Incurred sample reanalysis: different evaluation approaches on data obtained for spironolactone and its active metabolite canrenone.

BACKGROUND: The inherent reproducibility of a bioanalytical approach is usually sustained through incurred sample reanalysis (ISR). Questions relating to the number of ISRs, the right moment for performing reanalysis, the way of performing an appropriate statistical refinement of experimental data and actions to be taken in the case of failure are frequently raised. RESULTS: Data resulting from ISR following a bioequivalence study for spironolactone formulations are discussed. Reanalysis of samples was carried out twice: immediately after the end of the study and after a period that overcame the long-term stability study achieved during method validation. The Bland-Altman approach was used to assess experimental results. ISR was successful over the short reanalysis period for both compounds. Data produced through reanalysis after the long-term period indicated a systematic positive bias for the metabolite canrenone (although results supported reproducibility). The results obtained for spironolactone were affected by a strong negative systematic bias and failed to support reproducibility. The explanation deals with the continuous conversion of spironolactone to canrenone in plasma samples. However, reproducibility of the method may be sustained by comparing original and repeated differences between concentration values in samples by means of a paired t-test, Wilcoxon sign rank-sum test and linear regression. CONCLUSIONS: Different statistical approaches for making data comparisons are discussed and may be successfully applied during reanalysis of samples from a bioequivalence study. Results of the evaluations may differ in accordance with the statistical procedure being applied, thus a definitive conclusion requires consideration of all specific experimental circumstances arising during production of the processed data.

Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma. Use of incurred samples to test back-conversion.

Quantitative methods using LC-MS/MS allow achievement of adequate sensitivity for pharmacokinetic studies with clopidogrel; three such methods, with LLOQs as low as 5 pg/mL, were developed and fully validated according to the well established FDA 2001 guidelines. The chromatographic separations were performed on reversed phase columns Ascentis RP-Amide (15 cm x 2.1 mm, 5 µm), Ascentis Express C8 (10 cm x 2.1 mm, 2.7 µm) and Ascentis Express RP Amide (10 cm x 2.1 mm, 2.7 µm), respectively. Positive electrospray ionization in MRM mode was employed for the detection and a deuterated analogue (d3-clopidogrel) was used as internal standard. Linearity, precision, extraction recovery, matrix effects and stability tests on blank plasma spiked with clopidogrel and stored in different conditions met the acceptance criteria. During the analysis of the real samples from the first pharmacokinetic study, a significant increase (>100%) of the measured clopidogrel concentrations in the extracts kept in the autosampler at 10 °C was observed. Investigations led to the conclusion that most probably a back-conversion of one or more of the clopidogrel metabolites is occurring. The next methods were optimized in order to minimize this back-conversion. After a series of experiments, the adjustment of the sample preparation (e.g. processing at low temperature and introducing a clean-up step on Supelco HybridSPE-Precipitation cartridges) has proven to be the most effective in order to improve the stability of the extracts. Incurred samples of real subjects were successfully used in the validation of the last two analytical methods to evaluate the back-conversion, while tests using only the known metabolites could not detect this important problem.

High-throughput HPLC-MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications.

A sensitive high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of ibandronate in human plasma. In a previous study, we have analyzed alendronate in urine samples of subjects treated at therapeutic dosages, using a derivatization approach; a similar derivatization was adapted and improved to determine ibandronate in plasma. The bisphosphonate was isolated from the biological matrix by liquid-liquid extraction, and derivatized with trimethylsilyldiazomethane prior to separation on a reversed-phase column (Supelco Discovery HSC18) and detection on a quadrupole-linear ion trap mass spectrometer (API 4000 QTrap). Various parameters of extraction and derivatization were optimized in order to get adequate recovery, high derivatization yield and minimal ion suppression; a deuterated analogue, d3-ibandronate, was used as internal standard. The transitions 376.1-->114.2 and 379.1-->61.0 were acquired to monitor ibandronate and d3-ibandronate derivatives, respectively. A multiplexing LC system made possible the overlapping of two chromatographic runs, thus the interval between injections being reduced to only 2min, a very short analysis time for compounds of this class. The method was fully validated over the quantification range 0.2-175.0ng/ml, allowing an appropriate evaluation of the plasma concentrations of ibandronate, expected at therapeutic dosage, as proved by application to a pharmacokinetic study. A good linearity over the selected range (r>0.99), accuracy and precision within +/-15% of the target values and a recovery over 50% were obtained.