Quantitative analysis of steroid hormones in human hair using a column-switching LC–APCI–MS/MS assay
Introduction
The measurement of steroid hormones in hair is increasingly recognized as an important tool for the assessment of integrated hormone secretion over prolonged periods of time. While initially envisaged as a method for the detection of exogenous steroids, e.g. androgens in doping-related research [1], [2]; over the past years, hair analyses for endogenous hormones, particularly glucocorticoids, have been widely applied in fields of psychiatric and stress-related research [3]. However, despite an increasing use of hair analyses in such research, the analytical procedures for the detection of steroid hormones in hair have received comparatively little attention.
As most previous researches have focused on cortisol in hair, the mostly commonly employed methods have been immunoassays with chemiluminescence detection (CLIA) or enzyme-linked immunosorbent assays (ELISA) which were originally designed for the measurement of salivary cortisol [4], [5]. However, given cross-reactivity of other steroid hormones with the antibodies used in these assays, the specificity of these methods is relatively low which may result in an overestimation of the actual steroid content in hair samples. Moving away from immunoassay procedures, Gao et al. [6] reported the use of high performance liquid chromatography with fluorescence detection (HPLC-FLU) for the measurement of cortisol in human hair. However, the protocol employed by these authors requires a relatively large amount of hair matrix and extensive, time consuming pretreatment procedures. In addition, the protocol involves derivatization which is possible for cortisol but not for other steroid hormones. Besides HPLC-FLU, gas chromatography mass spectrometry (GC–MS) has been used to analysis steroid hormone concentrations in human hair samples [7]. However, while GC–MS methods show good specificity, they are characterized by time-consuming work and derivatization steps, long throughput times and require relatively large sample volumes. Besides these methods, other research has reported the use of high performance liquid chromatography tandem mass spectrometry (LC–MS) for the detection of different corticosteroids in hair [1], [2], [8]. While the employed methodologies provide good specificity and sensitivity and are thus preferable to the above methods, the respective protocols all use selected ion monitoring (SIM) mode. The limited data obtained in SIM mode make it more difficult to identify potential contaminants in biological matrices which may interfere with the determination of analytes. Even though this issue is also given in full scan mode, here more detail is provided which allows a better identification of endogenous contaminants and analytes.
Recently, the utility of on-line solid phase extraction (SPE) has been improved as an extraction technique for this type of analysis [9], [10], [11], [12], [13]. The on-line SPE method seems to be the best choice for improving method sensitivity, shortening pretreatment and analysis times as well as increasing the number of the samples that can be analyzed simultaneously. To our knowledge, no on-line SPE LC–MS/MS method for the simultaneous identification and quantitation of different steroids hormones in human hair has been described. Here, we thus present a novel method achieving high specificity and sensitivity at decreased throughput times with a column-switching mechanism for on-line SPE LC–MS/MS analysis.
Section snippets
Chemicals and reagents
Cortisol, cortisone, testosterone, progesterone, corticosterone, dehydroepiandrosterone (DHEA) and androstenedione were purchased from Sigma–Aldrich (Hamburg, Germany). Deuterated internal standard samples (cortisol-d4, cortisone-d7, testosterone-d5, progesterone-d9, corticosterone-d8, DHEA-d4, androstenedione-d3) were obtained from Biocrates Life Sciences AG (Innsbruck, Austria). LC–MS grade methanol was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Acetic acid and ammonium
LC–MS/MS method development
Mass spectra obtained by quantitative optimization showed a protonated molecular ion [M+H]+ for all steroids except for DHEA for which this was [M−H2O+H]+. Formation of a single dominant precursor ion proved to be superior for enhancing sensitivity compared to the formation of multiple ions for the same analyte. Fragmentation of these precursor ions yielded some product ions, the strongest ones were chosen for quantitation respectively. All main working parameters of the mass spectrometer were
Conclusions
Here we present a novel on-line SPE LC–MS/MS method for the simultaneous identification and quantitation of seven steroid hormones in human hair samples. Due to the low concentrations of these steroid hormones in hair, a highly sensitive method allowing complete separation of these compounds is required. Our results indicate that the presented LC–MS/MS protocol is able to achieve these objectives, providing a highly sensitive, selective and reliable method for the quantification of these
Acknowledgments
Mr. W. Gao would like to thank the scholarship under the State Scholarship Fund of China Scholarship Council (File No. 2010609117) as well as the Innovation Research Foundation of Southeast University for doctoral students (BC0905), China.
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