In this study, the development and validation of a method of analysis for 11,17,21,-trihydroxypregn-4-ene-3,20-dione (hydrocortisone, cortisol, HC) using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS) with atmospheric-pressure chemical ionization (APCI) is reported. This is the first report of the systematic development and validation of an HPLC-MS-MS method for the quantitation of HC in synthetic human urine with a deuterated internal standard. Prior to LC-MS-MS analysis, the only sample preparation used was the dilute-and-shoot technique prior to LC-MS-MS analysis. In this study, an analysis time of less than 3 min is achieved. The results show freedom of interference from other analytes such as analogous steroids. Validation parameters such as specificity/selectivity, limit of quantitation (LOQ), linearity, precision, accuracy, ruggedness, stability, and system suitability are evaluated for this method. The LOQ is 5 ng/mL with an 8% relative standard deviation (RSD). For calibration standard curves, an average linear response for a 3-day validation (R2 = 0.997) over the range of 5 to 500 ng/mL is obtained. The interday precision %RSDs are 7.2, 5.0, and 5.2 for 15, 75, and 300 ng/mL, respectively. Also, brief comparisons of the dilute-and-shoot and liquid-liquid extraction techniques for this analyte are discussed.
Rifamycins are a class of antibiotic compounds of which rifampicin is the most commonly prescribed. Conventional electron-impact mass spectrometry of rifampicin has not been found to provide useful data. Thermospray and electrospray mass spectrometry are studied as potential tools for the analysis of rifampicin, rifamycin SV, and rifamycin B. Using thermospray and electrospray ionization, all three compounds provide significant ion intensity for either the [MH]+ or [MNa]+ ions. In addition, combined high-performance liquid chromatography-thermospray mass spectrometry provides useful analytical data for a mixture of the three rifamycins.
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