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.
Quantitation and confirmation of the diazolo- and triazolobenzodiazepines in human urine by gas chromatography/mass spectrometry.
We report a reliable, rapid, sensitive, and quantitative method for the N- and O-trimethylsilyl (TMS) derivatives of the diazolo- and triazolobenzodiazepines after enzymatic incubation (2 h) and liquid-liquid extraction of 0.5-mL urine volumes. Each analyte was identified by gas chromatography/mass spectrometry through the retention time for the total ion current (TIC) and selected ion monitoring (SIM) of four ion currents. Quantitation of the diazolobenzodiazepines was obtained by the ratio of the base peak ion of the TMS analyte to that of the internal standard bromazepam in the concentration range 50-500 ng/mL (1-10 ng injected into the GC/MS). The limit of detection (LOD) at p less than 0.01 was 50 ng/mL for all the diazolobenzodiazepines. The assay was not quite as sensitive for triazolobenzodiazepines (5-20 ng injected in the GC/MS). The extraction efficiency of the assay ranged from 75 to 92% for all the analytes. The coefficient of variation (CV) for the diazolobenzodiazepines ranged from 5.4 to 9.4% for within-day runs and from 11.1 to 13.9% for between-day runs. For the triazolobenzodiazepines the values were 3.8 to 18.9% for a single day and 3.4 to 19.9% between days. The selected ion current ratio for each analyte was determined for a single day and over a one-week period. There was no statistical difference in the ratios during this time. The confirmation of diazolobenzodiazepines in urine by this method was relatively easy after screening by the immunoassay technique. Identification of triazolobenzodiazepines appeared to be more difficult by both the screening technique and the GC/EI/MS analysis.