Gas chromatographic determination of midazolam in low-volume plasma samples.
A gas chromatographic method for the sensitive determination of midazolam in plasma volumes as low as 40 microliters was developed, utilizing clinazolam as the internal standard. After liquid-liquid extraction at basic pH into 1-chlorobutane-dichloromethane (96:4) a 2- to 4-microliters portion of the reconstituted extract was injected under electronic pressure control onto a 12 m x 0.2 mm I.D. methyl silicone capillary column, and was exposed to a three-step temperature program from 120 to 310 degrees C, to separate the analytes from the plasma constituents. The compound of interest was identified and quantified by means of a mass-selective detector. The assay was linear from 10 to 500 ng/ml using 40 microliters of plasma (limit of quantification: 10 ng/ml) and was linear from 0.25 to 100 ng/ml using 500 microliters of plasma (limit of quantification: 0.25 ng/ml). The intra-day precision for the 40-microliters aliquots varied from 2.2 to 6.6%, the corresponding accuracy from -7.4 to -4.4%; the inter-day precision ranged from 5 to 7.2% and the corresponding accuracy from -7.2 to -5.1%.
Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma.
An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.
Sterol metabolism. XLI. Cholesterol A-ring autoxidations
Abstract Chromatographic and spectral evidence is adduced for the presence of cholest-5-en-3-one, cholest-4-en-3-one, and cholest-4-ene-3,6-dione in samples of cholesterol aged naturally in air or subjected to irradiation in air by 60 Co gamma radiation. These findings establish an additional mode of air oxidation of cholesterol to A-ring 3-ketones. Moreover, the oxidation by air of cholest-5-en-3-one induced by 60 Co gamma radiation yielded cholest-4-en-3-one, cholest-4-ene-3,6-dione, and the epimeric 3-oxocholest-4-ene-6-hydroperoxides. Cholest-4-en-3-one was not altered by irradiation in air, nor was cholesterol isomerized to cholest-4-en-3β-ol upon irradiation. From these observations it is deduced that the radiation-induced A-ring dehydrogenation of cholesterol yields initially cholest-5-en-3-one which upon isomerization yields cholest-4-en-3-one not further oxidized and which by a second oxidation yields the epimeric 3-oxocholest-4-ene-6-hydroperoxides which decompose to cholest-4-ene-3,6-dione.