GC/MS Method for the Determination of Flumazenil in Plasma and Urine

Flumazenil (FMZ, Flumazepil, Anexate, RO 15-1788) is an imidiazobenzodiazepine used in the reversal of benzodiazepine sedation and intoxication. Dosages of up to 1 mg reverse the coma and respiratory and CNS depression produced by benzodiazepines. Therapeutic dosages of FMZ produce plasma concentrations of up to 500 ng/mL. An analytical method was developed to determine FMZ concentrations in plasma and urine. Plasma and urine samples were spiked with FMZ from 5 to 500 ng/mL and flunitrazepam (FN, internal standard, 500 ng/ml). Specimens (3 ml) were extracted using 9 ml n-butyl chloride and 3 ml ammonium chloride buffer (pH 9.2). Extracts were dried under N2 in silanized glass, reconstituted in 25 μl methanol and 5 μl injected. GC/MS methodology utilized 15 m x 0.25 mm (ID) DB-5 (1.0 μm film) fused silica capillary column, with He carrier (29 cm/sec), splitless injection at 270°C, column 200-270°C @ 10°/min, separator oven 270°C, electron ionization (70 eV) with selective ion monitoring for FMZ (303 m/z) and FN (312 m/z). FMZ eluted at 7.37 min and FN at 8.19 min (relative index 0.8998). Correlation coefficients of concentration to peak area ratios were r = 0.9979 for plasma and r = 0.9943 for urine. Coefficients of variation were: plasma, 25 ng/ml, 13.2% (n = 5, x = 23 ± 1.3); plasma, 250 ng/ml, 6.6% (n = 6, x = 274 ± 7.4); urine, 25 ng/ml, 12.1% (n = 5, x = 23 ± 1.2); urine, 250 ng/ml, 10.7% (n = 5, x = 308 ± 14.7). This GC/MS method can determine FMZ concentrations in plasma and urine associated with therapeutic doses of FMZ