Monitoring of the deuterated and nondeuterated forms of levodopa and five metabolites in plasma and urine by LC-MS/MS.

To compare pharmacokinetics, metabolism and excretion of levodopa and a triply deuterated form, which is being developed as an improved treatment for Parkinson's disease, methods were needed for quantification of the deuterated and nondeuterated forms of levodopa and five metabolites in human plasma and urine. Results: The natural heavy isotopes in the nondeuterated compounds caused an absolute contribution of up to 100% in the response of the deuterated compounds. Similarly, heavy isotopes in the deuterated analytes contributed to the response of the internal standards, but this did not affect the reliability of the results. Conclusion: Deuterated and nondeuterated analytes can be quantified together by LC-MS/MS, but overestimation of the concentrations of the deuterated molecules may be unavoidable and a careful interpretation of the concentration data is essential.

Validation of an LC-MS/MS method for simultaneous quantification of venlafaxine and its five metabolites in rat plasma and its application in a pharmacokinetic study.

A sensitive, selective, and reliable LC-MS/MS method was developed and validated for simultaneous quantification of venlafaxine (VEN) and its 5 metabolites (ODV, NDV, NNDDV, OHV and NODDV) in rat plasma. The calibration ranges are 15.0 to 6000 ng/mL for VEN, 1.00 to 400 ng/mL for ODV, 5.00 to 2000 ng/mL for NDV, 1.00 to 400 ng/mL for NNDDV, 10.0 to 4000 ng/mL for OHV, and 0.200 to 20.0 ng/mL for NODDV. Briefly, 50 µL of rat plasma was extracted using liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE). The analytes were separated on an Agilent SB-Phenyl (50 mm × 4.6 mm, 3.5 µm) column using a binary gradient of 0.1% formic acid in water versus 0.1% formic acid in acetonitrile at a flow rate of 0.8 mL/min. The method was validated following FDA guidance for bioanalytical method validation. Validated method was successfully applied to a pharmacokinetic study of VEN orally administered to rats.

Sensitivity improvement of the LC-MS/MS quantification of carbidopa in human plasma and urine by derivatization with 2,4-pentanedione.

The reliable quantification of carbidopa in biological samples at low concentrations is challenging because of the polar and highly unstable nature of the compound. In this paper, LC-MS/MS methods are described for the determination of carbidopa in 50µL of human plasma and 25µL of human urine in the concentration ranges 1-1,000ng/mL and 100-50,000ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40°C. The product is a relatively non-polar molecule that is suitable for reversed-phase liquid chromatography (3.5min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalization. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5mg/mL sodium metabisulfite and 1mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5mg oral dose.

Simultaneous quantification of loxapine, loxapine N-oxide, amoxapine, 8-hydroxyloxapine and 7-hydroxyloxapine in human plasma using LC-MS/MS.

Loxapine is an antipsychotic medication used for the treatment of schizophrenia. In vivo, loxapine is metabolized to multiple metabolites. A high performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of loxapine and 4 of its metabolites, loxapine N-oxide, amoxapine (N-desmethyl loxapine), 8-hydroxyloxapine and 7-hydroxyloxapine, in human plasma to support regulated clinical development. During method development, several technical challenges such as poor chromatography, separation of structural isomers, and inadequate sensitivity were met and overcome. The final method utilized micro-elution solid phase extraction (SPE) to extract plasma samples (100µL), and the resulting extracts were analyzed using reversed phase LC-MS/MS using a turbo-ionspray interface in positive ionization mode with selected reaction monitoring (SRM). The method was fully validated according to the current regulatory guidance for bioanalysis over the calibration curve range 0.0500-50.0ng/mL for all analytes using 1/x2-weighted linear regression analysis. Based on three separate runs, the between-run precision and inter-day precision for all five analytes at all concentrations, including the LLOQ (lower limit of quantitation) quality control at 0.0500ng/mL, varied from 0.0% to 13.8%, while the accuracy ranged from 86.4% to 109.3% of nominal. The extraction recoveries of loxapine and the four metabolites were above 80%. Various forms of short-term and long-term stability were established in both solutions and matrix, including the stability of loxapine and the four metabolites in human plasma for up to 260days of storage at -20°C. This method has been used to support a regulated clinical study, which included the successful execution of incurred sample reanalysis (ISR) testing. To the best of our knowledge, this is the first published methodology in which these five analytes were quantified with a single extraction and injection.

Determination of modafinil, modafinil acid and modafinil sulfone in human plasma utilizing liquid-liquid extraction and high-performance liquid chromatography.

An assay was developed to determine concentrations of modafinil (dl-2-[(diphenylmethyl)sulfinyl]acetamide; Provigil) and its two major circulating metabolites, modafinil acid and modafinil sulfone, in human plasma. The assay utilized liquid-liquid extraction of the analytes and an internal standard, (phenylthio)acetic acid, from plasma into a mixture of hexane-dichloromethane-glacial acetic acid (55:45:2, v/v). The analytes were resolved isocratically on a narrow-bore phenyl column at a mobile phase flow-rate of 0.3 ml/min and were monitored by UV detection at 235 nm. The method reported herein reduces the required sample volume of previously reported methods from 1.00 to 0.200 ml of plasma while lowering the limit of quantification (LOQ). The linear range of the assay was from 0.100 to 20.0 microg/ml for each of the three compounds.