Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
39, Number 2, February 2001, pp. 59-64
Quantitative
Analysis of Hydrocortisone in Human Urine Using a High-Performance Liquid Chromatographic–Tandem
Mass Spectrometric–Atmospheric-Pressure Chemical Ionization Method
A.-E.F.
Nassar, N. Varshney, T. Getek, and L. Cheng
Bioanalytical R&D Department, Forest Laboratories, Inc., 220 Sea Lane, Farmingdale,
NY 11735
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.
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