Keith M. McErlane

High-performance liquid chromatography-mass spectrometry-mass spectrometry analysis of morphine and morphine metabolites and its application to a pharmacokinetic study in male Sprague–Dawley rats

A high-performance liquid chromatography tandem mass spectrometry-mass spectrometry (LC-MS-MS) assay was developed for the analyses of morphine, morphine glucuronides and normorphine in plasma samples from rats. The analytes were extracted by using C2 solid-phase extraction cartridges. The extraction recoveries were 100% for morphine, 84% for morphine-3-glucuronide, 64% for morphine-6-glucuronide and 88% for normorphine. Both intra- and inter-assay variabilities were below 11%. Using a plasma sample size of 100 microliters, the limits of detection were 13 nmol l-1 (3.8 ng ml-1) for morphine, 12 nmol l-1 (5.5 ng ml-1) for morphine-3-glucuronide, 26 nmol l-1 (12 ng ml-1) for morphine-6-glucuronide and 18 nmol l-1 (5.0 ng ml-1) for normorphine, at a signal-to-noise ratio of 3. The present assay was applied to a pharmacokinetic study in rats after intraperitoneal administration of morphine.

Comparison between capillary electrophoresis and high-performance liquid chromatography for the stereoselective analysis of carvedilol in serum

A high-performance liquid chromatographic (HPLC) assay using a chiral stationary phase was developed and validated for the determination of carvedilol enantiomers in human serum and was compared with a previously developed capillary electrophoresis (CE) method. The CE and the HPLC assay were compared by analyzing a series of serum samples containing racemic carvedilol in different concentrations using the two methods. The concentrations obtained by the two assays were not found to be significantly different indicating that CE and HPLC are comparable in terms of reproducibility and precision for the stereoselective analysis of carvedilol in human serum.

Systemic Administration of Monosodium Glutamate Elevates Intramuscular Glutamate Levels and Sensitizes Rat Masseter Muscle Afferent Fibers

Abstract There is evidence that elevated tissue concentrations of glutamate may contribute to pain and sensitivity in certain musculoskeletal pain conditions. In the present study, the food additive monosodium glutamate (MSG) was injected intravenously into rats to determine whether it could significantly elevate interstitial concentrations of glutamate in the masseter muscle and whether MSG administration could excite and/or sensitize slowly conducting masseter afferent fibers through N‐methyl‐d‐aspartate (NMDA) receptor activation. The interstitial concentration of glutamate after systemic injection of isotonic phosphate‐buffered saline (control) or MSG (10 and 50 mg/kg) was measured with a glutamate‐selective biosensor. The pre‐injection baseline interstitial concentration of glutamate in the rat masseter muscle was 24 ± 11 μM. Peak interstitial concentration after injection of 50 mg/kg MSG was 63 ± 18 μM and remained elevated above baseline for ∼18 min. In vivo single unit recording experiments were undertaken to assess the effect of MSG (50 mg/kg) on masseter afferent fibers. Injection of MSG evoked a brief discharge in one afferent fiber, and significantly decreased (∼25%) the average afferent mechanical threshold (n = 10) during the first 5 min after injection of MSG. Intravenous injection of ketamine (1 mg/kg), 5 min prior to MSG, prevented the MSG‐induced decreases in the mechanical threshold of masseter afferent fibers. The present results indicate that a 2‐ to 3‐fold elevation in interstitial glutamate levels in the masseter muscle is sufficient to excite and induce afferent mechanical sensitization through NMDA receptor activation. These findings suggest that modest elevations of interstitial glutamate concentration could alter musculoskeletal pain sensitivity in humans.

Hydromorphone metabolites: isolation and identification from pooled urine samples of a cancer patient.

1. Hydromorphone-3-glucuronide, dihydromorphine, dihydroisomorphine, dihydromorphine-3-glucuronide and dihydroisomorphine-3-glucuronide were isolated from a cancer patients urine and identified as metabolites of hydromorphone by comparison with synthetic standards using LC/MS/MS with gradient elution. 2. The relative urinary recovery of dihydroisomorphine-3-glucuronide was estimated to be 17-fold higher than previously reported. 3. Three new metabolites, including hydromorphone-3-sulphate, norhydromorphone and nordihydroisomorphine, were tentatively identified.

Development of a capillary electrophoresis assay for the determination of carvedilol enantiomers in serum using cyclodextrins

A capillary electrophoresis method using cyclodextrins as the chiral selectors was developed for the determination of carvedilol enantiomers in serum. Several types of cyclodextrins were evaluated. The effect of cyclodextrin concentration on enantiomer resolution was investigated. Best results were obtained using 10 mM hydroxypropyl-beta-cyclodextrin in the run buffer. The effect of voltage on efficiency was assessed. Other electrophoretic conditions were optimized. The method was validated for carvedilol enantiomers in serum. Linearity of detection was assessed over the concentration range of 50-4000 ng/ml of each enantiomer in serum. Intra- and inter-assay variability obtained were under 8% for both enantiomers.

Stereoselective analysis of the enantiomers of mexiletine by high-performance liquid chromatography using fluorescence detection and study of their stereoselective disposition in man

A sensitive, stereoselective high-performance liquid chromatographic assay was developed for the resolution of the enantiomers of mexiletine as their 2-naphthoyl derivatives on a Pirkle type 1A chiral phase column. Detection of the derivatives was accomplished with a fluorescent detector. Maximum recovery of the enantiomers from plasma was 83% and was observed when plasma proteins were precipitated with a mixture of barium hydroxide-zinc sulphate. The calibration curve in plasma was linear over the concentration range 5-750 ng/ml for each enantiomer (r2 = 0.999) and in urine the linear range was 0.25-7.5 micrograms/ml (r2 = 0.999) for each enantiomer. The minimum detectable quantity of each enantiomer in plasma was 5 ng/ml at a signal-to-noise ratio of 5:1, representing 100 pg injected. A preliminary pharmacokinetic study was undertaken in one healthy male volunteer following an oral dose of 300 mg of racemic mexiletine hydrochloride. The apparent elimination half-lives determined from the plasma data were 12.1 and 14.1 h for the R(-) and S(+) enantiomers, respectively. The cumulative urinary excretion amounts of R(-)- and S(+)-mexiletine were found to be 8.01 and 10.46 mg, respectively. The plasma data indicated that a cross-over of the enantiomer ratios occurred at approximately 8 h. The urinary excretion of the enantiomers was consistent with the pattern found in plasma.

The Pharmacokinetics of the Enantiomers of Mexiletine in Humans

1. This study examined the pharmacokinetics of the enantiomers of mexiletine in five healthy subjects who were each given a single, 300 mg, oral dose of racemic mexiletine hydrochloride. 2. The time course of the concentration ratio between the R(-) and the S(+) enantiomers (R/S) in plasma showed a progressive decrease, with a mean +/- S.D. ratio of 1.37 +/- 0.11 at 1 h and 0.64 +/- 0.11 at 48 h. Similarly, the R/S ratios in urine were 1.38 +/- 0.42 and 0.55 +/- 0.12 at 1 h and 72 h, respectively. 3. The terminal elimination half-life of S(+)mexiletine was 11.0 +/- 3.80 h, which was significantly greater (P less than 0.05) than that of the R(-) enantiomer, 9.10 +/- 2.90 h. S(+)Mexiletine also showed a significantly greater apparent volume of distribution (P less than 0.01) and renal clearance (P less than 0.05) than R(-)mexiletine. There was no significant difference in the apparent oral total drug clearance of the enantiomers. 4. The disposition of mexiletine enantiomers in man was stereoselective, and the differences observed between the enantiomers may be due largely to differences in their serum protein binding.

High-performance liquid chromatographic analysis of oxytetracycline in chinook salmon following administration of medicated feed

A high-performance liquid chromatographic assay was developed to detect oxytetracycline (OTC) in chinook salmon muscle tissue. A solid-phase extraction protocol was used to recover OTC and the internal standard, epitetracycline hydrochloride, from the salmon tissue samples. OTC was analyzed using a mobile phase of methanol-0.02 M phosphate buffer, pH 2.25 (60:190), an ultraviolet detection wavelength of 365 nm and 250 mm x 4.6 mm I.D. Ultrasphere ODS column. A linear calibration curve (r2 = 0.999) of OTC in salmon muscle tissue from 0.05 to 3.0 ppm was obtained. Using a signal-to-noise ratio of 5:1, the OTC detection limit was 0.5 ppm in salmon muscle tissue. OTC recovery (74.4%) and intra-assay variability (2.3%) were optimized for salmon muscle tissue. An in vivo feeding study was performed by administrating OTC-medicated feed for a period of 10 days, followed by a 42-day sampling period. The half-life for the elimination of OTC in chinook salmon muscle tissue was found to be 5.4 days.

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites. Hydromorphone was metabolized to norhydromorphone (apparent Km = 206 − 822 μM, Vmax = 104 − 834 pmol min−1 mg−1 protein) and dihydroisomorphine (apparent Km = 62 − 557 μM, Vmax = 17 − 122 pmol min−1 mg−1 protein) by human liver microsomes. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production. Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.

High-performance liquid chromatographic analysis of Romet-30® in salmon following administration of medicated feed

A sensitive and selective high-performance liquid chromatographic assay was developed for the simultaneous quantitation of sulphadimethoxine (SDM) and ormetoprim (OMP) in chinook salmon muscle tissue. SDM and OMP were extracted from tissue samples using a solid-phase extraction technique. Resolution of both drugs was accomplished using an Ultrasphere ion-pair column (250 x 4.6 mm I.D.) and a mobile phase of acetonitrile-methanol-0.1 M phosphate buffer, pH 4 (17:10:73) with ultraviolet detection at 280 nm. The calibration curve in salmon muscle tissue was linear over the concentration range 0.2-20 ppm for both SDM (r2 = 0.9974) and OMP (r2 = 0.9956). The minimum detectable quantity of SDM and OMP in salmon muscle tissue was 0.2 ppm at a signal-to-noise ratio of 5:1. An in vivo feeding experiment was undertaken where chinook salmon were administered Romet-30-medicated feed for a 10-day period, followed by a 42-day wash-out period. The rate of tissue uptake and decline of SDM and OMP was shown to differ.