Determination of L-ephedrine, pseudoephedrine, and caffeine in rat plasma by liquid chromatography-tandem mass spectrometry.

A rapid and simple liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of L-ephedrine, pseudoephedrine, and caffeine in male Fisher-344 rat plasma at nanogram-per-milliliter concentrations for use in support of toxicology studies. Only 25 µL of plasma is required, and extraction is performed using a simple, single-step protein precipitation. The method was validated over a range of 2.09 to 5460 ng/mL for L-ephedrine, 2.09 to 5050 ng/mL for pseudoephedrine and 2.03 to 5340 ng/mL for caffeine. A binary gradient elution at 0.3 mL/min was used with a Waters XBridge Phenyl (2.1 × 150 mm, 3.5 µm) column and a Waters XBridge Phenyl 2.1- × 10-mm guard column at ambient temperature. The mobile phase consisted of 10 mM ammonium acetate in water (pH 5.0) and methanol. Caffeine trimethyl-(13)C(3) was used as the internal standard. The method was evaluated for linearity, recovery, precision, accuracy, and stability, and it was successfully applied in toxicokinetic studies of ephedrine, administered alone, in combination with caffeine, and in the herbal source Ma Huang.

Determination of lovastatin hydroxy acid in female B6C3F1 mouse serum.

A liquid chromatographic-mass spectrometricmethod for the determination of lovastatin hydroxy acid in female B6C3F(1) mouse serum was developed for use in supporting toxicokinetic studies of animals dosed with the cholesterol lowering agent lovastatin. The method does not require an extensive sample cleanup and shows good correlation between serum matrix standards and solvent standards. The method was validated and used to analyze serum samples from a preliminary dose level range-finding study. The method was validated for a concentration range of approximatel 1.0 to 100 ng/mL in serum, and linearity was verified to ~2000 ng/mL. The stability of sample extracts was determined under various storage conditions and the stability of serum samples stored frozen was determined over a period of seven weeks. During the course of analyzing the animal samples, the serum was monitored for the presence of lovastatin not hydrolyzed to the hydroxy acid, but no attempt was made to quantify lovastatin. No unhydrolyzed lovastatin was noted in any of the serum samples from animals dosed with lovastatin.

Determination of methylene blue and leucomethylene blue in male and female Fischer 344 rat urine and B6C3F1 mouse urine.

A liquid chromatographic method for the determination of methylene blue and leucomethylene blue in male and female Fischer 344 rat urine and male and female B6C3F1 mouse urine was developed for use in supporting toxicokinetic studies, validated, and used to analyze urine samples for a preliminary dose level range-finding study. The method was validated for a concentration range of 10.0 to 20,000 ng/mL in urine. Samples up to 75,000 ng/mL demonstrated good recoveries when diluted into the range of the calibration curve. Six sets of calibration standards were prepared in F344 male rat urine for analysis to demonstrate reproducibility and ruggedness. The stability of sample extracts was determined under various storage conditions. During the course of analyses of the animal samples, a possible metabolic process was observed. Although Azure B is a significant impurity in methylene blue trihydrate, the amount of Azure B seen in urine samples collected from rodents dosed with methylene blue trihydrate is significantly greater than the amount seen in rodent urine spiked directly with methylene blue. This observation suggests possible N-demethylation of the methylene blue as a metabolic transformation to Azure B.