Methane negative chemical ionization analysis of 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones.

The methane negative chemical ionization (NCI) mass spectra of the medically important 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones generally consisted solely of M- and (M-H)- ions. Attempts to find the location of the H lost in the generation of the (M-H)- ion were unsuccessful, although many possibilities were eliminated. A Hammett correlation analysis of the relative sensitivities of a series of 7-substituted benzodiazepines suggested that the initial ionization takes place at the 4,5-imine bond. For certain benzodiazepines, the (M-H)- ion generated by methane NCI was 20 times more intense than the MH+ ion generated by methane positive chemical ionization (PCI). By using NCI, a sensitive and simple GC-MS assay for nordiazepam was developed that can quantitate this important metabolite of many of the clinically used benzodiazepines in the blood and brain of rats.

Determination of midazolam and two metabolites of midazolam in human plasma by gas chromatography--negative chemical-ionization mass spectrometry.

A method is described for measuring midazolam, a new anesthesia induction agent and hypnotic, and its hydroxymethyl and desmethyl metabolites in human plasma. Deuterated analogues of each compound are added to plasma as internal standards. The compounds are extracted from plasma with benzene containing 20% 1,2-dichloroethane and after removal of the extracting solvent are dissolved in a solution of bis-(trimethylsilyl)acetamide and acetonitrile. An aliquot of this solution is analyzed by gas chromatography--mass spectrometry with the mass spectrometer set to monitor in the gas chromatographic effluent the M.- ions of drug, metabolites and internal standards generated by methane electron-capture negative chemical ionization. For all three compounds, the limit of quantitation is 1 ng ml-1, and the precision (relative standard deviation) at a concentration of 5 ng ml-1 is less than 6%. Measurable amounts of the hydroxymethyl, but not the desmethyl, metabolite of midazolam could be found in the plasma of humans given either an intravenous or an oral dose of midazolam maleate.

Determination of imidazobenzodiazepine-3-carboxamide, a new anxiolytic agent, in human plasma by gas chromatography--negative chemical-ionization mass spectrometry.

A method is described for measuring imidazobenzodiazepine-3-carboxamide, a new anxiolytic agent, in human plasma. A tetradeuterated analogue of the analyte is used as the internal standard. The drug and its internal standard are (1) extracted from plasma at pH 9 with benzene containing 20% 1,2-dichloroethane, (2) derivatized with pentafluoropropionic anhydride in the presence of triethylamine and (3) the nitrile derivative of the analyte and internal standard are analyzed by gas chromatography (GC)-negative chemical-ionization mass spectrometry (CIMS) using methane as both GC carrier gas and CI reagent gas. The mass spectrometer is set to monitor the intense (M-HCl)- ions of imidazobenzodiazepine-3-nitrile and its tetradeuterated analogue at m/z 316 and m/z 320, respectively. Quantitation of an experimental plasma sample is based on the comparison of the m/z 316 to m/z 320 ion ratio in each sample to that obtained from the analyses of control plasma spiked with various amounts of the drug and a fixed amount of internal standard. The limit of quantitation of the method is approximately 100 pg ml-1 of plasma and the precision (relative standard deviation) at a plasma concentration of 1 ng ml-1 is 4%.

Determination of (-)-threo-chlorocitric acid in human plasma by gas chromatography-positive chemical-ionization mass spectrometry.

A method is described for measuring (-)-threo-chlorocitric acid in human plasma. Plasma is acidified to pH 1 to minimize lactonization and a 13C analogue of (-)-threo-chlorocitric acid is added as internal standard. The acidified plasma is then extracted with ethyl acetate containing 10% methanol. The ethyl acetate-methanol extract is back-extracted with acetate buffer (pH 5). This extract, following adjustment to pH 1, is reextracted with ethyl acetate. The residue after removal of the ethyl acetate is treated with ethereal diazomethane. The wet residue is reconstituted in ethyl acetate and a portion of this solution is analyzed by gas chromatography-chemical ionization mass spectrometry. The mass spectrometer is set to monitor m/z 269 [MH+ of trimethylated (-)-threo-chlorocitric acid] and m/z 270 [MH+ of trimethylated (-)-threo-[13C]chlorocitric acid] in the gas chromatographic effluent. The m/z 269 to m/z 270 ion ratio in a sample containing an unknown amount of (-)-threo-chlorocitric acid is converted to an amount of compound using a calibration curve. The calibration curve is generated by analyzing control plasma spiked with various known amounts of (-)-threo-chlorocitric acid and a fixed amount of (-)-threo-[13C]chlorocitric acid. The limit of quantitation is 0.1-0.6 micrograms ml-1, depending on the characteristics of the calibration curve generated with each set of samples. The precision (relative standard deviation) at a concentration of 2 micrograms ml-1 is 3.3%.

Quantitation of the enantiomers of rimantadine in human plasma and urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric procedure has been developed for the quantitation in plasma and urine of the enantiomers of rimantadine, an antiviral drug effective against type A influenza. The assay utilizes derivatization with an optically active reagent, selective ion monitoring, methane negative-ion chemical ionization (NICI) mass spectrometry and stable isotope dilution. The method has been used to measure concentrations of each rimantadine enantiomer over a range of 2.5-250 and 12.5-1250 ng/ml in the plasma and urine, respectively, of four male volunteers administered rimantadine. In plasma and urine, no differences were observed in the disposition of the unconjugated enantiomers. In urine, one enantiomer, but not both, was released following enzymatic hydrolysis.