate injection in healthy Chinese volunteers.

CES1A -816C as a genetic marker to predict greater platelet clopidogrel response in patients with percutaneous coronary intervention.

Abstract: This study was designed to determine whether CES1A −816A/C polymorphism could be associated with altered clopidogrel response. Recruited patients were pretreated with 300 mg clopidogrel loading dose before undergoing percutaneous coronary intervention for stenting and genotyped with CYP2C19 *2, *3, or *17, and CES1A −816A/C, respectively. Adenosine diphosphate–induced maximum platelet aggregation (MPA) was determined on day 3 after initiation of daily clopidogrel maintenance doses. The clinical primary end point was the 1-year incidence of definite stent thrombosis (ST). Multivariable linear regression revealed that the CES1A −816A/C polymorphism was independently associated with MPA measures with an absolute &bgr; value of 6.76. Of 617 patients, a subcohort of 249 patients not carrying CYP2C19 *2, *3, or *17 were categorized into 3 groups based on the −816A/C genotype. The median MPA value was lower in 125 carriers of the −816C variant than in 124 noncarriers (21.5% vs. 31.7%, P = 0.001). The 1-year definite ST occurred in 7 patients in that subcohort, and only 1 ST case was one of carriers of the −816 A/A that was associated with higher MPA values. The CES1A −816C would be used to predict greater platelet response to clopidogrel than the CES1A −816A in percutaneous coronary intervention–treated patients not carrying CYP2C19 variants.

Increased Risk for Developing Major Adverse Cardiovascular Events in Stented Chinese Patients Treated with Dual Antiplatelet Therapy after Concomitant Use of the Proton Pump Inhibitor

Background Some clinical studies have demonstrated that the proton pump inhibitor (PPI) could decrease clopidogrel platelet response and increase major adverse cardiovascular events (MACE) in white or black subjects. However, that remains to be determined in Chinese patients. In this study, we sought to determine whether there could be an increased risk for developing MACE after concomitant use of dual antiplatelet therapy (DAT) and a PPI in Chinese patients treated with percutaneous coronary intervention (PCI) and stenting. Methods This study was a 5-year, single-center, retrospective cohort analysis of eligible patients (n = 6188) who received DAT and a PPI concomitantly (defined as PPI users) before discharge and/or 12-month follow-up after discharge as compared with those who received DAT alone (also defined as non-PPI users, n = 1465). The incidence of recurrent MACE, such as myocardial infarction (MI), definite stent thromboses (ST), or cardiovascular death, was compared between the PPI users and non-users. Results PPI users had a significantly higher incidence of the MACE than non-users (13.9% vs. 10.6%; adjusted HR: 1.33; 95% CI: 1.12 – 1.57, P = 0.007). Stratified analysis revealed that concurrent use of DAT and a PPI was associated with a significantly increased risk for developing ST compared with DAT alone (1% vs. 0.4%; adjusted HR: 2.66, 95% CI: 1.16 – 5.87, P = 0.012). However, there were no significant differences in the risk of MI, cardiovascular death and other adverse events, regardless of combination of clopidogrel and a PPI. Conclusions The study further suggests that concomitant use of DAT and a PPI may be associated with an increased risk for developing MACE, in particular definite ST, in Chinese PCI patients after discharge as compared with use of DAT alone.

Pharmacokinetics of betamethasone and betamethasone 17-monopropionate in Chinese healthy volunteers after intramuscular injection of betamethasone phosphate/betamethasone dipropionate.

The aim of this study was to evaluate the pharmacokinetic profiles of betamethasone (BOH, CAS 378-44-9) and betamethasone 17-monopropionate (B17P), the active metabolites of betamethasone phosphate (BSP) and betamethasone dipropionate (BDP), respectively, after administration of betamethasone i.m. (BSP 2 mg and BDP 5 mg). After ten healthy volunteers had received a single-dose intramuscular adminitration of betamethasone i.m., blood samples were collected pre-dose and for 336 h postdose. The plasma levels of B17P and BOH were measured by liquid chromatography-tandem mass spectrometry (LC-MS/ MS). When compared to BOH, B17P exhibited a longer time to maximum concentration (15.0 +/- 9.0 h vs. 2.8 +/- 1.7 h), a lower Cmax (0.6 +/- 0.2 ng/mL vs. 14.5 +/- 3.7 ng/mL), and a much longer half-life (80.8 +/- 22.7 h vs. 9.6 +/- 3.6 h). Betamethasone i.m. produced rapid onset and sustained action through an initial rapid-increased plasma concentration of BOH and a sustained plasma concentration of B17P, respectively.

Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Trimetazidine in Human Plasma

A sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of trimetazidine (CAS 13171-25-0) in human plasma, using pseudoephedrine as internal standard (IS). Plasma samples were simply pretreated with methanol for deproteinization. The chromatographic separation was performed on a C18 column with a mobile phase of 3 mmol/L ammonium acetate solution-methanol (15:85, v/v) at a flow rate of 0.3 mL/min. The chromatographic separation was achieved in less than 3.2 min. The linearity was established over the concentration range of 1-100 ng/mL. Both intra- and inter-batch standard deviations were less than 9.5%. The method was successfully applied to study the relative bioavailability of trimetazidine hydrochloride tablets in healthy Chinese volunteers and the pharmacokinetic parameters of the reference and test tablets were compared.

Efffect of the ABCC3 –211C/T polymorphism on clopidogrel responsiveness in patients with percutaneous coronary intervention

Multidrug resistance protein 3 (MPR3), encoded by the ATP‐binding cassette, subfamily C (CFTR/MRP), member 3 (ABCC3) gene, functions as an important drug efflux transporter. The ABCC3 –211C/T polymorphism is associated with decreased MRP3 mRNA expression, and low MRP3 mRNA expression is associated with increased clopidogrel response in patients. The aim of the present study was to determine whether the –211C/T polymorphism is associated with altered antiplatelet effects and clinical outcomes in clopidogrel‐treated patients. A subcohort of 249 patients not carrying the CYP2C19*2, *3 or *17 variant was identified from a total of 617 consecutive clopidogrel‐treated patients undergoing percutaneous coronary intervention and then categorized into three groups on the basis of their ABCC3 –211C/T genotype. Baseline data, clinical characteristics and DNA samples were collected for all patients. Light transmittance aggregometry was used to determine ADP‐induced maximum platelet aggregation (MPA) in blood samples obtained from patients on Day 3 after starting daily clopidogrel maintenance doses. Genotyping of CYP2C19*2, *3 and *17 variants and the ABCC3 –211C/T polymorphism was performed using matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. The primary clinical end‐point was a definite stent thrombosis (ST) episode, whereas secondary end‐points were other major adverse cardiovascular events within 12 months after stenting. There were no differences in MPA values according to ABCC3 –211C/T genotype. A multiple linear regression model revealed that the ABCC3 –211C/T polymorphism was not independently associated with ADP‐induced MPA measurements; a multiple logistic regression model revealed that carrying the ABCC3 –211C allele was not associated with the risk of developing an ST event in clopidogrel‐treated patients not harbouring CYP2C19*2, *3 and *17 variants. In conclusion, the ABCC3 –211C/T polymorphism seems not to be associated with altered antiplatelet effects and clinical outcomes in clopidogrel‐treated patients.

Bioequivalence of two formulations of glucosamine sulfate 500-mg capsules in healthy male chinese volunteers: An open-label, randomized-sequence, single-dose, fasting, two-way crossover study

BACKGROUND Glucosamine sulfate is used for the treatment of arthrosis, especially osteoarthritis of the knee joint. The available evidence suggests differences in its pharmacokinetics in Chinese subjects compared with non-Chinese subjects. OBJECTIVE The aim of this study was to compare the pharmacokinetics and relative bioavailability of a test and reference formulation of glucosamine sulfate 500 mg after single oral administration in healthy Chinese volunteers. METHODS This open-label, randomized-sequence, single-dose, 2-way crossover study was performed at the First Hospital of Nanjing Medical University, Nanjing, China. Eligible subjects were healthy male volunteers who were randomly assigned at a 1:1 ratio to receive a single 500-mg dose of the test or reference capsule formulation, followed by a 1-week washout period and administration of the alternate formulation. The study drugs were administered after a 12-hour overnight fast. Glucosamine sulfate was assayed using a liquid-chromatography tandem mass spectrometry method. For analysis of pharmacokinetic properties, including C(max), AUC(0-t), and AUC(0-infinity)), blood samples were obtained at intervals over a 14-hour period after study drug administration. The formulations were considered bioequivalent if the log-transformed ratios of C(max) and AUC were within the predetermined equivalence range (70%-143% for C(max) and 80%-125% for AUC) as established by the State Food and Drug Administration (SFDA) of China. Tolerability was assessed by monitoring vital signs and laboratory tests (hematology, blood biochemistry, hepatic function, and urinalysis), and by questioning subjects about adverse events (AEs). RESULTS Twenty-two healthy male Chinese subjects were enrolled (mean [range] age, 24 [22-26] years; weight, 63.9 [58.5-69.3] kg; height, 172 [167-177] cm); all completed the study. No period or sequence effect was observed. The 90% CIs for the log-transformed ratios of C(max), AUC(0-t), and AUC(0-infinity)) were 93.4 to 127.3, 92.4 to 114.5, and 92.7 to 114.6, respectively (all, P = NS). The AUC(0-infinity) of the test and reference formulations was 1.83 (0.66) and 1.77 ( 0.72) microg/h/mL, respectively. No AEs were observed or reported during the study. CONCLUSIONS In this small study in healthy male Chinese volunteers, a single 500-mg dose of the test formulation met the SFDAs regulatory definition for bioequivalence to the reference formulation. Both formulations were well tolerated.