Winnie L. Gee

Sensitive analysis of anti‐HIV drugs, efavirenz, lopinavir and ritonavir, in human hair by liquid chromatography coupled with tandem mass spectrometry

A highly sensitive and selective method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed and validated for the measurement of three antiretroviral agents, efavirenz, lopinavir and ritonavir, in human hair. Hair samples from adherent HIV-infected patients on antiretroviral therapies were cut into about 1 mm length segments and drugs were extracted by first shaking the samples with methanol in a 37 degrees C water bath overnight (>14 h), followed by methyl tert-butyl ether/ethyl acetate (1:1) extraction under weak alkaline conditions. The extracted lopinavir and ritonavir were separated by reversed-phase chromatography and detected by tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring (MRM), while efavirenz was monitored in negative ionization MRM mode. This method was validated from 0.01 to 4.0 ng/mg hair for ritonavir and 0.05-20 ng/mg hair for lopinavir and efavirenz by using 2 mg of a human hair sample. The interday and intraday assay precision (coefficients of variation, CV) for spiked quality control (QC) samples at low, medium and high concentrations were within 15% and accuracy ranged from 89% to 110%. Assay reproducibility was also demonstrated by analysis of incurred hair QC samples (CV <14%). No significant matrix ionization suppression was observed. This developed method allowed for the monitoring of these target medications in the hair samples of HIV-infected women on antiretroviral therapy in an observational study using small amounts of hair.

A simple LC–MS/MS method for determination of kynurenine and tryptophan concentrations in human plasma from HIV-infected patients

BACKGROUND Indoleamine 2,3-dioxygenase, catalyzing tryptophan (Trp) metabolism through the kynurenine (Kyn) metabolic pathway, plays important roles in immune suppression and the CNS. In this article, we report a simple, rapid and specific LC-MS/MS method for accurate determination of Kyn and Trp concentrations in human plasma from HIV-infected patients. RESULTS The human plasma sample (100 µl) was mixed with Kyn-d4 and Trp-d5 internal standards and then precipitated with trifluoroacetic acid. The supernatant was directly analyzed by LC-MS/MS. The assay using surrogate matrix calibrators was validated for precision, accuracy, matrix effect, extraction efficiency and stability. Some assay validation issues for endogenous substance bioanalysis using an LC-MS/MS method are discussed. CONCLUSION A simple, specific and reproducible LC-MS/MS method has been developed and validated for measuring Kyn and Trp in human plasma samples.

Effects of formulation and food on the absorption of hydrochlorothiazide and triamterene or amiloride from combination diuretic products.

The absorption of three combination formulations of hydrochlorothiazide and either triamterene or amiloride was studied over a 5-year period in seven separate investigations under varying conditions of food and fasting. The most widely prescribed combination, containing 25 mg of hydrochlorothiazide and 50 mg of triamterene, demonstrated impaired absorption in the fasting state that was partially corrected by the addition of a breakfast high in fat. The increase in the fat content of the food appeared to correlate directly with the amount of both drugs absorbed from this formulation. The second formulation studied, a new combination formulation of 50 mg of hydrochlorothiazide and 75 mg of triamterene, demonstrated acceptable absorption in the fasting state that was not altered by the concurrent administration of a high-fat breakfast. The absorption of the third formulation, a combination of 50 mg hydrochlorothiazide and 5 mg amiloride, was acceptable in the fasting state and demonstrated a slight reduction in the absorption of the amiloride component when administered concurrently with a high-fat meal. The clinical and biopharmaceutic implications of these observations are discussed.

High Performance Liquid Chromatographic Assay for Basic Amine Drugs in Plasma and Urine Using a Silica Gel Column and an Aqueous Mobile Phase. II. Chlorpheniramine

Abstract A high performance liquid chromatographic [HPLC] method that involves the use of a silica gel column and an aqueous mobile phase for quantitation of chlorpheniramine in plasma and urine is presented. Alkalinized samples are cleaned by extraction with pentane [containing 1% CH3CN], and the extraction is followed by evaporating the solvent and reconstituting the residue in a small amount of mobile phase. An aliquot of this solution is analyzed by an HPLC system with an Ultrasphere Si Column, an aqueous mobile phase at pH 7 containing 60% CH3CN and 7.5 mM [NH4]2HPO4, and UV detection at 200 nm. Although the average recovery of extraction is 58% ± SD 10%, the detection limit for the method is 0.7 ng/ml in plasma and 100 ng/ml in urine [s/n = 3] for 0.5 ml samples. The coefficients of variation [CV] on the results of samples run to measure interday and intraday precision and the bias on control samples were all 10% or less. We have used the method in a bioavailability study of a controlled release for...

Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to quantify S-(-) and R-(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid-liquid extraction for sample clean-up and employed 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C18 column using a mixture of acetonitrile-water-phosphoric acid-triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at lambda(ex)=280 nm and lambda(em)=325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.

Absence of a significant pharmacokinetic interaction between hydrochlorothiazide and triamterene when coadministered

Hydrochlorothiazide, triamterene, and hydroxytriamterene sulfate were monitored in the plasma and urine of 24 healthy young men taking single doses of a liquid preparation containing both hydrochlorothiazide and triameterene, liquid preparations containing either of these drugs alone, and a combination tablet recently formulated with a dose ratio of hydrochlorothiazide: triamterene (1∶1,5) found to give optimal potassium-sparing effect. In contradiction to a recent publication, no interaction between the drugs affecting the bioavailability or renal clearance of either could be demonstrated. The previous report of drug-drug interaction probably arose from formulationrelated problems with bioavailability from the two capsule and two tablet products which had been studied. A well-formulated hydrochlorothiazide-triamterene combination tablet promotes plasma concentrations and urinary excretion of hydrochlorothiazide, triamterene, and hydroxytriamterene sulfate which are virtually identical to those seen after either a combination liquid dosage form or simple liquid forms containing only one of the two drugs.

Regulated drug bioanalysis for human pharmacokinetic studies and therapeutic drug management

Regulated drug bioanalysis (i.e., determination of drug concentrations in biological matrices for regulated studies) usually refers to animal toxicokinetics, bioavailability/bioequivalence and clinical pharmacokinetic studies. However, there is another important regulated drug bioanalysis - therapeutic drug management (TDM). In the USA, TDM is regulated by Clinical Laboratory Improvement Amendments. In this article, we review and compare human pharmacokinetic sample analysis and TDM sample analysis. The US FDA/Bioanalytical Method Validation Guidance and the American Association for Clinical Chemistry/TDM Roundtable Recommended Generic Assay Validation Guidance are also compared. Some regulated drug bioanalysis issues, such as terminology, validation concepts and acceptance criteria, are discussed. Fostering interaction between bioanalysts from pharmaceutical science and clinical chemistry and reducing the regulatory gaps between different agencies for drug bioanalysis is our objective.

Ion-pair liquid chromatographic analysis of phenylpropanolamine in plasma and urine by post-column derivatization with O-phthalaldehyde

Abstract A high pressure Liquid chromatographic analysis of phenylpropano Lamine in plasma and urine by post-column derivatzaition with o-phthalaldehyde is described. Plasma samples are extracted with methylene chloride under alkaline conditions. Urine is diluted with mobile phase without extraction. Using fluorescence detection, the method is sufficiently sensitive (2 ng/ml in 0.5 ml of plasma and 0.5 mcg/ml in 0.2 ml of urine) so that phenylpropano lamine concentrations in plasma or urine may be measured for up to 24 hours following a 75 mg oral dose. Coefficients of variation for inter-day and intra-day precision are less than 10%.

Reduced cardiovascular effects of methamphetamine following treatment with selegiline

Selegiline is a specific MAO-B inhibitor. As MAO-B has been shown to be significantly involved in the metabolism of dopamine in certain regions of the primate brain, selegiline has been proposed for use in the treatment of drug addiction. Selegiline is also metabolized in vivo to l-methamphetamine. Therefore, when given in combination with psychostimulants such as d-methamphetamine, there is the potential for adverse effects. To study this possibility, squirrel monkeys were treated with chronic selegiline and tested with two doses of d-methamphetamine (0.1 and 1.0 mg/kg, i.v.). Following at least 7 days of treatment with once daily 0.3 mg/kg i.m. selegiline, the effects of methamphetamine on blood pressure and heart rate were no different than the effects of methamphetamine observed prior to selegiline treatment. However, following at least 10 days of treatment with 1.0 mg/kg i.m. selegiline, the effects of methamphetamine on blood pressure and heart rate were significantly reduced. Both methamphetamine and amphetamine were detected in plasma following chronic selegiline treatment. When monkeys were given an acute selegiline injection prior to methamphetamine, reduced cardiovascular effects were also seen. These results indicate that selegiline can be used safely even in combination with methamphetamine, as the cardiovascular effects of the drug combination were no greater than either drug alone, and were actually reduced at the higher selegiline dose.

High-Performance Liquid Chromatographic Assay for Meptazinol and Metabolite in Plasma and Urine Using a Silica Gel Column and an Aqueous Mobile Phase

Abstract An HPLC method for quantitating meptazinol, a synthetic narcotic analgesic, and its glucuronide in plasma and urine is presented. the method involves extraction from plasma with methylene chloride under alkaline conditions (pH = 9.7). Urine samples are injected without further preparation following addition of internal standard. the conjugate metabolite of meptazinol was hydrolyzed with β-glucuronidase. Samples are analyzed using an Ultrasphere Si column with an aqueous mobile phase of 65% acetonitrile and 5 mM dibasic ammonium phosphate at pH = 7.0. the flow rate is 1.0 ml/min. Fluorescent detector settings were 275 nm and 315 nm for excitation and emission wavelengths, respectively. Detection limits were 2 and 5 ng/ml in plasma and 50 and 70 ng/ml in urine for meptazinol and the hydrolyzed glucuronide, respectively. the coefficients of variation for interday and intraday precision were less than 10%. This method has been used in pharmacokinetic studies of i.v. and i.m. formulations involving 13...