RAPID HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY APCI METHOD FOR DETERMINATION OF NITROGEN CONTAINING COMPOUNDS IN PLASMA

We report the development and validation of a method of analysis for nitrogen containing compounds using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). This LC/MS-MS procedure has been developed for quantitation of the analyte in rat plasma. The analyte and internal standard were isolated from the plasma matrix using deproteination with perchloric acid as the sole sample preparation involved prior to LC-MS/MS analysis. Analysis time of less than 2 minutes was achieved. The derivatization steps required for typical gas-chromatography/mass spectrometry (GC/MS) analyses are eliminated. The validation parameters such as specificity (selectivity), limit of quantitation, linearity, precision, accuracy, ruggedness, stability and system suitability were evaluated for this method. The results showed freedom from interference from other analytes. Retention time precision was less than 2% RSD for analyte and internal standard. The limit of quantitation (LOQ) was 2 µg/L with 9% RSD. For calibration standard curves, an average linear response for three days validation (R2 = 0.998) over the range 2 to 2000 µg/L was obtained. The interday precision % RSDs were 7.5, 4.6 and 4.1 for 6, 60 and 600 µg/L, respectively. Analysis accuracy was assessed by spiking actual rat plasma samples with analyte. The spike levels were 3 and 300 µg/L, with recoveries of 87% and 88% respectively.