E.J. Woolf

Best practices during bioanalytical method validation for the characterization of assay reagents and the evaluation of analyte stability in assay standards, quality controls, and study samples

Characterization of the stability of analytes in biological samples collected during clinical studies together with that of critical assay reagents, including analyte stock solutions, is recognized as an important component of bioanalytical assay validation. Deficiencies in these areas often come to light during regulatory inspections. Best practices, based on current regulatory guidance, for the assessment of these issues as they pertain to ligand binding and chromatographic assays are covered in this review. Additionally, consensus recommendations reached during the recent AAPS/FDA Workshop on bioanalytical assay validation are highlighted.

A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit

AIMS A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. MATERIALS & METHODS An average blood volume of 10.6 μl was absorbed by the samplers across the different HCTs investigated (20-65%). RESULTS No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. CONCLUSION The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

Determination of L-735 524, an human immunodeficiency virus protease inhibitor, in human plasma and urine via high-performance liquid chromatography with column switching

A method for the determination of an HIV protease inhibitor, L-735 524, in human plasma and urine is described. Isolation of the analyte and the internal standard from the matrices was achieved via multiple liquid-liquid extractions with methyl tert.-butyl ether. The analyte lacks significant UV absorption at wavelengths greater than 220 nm, hence a column switching system using a cyano and C18 column was used to further purify the extracts prior to UV detection at 210 nm. The assay has been found to be linear and has been validated over the concentration range of 5 to 500 ng/ml, when 1-ml aliquots of plasma or urine were extracted. The assay has been utilized to support human pharmacokinetic studies.

Single- and Multiple-Dose Pharmacokinetics of Etoricoxib, a Selective Inhibitor of Cyclooxygenase-2, in Man

The single‐ and multiple‐dose pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase‐2, were examined in two clinical studies. Single‐dose pharmacokinetics—including dose proportionality, absolute bioavailability of the highest dose‐strength (120‐mg) tablet, and the effect of a high‐fat meal on the bioavailability of that tablet—were investigated in a two‐part, open, balanced crossover study in two panels of healthy subjects (12 per panel). Steady‐state pharmacokinetics were investigated in an open‐label study in which 24 healthy subjects were administered 120‐mg single and multiple (once daily for 10 days) oral doses of etoricoxib tablets. The pharmacokinetics of etoricoxib were found to be consistent with linearity through doses at least twofold greater than the highest anticipated clinical dose of 120 mg. Etoricoxib administered as a tablet was rapidly and completely absorbed and available; the absolute bioavailability was estimated to be 100%. A high‐fat meal decreased the rate of absorption without affecting the extent of absorption of etoricoxib; therefore, etoricoxib can be dosed irrespective of food. Steady‐state pharmacokinetics of etoricoxib, achieved following 7 days of once‐daily dosing, were found to be reasonably predicted from single doses. The accumulation ratio averaged 2.1, and the corresponding accumulation t1/2 averaged 22 hours, supporting once‐daily dosing. Etoricoxib was generally well tolerated.

Determination of celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection.

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO2 (150x4.6 mm, 5 microm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25-2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.

Dose proportionality of oral etoricoxib, a highly selective cyclooxygenase-2 inhibitor, in healthy volunteers.

To assess dose proportionality of etoricoxib across the anticipated clinical dose range, a single panel of 12 healthy subjects was administered single oral doses of etoricoxib of 5,10, 20, 40, and 120 mg in an open, two‐part, five‐period crossover study. Plasma samples were collected after each dose and analyzed for etoricoxib concentrations. The pharmacokinetics of etoricoxib appear to be linear over the entire dose range examined, from 5 to 120 mg. Etoricoxib was found to be well tolerated across the 5 to 120 mg dose range.

Merck’s perspective on the implementation of dried blood spot technology in clinical drug development – why, when and how

This paper communicates Mercks thoughts on why, when and how to use dried blood spot (DBS) technology in a clinical setting, and provides a strategic approach, emphasizing the necessary steps, for successful clinical implementation of this microsampling technique. PK consideration based on relevant in vitro data, that is, blood-to-plasma ratio, hematocrit, plasma unbound fraction and/or blood cell partition, is suggested to be part of the decision tree on when to choose DBS as a surrogate matrix for PK analysis. A quick feasibility assessment addressing analytical challenges, including sensitivity, hematocrit impact and storage stability, needs to be evaluated before initiating DBS studies. Special attention should be paid to the clinical sample collection procedures to ensure data quality. Bridging studies are required to establish the correlation between plasma and DBS data to ensure that pooling of data from the various clinical studies can be used in population PK or PK/PD assessment. Seeking regulatory feedback and guidance on a case-by-case basis is recommended.

Determination of rofecoxib, a cyclooxygenase-2 specific inhibitor, in human plasma using high-performance liquid chromatography with post-column photochemical derivatization and fluorescence detection

A method for the determination of rofecoxib in human plasma is described. After the addition of an internal standard, buffered (pH 5) plasma samples are extracted with hexane-methylene chloride (50:50, v/v). The extracts are evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to undergo a stilbene-phenanthrene-like photocyclization reaction with the resulting formation of a highly fluorescent species. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. The assay has been validated in the concentration range of 0.5-100 ng/ml using 1-ml samples. The method has been successfully utilized to support human clinical pharmacokinetic studies.

Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection

Methods for the quantitative determination of efavirenz in human plasma and the qualitative assessment of the stereochemical integrity of efavirenz in post-dose human plasma samples are described. After the addition of an internal standard, plasma samples were extracted with hexane-methylene chloride (65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to form fluorescent products; the major fluorescent product was isolated and identified as a substituted quinoline. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. Reverse phase chromatography was used for the quantitative assay, whereas chromatography with a column containing a chiral stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment. The quantitative assay has been validated in the concentration range of 50-1000 ng/ml using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and 104% of nominal over the range of the assay. The selective detection method reduces the likelihood of interference by co-administered medications or endogenous species. The stereochemical configuration of efavirenz was confirmed to remain intact in post-dose human plasma samples. The quantitative method has been successfully utilized to support a study in which a possible drug interaction between co-administered HIV protease inhibitors and efavirenz was evaluated.

Pharmacokinetics of Etoricoxib in Patients with Renal Impairment

The effect of renal insufficiency on the pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase‐2, was examined in 23 patients with varying degrees of renal impairment (12 moderate [creatinine clearance between 30 and 50 mL/min/1.73 m2], 5 severe [creatinine clearance below 30 mL/min/1.73 m2], and 6 with end‐stage renal disease requiring hemodialysis) following administration of single 120‐mg oral doses of etoricoxib. Even the most severe renal impairment was found to have little effect on etoricoxib pharmacokinetics. The low recovery of etoricoxib in dialysate (less than 6% of the dose) supports that hemodialysis also has little effect on etoricoxib pharmacokinetics, and binding of etoricoxib to plasma proteins was generally unaffected by renal disease. Single doses of etoricoxib were generally well tolerated by patients with renal impairment. Based on pharmacokinetic considerations, dosing adjustments are not necessary for patients with any degree of renal impairment. However, because patients with advanced renal disease (creatinine clearance below 30 mL/min/1.73 m2) are likely to be very sensitive to any further compromise of renal function, and there is no long‐term clinical experience in these patients, the use of etoricoxib is not recommended in patients with advanced renal disease.