Chromatographic separation and sensitive determination of teriflunomide,an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide (m/z 269.0 → 82.0) and IS (m/z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.     

Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects

A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00–500 ng/mL, corresponding to a concentration range of 0.0300–15.0 μg/mL in 50 μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2 g strontium ranelate.     

Basing the Analysis of Comparative Bioavailability Trials on an Individualized Statistical Definition of Equivalence

The conventional definition of bioequivalence in terms of population means only, is criticized for lacking relevance to the individual subject. Both approaches to bioequivalence assessment proposed here for avoiding this shortcoming, focus on the probability of an event induced by the response of a randomly selected subject to two formulations of a given active agent. The first approach leads to converting the basic idea underlying the well-known 75-rule into an exact statistical procedure. The second approach is of a parametric nature. It reduces bioequivalence assessment to testing against the alternative hypothesis that the standardized expected value of a Gaussian distribution is contained in a short interval around zero. For this problem, an exact optimal solution is provided as well.     

长半衰期药物无清洗期时的生物等效性研究

目的：探讨长半衰期药物（t1／2〉24h）在无清洗期时生物等效性研究中的AUC和Cmax的计算,通过无清洗期的实验数据推算出正常清洗期的数据。方法：利用SPSS软件,建立二室模型口服药物在无清洗期时的半衰期为100小时的生物等效性模型,通过优化AUC和Cmax的计算方法,降低药物残留对第二周期药物浓度的影响,进而增加AUC和Cmax的计算的精确性,最后用较精确的方法推算出正常清洗期的AUC和Cmax,利用精确的数据进行生物等效性的进一步验证。结果：在无清洗期的状态下,取样时间在大于0．8个半衰期时,平均值法计算的Auc和Cmax的结果误差小于5％,变异系数小于25％,较为精确,生物等效性研究进一步验证了这一观点。结论：在无清洗期的情况下,生物等效性研究最小的采样时间为0．8个半衰期。     

Extension of the SAEM algorithm for nonlinear mixed models with 2 levels of random effects

This article focuses on parameter estimation of multilevel nonlinearmixed-effects models (MNLMEMs). These models are used to analyzedata presenting multiple hierarchical levels of grouping (clusterdata, clinical trials with several observation periods, ...).The variability of the individual parameters of the regressionfunction is thus decomposed as a between-subject variabilityand higher levels of variability (e.g. within-subject variability).We propose maximum likelihood estimates of parameters of thoseMNLMEMs with 2 levels of random effects, using an extensionof the stochastic approximation version of expectation–maximization(SAEM)–Monte Carlo Markov chain algorithm. The extendedSAEM algorithm is split into an explicit direct expectation–maximization(EM) algorithm and a stochastic EM part. Compared to the originalalgorithm, additional sufficient statistics have to be approximatedby relying on the conditional distribution of the second levelof random effects. This estimation method is evaluated on pharmacokineticcrossover simulated trials, mimicking theophylline concentrationdata. Results obtained on those data sets with either the SAEMalgorithm or the first-order conditional estimates (FOCE) algorithm(implemented in the nlme function of R software) are compared:biases and root mean square errors of almost all the SAEM estimatesare smaller than the FOCE ones. Finally, we apply the extendedSAEM algorithm to analyze the pharmacokinetic interaction oftenofovir on atazanavir, a novel protease inhibitor, from theAgence Nationale de Recherche sur le Sida 107-Puzzle 2 study.A significant decrease of the area under the curve of atazanaviris found in patients receiving both treatments.     

Determination of 2-hydroxyflutamide in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS): Application to a bioequivalence study on Chinese volunteers

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)–ESI-MS/MS method was developed for the determination of 2-hydroxyflutamide in human plasma using tegafur as the internal standard. The plasma sample was pretreated with methanol for protein precipitation and the analytes were separated on an Ultimate C18 column (5 μm, 2.1 mm × 50 mm, MD, USA) with the mobile phase consisted of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a negative multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 290.90–204.8 for 2-hydroxyflutamide and 198.9–128.8 for tegafur. Linear calibration curves were obtained in the concentration range of 1.742–1452 ng/ml with a lower limit of quantification of 1.742 ng/ml. The intra- and inter-batch precision values were less than 8.1% and 5.6%, respectively. The established method was successfully applied to a bioequivalence study of two flutamide preparations (250 mg) in 20 healthy male volunteers.     

An Improvement of the Westlake Symmetric Confidence Interval

A common statistical method for assessing bioequivalence of two formulations of a chemical substance is the symmetric confidence interval of WESTLAKE (1972). As mentioned by WEST -LAKE (1981) and SCHUIRMAN (1981) a more powerful method consists of two one-sided t-tests. An (1-α)-confidence interval consistent with the two one-sided t-tests procedure is given by [min(α, 0), max (0, b)] where [a, b] is the conventional (1–2α)-confidence interval of the t-test. This “central” confidence interval is always a strict subset of the symmetric confidence interval and thus has more power in proving bioequivalence. The central confidence interval has properties comparable with those of the conventional one-sided confidence intervals.     

孟鲁斯特钠咀嚼片在健康人体内药动学和生物等效性

目的:研究两种(仿制新药与市售)孟鲁司特钠咀嚼片在人体内生物等效性。方法:采用单中心、随机、开放、双周期自身交叉试验设计,20名健康男性志愿者分2周期分别口服受试制剂和参比制剂各10 mg,HPLC法测定血浆中孟鲁司特钠咀嚼片的浓度,用DAS2.1.1软件计算人体药动学参数并进行生物等效性评价。结果:受试制剂和参比制剂两药的主要药代动力学参数AUC0-t分别为(17.94±6.19)μg h/ml和(17.37±4.73)μg h/ml,AUC0-∞分别为(18.26±6.16)μg h/ml和(17.64±4.66)μg h/ml,Cmax分别为(5.58±1.95)μg/ml和(5.54±1.65)μg/ml,Tmax分别为(2.03±0.97)h和(1.93±0.69)h,t1/2分别为(1.20±0.17)h和(1.19±0.13)h。受试制剂的平均相对生物利用度为(101.5±6.56)%。结论:受试制剂和参比制剂具有生物等效性。