LC-MS/MS determination of dutasteride and its major metabolites in human plasma.

Dutasteride is a specific and selective inhibitor of both 5α-reductase isoforms used mainly in benign prostatic hyperplasia and lower urinary tract symptoms. Although the drug is extensively metabolized in humans, data on the concentrations of its main metabolites are lacking. There is also a lack of data on dutasteride stability in frozen plasma samples. Our method was used to determine dutasteride and its active metabolites: 4'-hydroxydutasteride, 6ß-hydroxydutasteride, and 1,2-dihydrodutasteride in plasma after a single administration of 0.5 mg of dutasteride. We also assessed the long-term stability (two years in the freezer) of dutasteride in clinical samples. The developed method covered the range of 0.1-3.5 ng/mL for dutasteride and 0.08-1.2 ng/mL for 1,2-dihydrodutasteride, 4'-hydroxydutasteride, 6ß-hydroxydutasteride. It was proved to be reliable as it met all validation criteria required by the European Medicine Agency for bioanalytical methods. 4'-hydroxydutasteride and 1,2-dihydrodutasteride concentrations in plasma were higher than 6ß-hydroxydutasteride. Dutasteride was stable in the freezer for up to 2 years in clinical samples. Thus within 1014 days of storage (below - 65 °C), samples can be reanalyzed without the risk of unreliable results.

10th Anniversary of a Two-Stage Design in Bioequivalence. Why Has it Still Not Been Implemented?

PURPOSE: In 2010 the European Medicines Agency allowed a two-stage design in bioequivalence studies. However, in the public domain there are mainly articles describing the theoretical and statistical base for the application of the two-stage design. One of the reasons seems to be the lack of practical guidance for the Sponsors on when and how the two-stage design can be beneficial in bioequivalence studies. METHODS: Different variants with positive and negative outcomes have been evaluated, including a pivotal study, pilot + pivotal study and two-stage study. The scientific perspective on the two-stage bioequivalence study has been confronted with the industrial one. RESULTS: Key information needed to conduct a bioequivalence study - such as in vitro data and pharmacokinetics - have been listed and organized into a decision scheme. Advantages and disadvantages of the two-stage design have been summarized. CONCLUSION: The use of the two-stage design in bioequivalence studies seems to be a beneficial alternative to the 2 × 2 crossover study. Basic information on the properties of the active substance and the characteristics of the drug form are needed to make an initial decision to carry out the two-stage study.

Incurred Sample Reanalysis: Time to Change the Sample Size Calculation?

Reliable results of pharmacokinetic and toxicokinetic studies are vital for correct decision making during drug discovery and development. Thus, ensuring high quality of bioanalytical methods is of critical importance. Incurred sample reanalysis (ISR)-one of the tools used to validate a method-is included in the bioanalytical regulatory recommendations. The methodology of this test is well established, but the estimation of the sample size is still commented on and contested. We have applied the hypergeometric distribution to evaluate ISR test passing rates in different clinical study sizes. We have tested both fixed rates of the clinical samples-as currently recommended by FDA and EMA-and a fixed number of ISRs. Our study revealed that the passing rate using the current sample size calculation is related to the clinical study size. However, the passing rate is much less dependent on the clinical study size when a fixed number of ISRs is used. Thus, we suggest using a fixed number of ISRs, e.g., 30 samples, for all studies. We found the hypergeometric distribution to be an adequate model for the assessment of similarities in original and repeated data. This model may be further used to optimize the sample size needed for the ISR test as well as to bridge data from different methods. This paper provides a basis to re-consider current ISR recommendations and implement a more statistically rationalized and risk-controlled approach.

Bioanalytical method validation: new FDA guidance vs. EMA guideline. Better or worse?

Bioanalysis concerns the identification and quantification of analytes in various biological matrices. Validation of any analytical method helps to achieve reliable results that are necessary for proper decisions on drug dosing and patient safety. In the case of bioanalytical methods, validation additionally covers steps of pharmacokinetic and toxicological studies - such as sample collection, handling, shipment, storage, and preparation. We drew our attention to the difference of both the newest FDA Guidance and the EMA Guideline on bioanalytical method validation. We aimed to point out advantages of both documents from the laboratory perspective. The FDA and the EMA documents are similar, but not identical. The EMA describes the practical conduct of experiments more precisely, while the FDA presents reporting recommendations more comprehensively. There are also differences in recommended validation parameters. We hope that the International Council for Harmonisation will combine advantages of both documents to avoid confusing differences in terminology as well as the unnecessary effort of being compliant with two or more guidelines.

Extended 3D and 4D cumulative plots for evaluation of unmatched incurred sample reanalysis.

AIM: Incurred sample reanalysis (ISR) helps ensure the reliability of pharmacokinetic studies. An appropriate graph may facilitate the evaluation of an unmatched reanalyses or a failed ISR test. METHODS: We evaluated different ways of visualizing multidimensional ISR data using an extended cumulative ISR plot. RESULTS: 3D and 4D cumulative ISR plots enable comprehensive data analysis using a single plot. We propose to use color for percentage difference classes in bar and XY-scatter plots. For the latter the shape of symbols may represent analyte concentration class, study phase, analyst or subject. CONCLUSION: The extended 3D and 4D cumulative ISR plots facilitate in-study monitoring and post-study inspection of data. It helps find the root cause of unmatched ISR, thus increasing reliability of bioanalytical data.

Incurred sample reanalysis: adjusted procedure for sample size calculation.

AIM: The incurred sample reanalysis (ISR) helps to assure bioanalysis reliability. The regulatory guidelines recommend the reanalysis of up to 10% of the study samples for this test, but not all reanalyses are necessary to evaluate the test result. MATERIALS & METHODS: We have optimized ISR sample size calculation to eliminate negligible reanalyses. RESULTS: Adjusted procedure eliminates negligible reanalyses - up to 66% of currently analyzed ISRs - without affecting the test outcome. CONCLUSION: The procedure is universal as it may be applied in the studies compliant with EMA and US FDA requirements, for both small and large molecules. It may assist the evolution of bioanalytical method validation as the current ISR sample size recommendations seem to be ill-matched with the acceptance criteria.

Comprehensive graphical presentation of data from incurred sample reanalysis.

AIM: Incurred sample reanalysis (ISR) contributes to the reliability of pharmacokinetic studies. Despite regulatory guidelines having adopted ISR methodology, graphical presentation of data has been overlooked. MATERIALS & METHODS: Different graphs were tested for datasets including limited, standard and large numbers of ISR pairs. The datasets covered both passed and failed cases. RESULTS: We have developed a combination of complementary plots enabling the visual inspection of ISR data quality: %difference versus mean concentration and cumulative ISR plot. The former shows individual ISR datapoints and concentration-dependent trends, while the latter presents the contribution of individual pairs to the overall result as well as time-dependent trends. CONCLUSION: The proposed visualization of ISR data shows at a glance whether acceptance criteria for each sample and whole experiment are met or not. Standardized graphical presentation of ISR outcomes may increase quality of bioanalytical data.

HPLC-UV ASSAY OF IMATINIB IN HUMAN PLASMA OPTIMIZED FOR BIOEQUIVALENCE STUDIES.

lmatimb is an anticancer drug approved for the treatment of a number of cancers, mostly used in chronic myeloid leukemia. Numerous bioanalytical methods using high performance liquid chromatography coupled to ultraviolet detection point at the importance and necessity of the therapeutic drug monitoring of imatinib. Unfortunately, these methods are not optimized for single dose pharmacokinetic studies such as bioe- quivalence. In this study, attention was turned mostly to the analysis time, linearity range and interferences by endogenous components of the matrix and exogenous substances - especially metabolites. The method enables the quantification of imatinib in the presence of the main metabolite (N-desmethyl imatinib). Its potential back- conversion was examined during storage for 4 h at ambient temperature as well as for 239 days at -20°C. The sample preparation based on the liquid-liquid extraction was combined with a short analysis time of 7 min. Therefore, the method was suitable for analyzing large numbers of samples in a short time. The linearity range of 40-4000 ng/mL was optimized for human pharmacokinetic studies after a single 400 mg oral dose of ima- tinib. Successful application in a bioequivalence study confirmed the reliability of the method.

Validated HPLC-UV method for determination of naproxen in human plasma with proven selectivity against ibuprofen and paracetamol.

Estimating the influence of interfering compounds present in the biological matrix on the determination of an analyte is one of the most important tasks during bioanalytical method development and validation. Interferences from endogenous components and, if necessary, from major metabolites as well as possible co-administered medications should be evaluated during a selectivity test. This paper describes a simple, rapid and cost-effective HPLC-UV method for the determination of naproxen in human plasma in the presence of two other analgesics, ibuprofen and paracetamol. Sample preparation is based on a simple liquid-liquid extraction procedure with a short, 5 s mixing time. Fenoprofen, which is characterized by a similar structure and properties to naproxen, was first used as the internal standard. The calibration curve is linear in the concentration range of 0.5-80.0 µg/mL, which is suitable for pharmacokinetic studies following a single 220 mg oral dose of naproxen sodium. The method was fully validated according to international guidelines and was successfully applied in a bioequivalence study in humans. Copyright © 2015 John Wiley & Sons, Ltd.

Determination of duloxetine in human plasma with proven lack of influence of the major metabolite 4-hydroxyduloxetine.

OBJECTIVES: Minimizing the impact of major or unstable metabolites on the determination of a drug substance represents a leading task in the development and validation of bioanalytical methods. "Incurred samples reanalysis" provides relevant information too late; therefore, carefully selected tests on known metabolites should precede the pharmacokinetic studies. DESIGN AND METHODS: This paper describes a simple and rapid HPLC-UV method for the determination of duloxetine, a potent serotonin and norepinephrine reuptake inhibitor, in the presence of its major metabolite, i.e. 4-hydroxyduloxetine glucuronide. Analyte and fluoxetine (internal standard) were extracted from human plasma by liquid-liquid extraction. RESULTS: No influence of the major metabolite was observed on the reliability of the new method. There was also lack of evidence of the major metabolite back-conversion to the parent drug substance. The validation demonstrated high precision of the new method. All validation parameters met the acceptance criteria of bioanalytical regulations. CONCLUSIONS: The new method enabled the reliable determination of duloxetine in the presence of its major metabolite in the human plasma. The method might be applied to pharmacokinetic studies in humans, including bioequivalence and therapeutic drug monitoring.

Validated HPLC method for determination of temozolomide in human plasma.

The aim of the study was to develop a bioanalytical method for the determination of temozolomide (TMZ) in human plasma. Plasma concentration of TMZ was determined on a C18 column after liquid-liquid extraction. Isocratic elution was applied with the mixture of aqueous acetic acid and methanol. Theophylline was used as the internal standard. To prevent chemical degradation of TMZ at physiological pH, plasma samples were acidified to pH < 3. All validation parameters met the acceptance criteria. Calibration curve, prepared using freshly spiked plasma samples, was linear within the range of 0.10-20.00 microg/mL. The method was found to be sufficiently accurate and precise over the studied range of concentrations. TMZ was stable in the acidified plasma samples for at least 50 days at < or = -14 degrees C and < or = -65 degrees C. The method recovery of TMZ from human plasma was consistent and ranged 37.1-41.1%. The developed method is suitable for pharmacokinetic studies in humans after oral administration of TMZ.

Bioequivalence study of 500 mg cefuroxime axetil film-coated tablets in healthy volunteers.

The aim of the study was to investigate the bioavailability of a generic product of 500 mg cefuroxime axetil film-coated tablets (test) as compared to that of a branded product (reference) at the same strength to determine bioequivalence and to apply for regulatory approval. The secondary objective of the study was to evaluate tolerability of both products. A double blinded, randomized, crossover, two-period, single-dose, comparative study was conducted in Caucasian healthy volunteers in fasting conditions. A single oral dose administration of the test or reference product was followed by 7-day wash-out period. The cefuroxime concentration was determined using a validated HPLC-UV method. The results of the single-dose study in healthy volunteers indicated that the film-coated tablets of Tarsime 500 mg manufactured by Tarchominskie Zaklady Farmaceutyczne Polfa S.A. (test product) are bioequivalent to those of Zinnat manufactured by GlaxoSmithKline Export Ltd. (reference product). Both products were well tolerated.