[Show abstract][Hide abstract] ABSTRACT: Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D(2) (1α(OH)D(2)), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D(2) (1,25(OH)(2)D(2)). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)(2)D(2) suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC-MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0-1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)(2)D(2) rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D(2).
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 01/2011; 879(2):139-45. DOI:10.1016/j.jchromb.2010.11.025 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent K(m) and V(max) values of 1.14 +/- 0.90 microM and 11.3 +/- 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 +/- 6.4 microM and 258 +/- 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations < or =1 microM. At substrate concentrations > or =10 microM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 microM OMF and 2 microM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC(50) data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.
Drug Metabolism and Disposition 06/2007; 35(6):841-7. DOI:10.1124/dmd.106.014472 · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A high-performance liquid chromatography (HPLC) method with UV detection at 232 nm was developed and validated for the simultaneous determination of triamcinolone acetonide (TAA) and oxymetazoline hydrochloride (OXY) in nasal spray formulations. The chromatographic system consisted of a micro Bondapak CN column (150 mm x 3.9 mm), 5 microm particle size with a mobile phase composition of acetonitrile:ammonium acetate (pH 5.0, 20mM) (10:90, v/v) at a flow rate of 1.0 mL/min. Calibration curves were linear for both TAA and OXY in the concentration range of 2.5-25.0 microg/mL. The limit of detection and quantitation were 0.29 and 0.88 microg/mL for OXY and 0.24 and 0.73 microg/mL for TAA. The described method was further applied to the analysis and stability studies of two nasal spray formulations I and II prepared from TAA and OXY commercial nasal spray products. The stability of OXY and TAA in the commercial products and the nasal formulations I and II were analyzed after 30 days at room temperature and 30 days at 40 degrees C/60% relative humidity. The results of the stability study showed that OXY and TAA in the commercial nasal spray products and the nasal formulations I and II were stable at 20-25 degrees C (room temperature) but TAA was unstable at 40 degrees C/60% relative humidity. TAA exhibited more than 10% loss at 14 days in both the nasal formulations and in the commercial products. OXY showed increased degradation at 40 degrees C/60% relative humidity but <10%.
Journal of Pharmaceutical and Biomedical Analysis 03/2006; 40(5):1273-80. DOI:10.1016/j.jpba.2005.09.018 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 enzymes (CYP450) and flavin-containing monooxygenses (FMO) are membrane-bound metabolizing enzymes catalyzing the metabolism of a variety of xenobiotics. Oxidative metabolism of xenobiotics by CYP450 or FMO often generate the same metabolites. Consequently, elucidation of the enzymes responsible for biotransformation is required to appropriately direct the medicinal chemistry campaign and to understand the potential drug-drug interaction (DDI) risks. In this work, a reaction phenotyping assay was developed to differentiate metabolism by CYP450 or FMO enzymes in liver microsomes. Benzydamine was employed as a marker substrate for both CYP450 mediated pathway (N-demethylation) and FMO catalyzed metabolism (N-oxidation). In an example of this approach, the assay was implemented to support a discovery project in which the major metabolite of the lead chemical scaffold was the N-oxidation of a tertiary amine in vitro across species (i.e., human, rat, dog, and monkey) and in vivo in rat and dog. Species differences were observed between human and preclinical species in the formation of N-oxide metabolite. In human and monkey liver microsomes, the N-oxidation was almost completely inhibited by 1-aminobenzotriazole (1-ABT), indicating the dominant presence of the CYP450 mediated metabolic pathway. In contrast, in dog and rat liver microsomes, the N-oxidation was only partially blocked by either 1-ABT or FMO inhibition (via heat inactivation or by methimazole), indicating dual CYP450 and FMO pathways were involved in the formation of N-oxide metabolite. This finding provided the appropriate direction for in vitro CYP450 reaction phenotyping studies of the lead candidate to assess potential DDI.
17th North American Regional International society for the study of xenobiotics Meeting;