Quantitative analysis of hydrocortisone in human urine using a high-performance liquid chromatographic-tandem mass spectrometric-atmospheric-pressure chemical ionization method.
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.
Nassar
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AE
Bioanalytical R&D Department, Forest Laboratories, Inc, Farmingdale, NY 11735, USA.
Varshney
N
N
Getek
T
T
Cheng
L
L
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006854:Hydrocortisone / Q000652:urine*; D013058:Mass Spectrometry / Q000379:methods*; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D006854:Hydrocortisone