Quantitative Analysis of Hydrocortisone in Human Urine Using a High-Performance Liquid Chromatographic-Tandem Mass Spectrometric-Atmospheric-Pressure Chemical Ionization Method

Abstract

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.

Quantitation of lanosterol and its major metabolite FF-MAS in an inhibition assay of CYP51 by azoles with atmospheric pressure photoionization based LC-MS/MS

“…In the case of steroids the fragment pattern is very similar to EI or to particle beam spectra of sterols as published by Byskov et al [21]. CID spectra similar to ours have previously been published for 6␤-OH-hydrocortisone and testosterone [22,23]. The fragments of the sterols presented in Figure 1-3 are calculated as described for particle beam spectra of FF-MAS and lanosterol where a side chain fragmentation was observed [21].…”

New Spectrofluorimetric Method for Determining Serotonin: Application to Human Urine

“…However, in the case of tumors producing only small amounts of serotonin, the predictive values of an increased urinary 5-HIAA is low (Meijer, Kema, Volmer, Willemse, & De Vries, 2000). In addition, the concentration of 5-HIAA in urine may be incresead by consumption of foods rich in 5-HT (Nassar, Varshney, Getek, & Cheng, 2001;Patel, Arundell, Parker, & Yeoman, 2005). It has been shown that platelet serotonin is the most sensitive indole marker for the diagnostic of caracinoid tumors, especially those with low serotonin production (Meijer & al., 2000;Kema et al, 2001;Kema et al, 1992).…”

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

“…Chromatographic methods accurately measure UF, not only reducing the interference for the F quantification, but also allow quantification of the E [13][14][15]. Moreover, liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS) [15][16][17] have the disadvantage of being expensive and are not available in all laboratories. Liquid chromatography with ultraviolet detection (LC-UV) coupled with solid phase extraction (SPE), to eliminate interfering compounds, is a practical and cheaper technique [18,19].…”