Application of two-dimensional high-performance liquid chromatography--mass spectrometry with particle beam interface.

The use of a particle beam interface (PBI) in LC-MS has faded because of its poor resolution and low sensitivity for many compounds. It also does not perform well in reversed-phase systems with a high percentage of water. This manuscript describes two-dimensional LC to overcome the problems of PBI, both in the identification of unknown samples and in the quantitative determination of metabolites of vitamin D in human plasma. For the identification of unknown components, the reversed-phase solvent in the first dimension was transferred to an isocratic normal solvent system in second dimension by column switching. Moreover, only the peaks of interest (components) in the first dimension were transferred into the second LC column for mass analysis. For the quantitative determination of the metabolite of vitamin D, the peak width of analyte in the second dimension was greatly narrowed, and interference was excluded such that high sensitivity and resolution resulted. The limit of quantification for the test metabolite of vitamin D in human plasma can reach 0.050 ng/ml with injection volume of 50 microl.

Quantitative determination of clonazepam in plasma by gas chromatography-negative ion chemical ionization mass spectrometry.

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the quantitative analysis of clonazepam in human plasma is described. Clonazepam (M(r) = 315) was derivatized by N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. A pre-conditioning procedure involving injection of a silyl-8 and ethyl acetate extraction solution from plasma reduces the interaction between clonazepam-TMS and the analytical system. The routine limit of quantification was set to be 0.25 ng/ml with an injection volume of 2 microliters and a sample volume of 1 ml. The signal-to-noise ratio was greater than five. The detection limit for clonazepam can reach 0.1 ng/ml. The isotope clonazepam-d5 was used as the internal standard.

Determination of flumazenil in plasma by gas chromatography-negative ion chemical ionization mass spectrometry.

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the determination of flumazenil in plasma is described. The GC of flumazenil (M(r) 303) is considered to be difficult as it is readily adsorbed in the GC column. Therefore, preconditioning the GC column with reconstituted extract from plasma and Silyl-8 was required to cover the active sites on the column. Monitoring the maximum mass peak (m/z 275) of the flumazenil resulted in a tenfold enhancement of sensitivity and signal-to-noise ratio (concentration = 1 ng/ml). Isotopically labeled flumazenil-d3 (M(r) 306, m/z 278) was used as the internal standard. The detection limit for flumazenil was found to be 0.1 ng/ml with an injection volume of 2 microliters. The signal-to-noise ratio was about 10. The routine quantification limit was set at 2 ng/ml for dog plasma and 1 ng/ml for human plasma. The sample volumes in both instances were 1 ml.

Determination of chlordiazepoxide in mouse plasma by gas chromatography-negative-ion chemical ionization mass spectrometry.

A gas chromatographic-negative-ion chemical ionization mass spectrometric (GC-NCIMS) method for the determination of chlordiazepoxide (Librium) in mouse plasma is described. Chlordiazepoxide was not suitable for GC analysis because of its thermal instability. The specific derivatization technique described permits the analysis using GC-MS with high sensitivity and selectivity. The use of halazepam as an internal standard instead of an isotopic internal standard decreases the cost and time of the analysis. The detection limit for chlordiazepoxide was 0.1 ng/ml with an injection volume of 1 microliter at a signal-to-noise ratio > 5. The limit of quantification was 5 ng/ml.

Gas chromatographic-mass spectrometric method for the determination of flurazepam and its major metabolites in mouse and rat plasma.

A capillary gas chromatographic-negative chemical ionization (NCI) mass spectrometric method for the determination of flurazepam and its metabolites N-1-hydroxyethyl-flurazepam and N-1-desalkyl-flurazepam in mouse and rat plasma was described. Derivatization of the metabolites of flurazepam with BSTFA allowed a highly stable, accurate, and sensitive GC-MS analysis. The use of a single internal standard (halazepam) for the quantification of all compounds saved cost and time. The detection limits were 0.1 ng/ml for N-hydroxyethyl-flurazepam-TMS (M(r) = 404), 0.5 ng/ml for desalkyl-flurazepam-TMS (M(r) = 360), and 0.5 ng/ml for flurazepam (M(r) = 387) with an injection volume of 1 microliter at a signal-to-noise ratio greater than 5. The quantitation limit was set to 10 ng/ml for all compounds.

Analysis of doxylamine in plasma by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the quantitative determination of doxylamine in plasma is described. The drug levels of doxylamine in plasma were monitored after the oral administration of a single 25-mg tablet of doxylamine succinate to each of 20 male volunteers. The compound was extracted from the plasma samples, concentrated under a nitrogen stream, and analyzed by HPLC using normal-phase chromatography with detection at 254 nm. The detection limit is approximately 5 ng/ml.