LC-MS/MS method for quantification of 3,4-dihydroxyphenylglycol, a norepinephrine metabolite in plasma and brain regions.

Aim: To evaluate suitability of the LC-MS/MS method to quantify 3,4-dihydroxyphenylglycol (DHPG) that is used as a biomarker for monoamine oxidase (MAO) inhibition. Methods: DHPG was extracted using alumina basic cartridges and quantified on a triple quadrupole mass spectrometer using negative electrospray ionization, without the use of derivatization reagents. Results: Modulation of DHPG levels was observed following administration of selective and nonselective MAO inhibitors and results were in correlation with historical MAO inhibition potential of compounds. Conclusion: The proposed method is sensitive enough to measure plasma DHPG levels and DHPG can be used as a biomarker to assess MAO inhibition potential of new therapeutic agents.

Methoxsalen as an in vitro phenotyping tool in comparison with 1-aminobenzotriazole.

The objective is to evaluate methoxsalen as an in vitro phenotyping tool in comparison to ABT as a nonspecific inactivator of P450 mediated metabolism. The reversible inhibition of methoxsalen and ABT against the P450, FMO, AO, MAO-A and -B, enzymes were evaluated using standard marker probe reactions. The time-dependent inhibition of P450 enzymes was evaluated in human liver microsomes. CES1 activities were determined by monitoring the depletion of known substrate, the clopidogrel. The metabolism of P450 substrates in the presence and absence of methoxsalen or ABT was evaluated in human liver microsomes. Methoxsalen is a direct inhibitor and inhibited the activities (>90%) of all enzymes at a concentration of 300 µM except for CYP2C9. Methoxsalen is also a potent time-dependent inhibitor of all P450 enzymes except for CYP2C19 (moderate) at a concentration of 300 µM. Methoxsalen inhibited the metabolism of P450 substrates in the pre-incubation mode. ABT is a potent TDI of several P450 except for CYP2C19 (47%) and CYP2C9 (27%). The results indicate that methoxsalen is a potent pan P450 inhibitor than ABT and can be a better tool in distinguishing P450 mediated metabolism form non-P450 metabolism in human liver microsomes.

Development and validation of sensitive LC-MS/MS method for the quantification of SUVN-502 and its metabolite and its application for first in human pharmacokinetic study.

A sensitive and rapid LC-MS/MS method was developed and validated for the quantification of SUVN-502 and M1 of SUVN-502, a 5-HT6 receptor antagonist for the treatment of dementia associated with Alzheimer's disease. Following solid-phase extraction, SUVN-502 and M1 of SUVN-502 and IS were eluted with 10mM ammonium acetate (pH 4.0) and acetonitrile using a rapid gradient program on reverse phase column. Multiple reaction monitoring mode was used to monitor the respective transitions of m/z 478.2→377.7 for SUVN-502 and m/z 464.1→377.7 for M1 of SUVN-502. The assay exhibited a linear dynamic range of 10-10000pg/mL for SUVN-502 and 20-20000pg/mL for M1 of SUVN-502 in human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The within batch accuracy and precision were within acceptable limits. All the other validation parameters were within the acceptable limits. The validated method was applied to analyze human plasma samples obtained from a human pharmacokinetic study consisting single and multiple ascending doses.

Incurred sample reanalysis of fingolimod and fingolimod phosphate in blood: stability evaluation and application to a rat pharmacokinetic study.

BACKGROUND: Incurred sample reanalysis (ISR) is an in-study validation parameter, which reinforces that the validated bioanalytical methods are reproducible. ISR of whole blood samples is complex when the test compounds can interconvert, ex vivo. Fingolimod and fingolimod phosphate are highly distributed in the blood cellular components and undergo rapid interconversion, both in vivo and ex vivo. An LC-MS/MS method capable of simultaneous quantification of fingolimod and fingolimod phosphate with the controlled sample preparation procedure is essential. RESULTS: The ex vivo analyte interconversion in blood was controlled by lysing the blood cells. CONCLUSION: Lysis of blood samples not only controlled the interconversion but also rendered homogeneity to the sample, which led to acceptable ISR results from the study.

Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the Km values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values.

Skin sample preparation by collagenase digestion for diclofenac quantification using LC-MS/MS after topical application.

BACKGROUND: Skin is the target site to evaluate the pharmacokinetic parameters of topical applications. Sample preparation is one of the influential steps in the bioanalysis of drugs in the skin. Evaluation of dermatopharmacokinetics at preclinical stage is challenging due to lack of proper sample preparation method. There is a need for an efficient sample preparation procedure for quantification of drugs in the skin using LC-MS/MS. RESULTS: The skin samples treated with collagenase followed by homogenization using a bead beater represents a best-fit method resulting in uniform homogenate for reproducible results. CONCLUSION: A new approach involving enzymatic treatment and mechanical homogenization techniques were evaluated for efficient sample preparation of skin samples in the bioanalysis.

Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays.

1. Chemical inhibition is the widely used method in reaction phenotyping assays for estimation of specific enzyme contribution to a given metabolic pathway. The results from phenotyping assays depend on the selectivity of chemical inhibitor and the concentration of inhibitor used in the incubation. 2. The higher protein concentrations used in the in vitro phenotyping assays will impact the inhibitory potency of chemical inhibitors. The objective of the study is to evaluate comprehensively the selectivity of chemical inhibitors and to guide in selecting appropriate concentration of the chemical inhibitors to be used in the phenotyping assays based on unbound fractions. 3. Selectivity of chemical inhibitors against nine major CYP450 isoforms was determined in liver microsomes using standard probe substrates. The unbound fractions of the selective inhibitors were determined in human liver microsomes using high-throughput equilibrium dialysis. Combining unbound inhibitor concentrations that are required to inhibit the CYP450 activities by 90% and unbound fractions of the chemical inhibitors in liver microsomes appropriate total concentrations of the inhibitors to be used in the phenotyping assays were reported. 4. The findings suggest that non-specific binding of the chemical inhibitors need to be taken into account while selecting concentrations for phenotyping assays.