John A. Dunn

Pharmacokinetics of Bupropion and its Metabolites in Cigarette Smokers versus Nonsmokers

Bupropion is an antidepressant agent that is also effective as an aid to quit cigarette smoking. A single 150‐mg tablet of sustained‐release bupropion hydrochloride was administered to two groups of volunteers, cigarette smokers and nonsmokers, who were matched for race, gender, body frame, age, and weight. Pharmacokinetic parameters were calculated for bupropion, and three major metabolites (hydroxybupropion and the aminoalcohol isomers, threohydrobupropion and erythrohydrobupropion, expressed as a composite total). Mean (±SD) values of area under the concentration‐time curve from time 0 extrapolated to infinity (AUC0–∞), maximum concentration (Cmax), time to reach Cmax (tmax), and half‐life (t1/2) of bupropion in smokers and nonsmokers, respectively, were 1,164 ± 220 ng · hr/mL and 1,161 ± 292 ng · hr/mL; 144 ± 28 ng/mL and 143 ± 39 ng/mL; 3.00 ± 0.50 hours and 2.88 ± 0.49 hours; and 19 ± 5 hours and 18 ± 3 hours. No clinically significant differences between smokers and nonsmokers or between male and female volunteers were observed for the pharmacokinetics of bupropion or its metabolites. The absence of pharmacokinetic differences indicates that dosage adjustments are not necessary when bupropion is prescribed to male and female cigarette smokers.

Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry

A method employing high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS) has been developed and validated for the simultaneous determination of clinically relevant levels of zidovudine (AZT) and lamivudine (3TC) in human serum. The method incorporates a fully automated ultrafiltration sample preparation step that replaces the solid-phase extraction step typically used for HPLC with UV detection. The calibration range of the dual-analyte LC-MS/MS method is 2.5-2,500 and 2.5-5,000 ng ml-1 for AZT and 3TC, respectively, using 0.25 ml of human serum. The lower limit of quantification was 2.5 ng ml-1 for each analyte, with a chromatographic run time of approximately 6 min. Overall accuracy, expressed as bias, and inter- and intra-assay precision are < +/- 7 and < 10% for AZT, and < +/- 5 and < 12.1% for 3TC over the full concentration ranges. A cross-validation study demonstrated that the LC-MS/MS method afforded equivalent results to established methods consisting of a radioimmuno-assay for AZT and an HPLC-UV method for 3TC. Moreover, the LC-MS/MS was more sensitive, allowed markedly higher-throughput, and required smaller sample volumes (for 3TC only). The validated method has been used to support post-marketing clinical studies for Combivir a combination tablet containing AZT and 3TC.

Nucleoside analogues achieve high concentrations in seminal plasma: relationship between drug concentration and virus burden.

Human immunodeficiency virus (HIV) can be transmitted in semen from a man to his sexual partners. Antiretroviral drugs are likely to affect the amount of HIV-1 in semen and perhaps transmission of the virus. The concentrations of zidovudine, lamivudine, and HIV-1 RNA in blood and seminal plasma were measured in 9 HIV-positive men over </=2 years. Median (25th-75th percentiles) zidovudine blood and seminal plasma concentrations were 64.2 (range, 48.4-206.9; n=82) and 292.5 (range, 194.3-438.4; n=79) ng/mL, respectively. Median lamivudine blood and seminal plasma concentrations were 391.3 (range, 175.3-793.8; n=82) and 2701.8 (range, 1460.5-4320.2; n=79) ng/mL, respectively. The concentration of HIV-1 RNA in seminal plasma was monitored as a potential surrogate marker for infectiousness. RNA became undetectable (<400 copies/mL) in the blood and seminal plasma of 8/9 subjects after initiation of therapy and remained undetectable in 6/9 subjects. These data show that zidovudine and lamivudine achieve high concentrations in seminal plasma and significantly reduce HIV-1 RNA. The effects of antiviral therapy on HIV-1 in semen and on the sexual transmission of HIV-1 require further study.

Simultaneous determination of lamivudine and zidovudine concentrations in human seminal plasma using high-performance liquid chromatography and tandem mass spectrometry

A HPLC-MS-MS method was developed and validated to measure lamivudine and zidovudine simultaneously in small volumes of human seminal plasma. Sample preparation was simple and rapid, requiring 25 microl of sample, the use of isotopically labeled lamivudine and zidovudine as internal standards and ultrafiltration through a molecular mass cut-off membrane. Lamivudine and its internal standard were separated from zidovudine and its internal standard with isocratic HPLC. Detection was carried out using tandem mass spectrometry. This validated method was used to analyze seminal samples obtained from six HIV-positive patients prescribed lamivudine and zidovudine.

Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC-MS-MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10-5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).

Lack of effect of cimetidine on the pharmacokinetics of sustained-release bupropion.

The objective of this study was to assess whether cimetidine affects the pharmacokinetics of sustained‐release (SR) bupropion hydrochloride and the active metabolite, hydroxybupropion. This randomized, open‐label, two‐period crossover study was conducted in 24 healthy volunteers 18 to 45 years of age. ANOVA showed that administration of two 150 mg bupropion SR tablets with one 800 mg cimetidine tablet following an overnight fast did not change values for AUC∞, Cmax, tmax, t1/2, and CL/F (CL/F calculated for bupropion only) for either bupropion or hydroxybupropion as compared with two 150 mg bupropion SR tablets alone. In this study, it appears that there is no effect of cimetidine on the pharmacokinetics of bupropion SR.

Determination of amprenavir, a HIV-1 protease inhibitor, in human seminal plasma using high-performance liquid chromatography-tandem mass spectrometry.

A HPLC-MS-MS method to measure amprenavir in human seminal plasma has been developed and validated. The procedure uses stable, isotopically labeled 13C6-amprenavir as an internal standard and 100 microl of sample. The method is accurate (bias less than or equal to 7.2%) and precise (within- and between-day RSDs less than or equal to 4.2%) over the dynamic range of 30-4,000 ng/ml. Recently, this simple and sensitive method was used to determine amprenavir concentrations in seminal samples collected from HIV-1 positive subjects receiving amprenavir antiretroviral therapy as part of a multicenter clinical trial.

The quest for assay robustness across the life cycle of a bioanalytical method

White has been an analytical chemist for 28 years, with a range of knowledge and experience across multiple disciplines including pharmaceuticals, foodstuffs, environmental and forensic analysis. Steve works within the regulated bioanalytical group at GSK supporting both preclinical and clinical bioanalysis. His responsibilities have included leadership of bioanalytical, dose formulation and sample management teams as well as assisting with the set up of the Anti-Doping Testing Laboratory for the London 2012 Olympics. In his current role, Steve is part of the UK bioanalytical leadership team with a focus on regulatory & compliance issues. Steve is the small molecule rep for GSK within the European Bioanalysis Forum (EBF), a member of the EBF Steering Committee and also contributed to the Global Bioanalysis Consortium S2 team. The successful validation or qualification of an assay plus incurred sample reproducibility gives the bioanalyst confidence of assay suitability. However, these elements alone do not give the full picture of how reliable an assay may be during routine production use for analysis of unknown study samples. The robustness of an assay can be understood by applying key performance indicators to monitor assay performance pre- and post-transfer between laboratories, plus a cross-validation step during assay transfer. The measurement of assay robustness (beyond the context of cross-validation) should not be considered as yet another regulatory layer, but more as a means for the bioanalyst to better understand an individual assay within a large portfolio, when used for its intended purpose.

Integrating internal and external bioanalytical support to deliver a diversified pharmaceutical portfolio.

The portfolios of pharmaceutical companies have diversified substantially over recent years in recognition that monotherapies and/or small molecules are less suitable for modulating many complex disease etiologies. Furthermore, there has been increased pressure on drug-development budgets over this same period. This has placed new challenges in the path of bioanalytical scientists, both within the industry and with contract research organizations (CROs). Large pharmaceutical, biotechnology and small-medium healthcare enterprises have had to make important decisions on what internal capabilities they wish to retain and where CROs offers a significant strategic benefit to their business model. Our journey has involved asking where we believe an internal bioanalytical facility offers the greatest benefit to progressing drug candidates through the drug-development cycle and where externalization can help free up internal resources, adding flexibility to our organization in order to deal with the inevitable peaks and troughs in workload.