Endogenous steroid profiling in hair and nails with a newly developed LC-MS/MS method

Abstract

Steroid hormones are key regulators in many physiological processes such as physical or mental stress and fatigue. The analysis of endogenous steroid hormones in hair is used for the long-term assessment of those biomarkers. If hair is not available, nails can be used as an alternative matrix for the retrospective evaluation of substances. The aim of this study was to develop and validate a sensitive LC-MS/MS method for the quantification of steroid hormones in human hair and nails. An LC-MS/MS method was developed for the simultaneous identification and quantification of 12 steroid hormones (aldosterone, cortisone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, dehydroepiandrosterone, 17α-hydroxyprogesterone, dihydrotestosterone, progesterone) in hair and nails. Analysis was conducted on a QTRAP ® 6500+ mass spectrometer (Sciex, Darmstadt, Germany) in electrospray ionization positive mode, after LC separation (Phenomenex ® Kinetex ® XB-C 18 ) using a Prominence UFLC system (Shimadzu, Kyoto, Japan). The method was validated using 13 C 3 -steroid analytes as surrogate analytes. Thirty-nine authentic hair and nail samples were analyzed to investigate a possible correlation between steroid levels in hair and nails. Furthermore, nail samples from 31 right and left hands from mothers ( n = 12) (before and after birth) and 8 samples from their newborn infants were analyzed. Finally, hair samples ( n = 18) were measured to see if there is an influence of pigmentation on the steroid level. Paired samples were compared using Wilcoxon signed rank test and correlation coefficients were calculated using Spearman correlation. Methods for both matrices were successfully validated. The mean concentrations determined in the hair samples ( n = 39) were 19.8 pg/mg for cortisone, 4.4 pg/mg for cortisol, 2.7 pg/mg for androstenedione, 0.7 pg/mg for testosterone and 3.0 pg/mg for progesterone. In nails, the mean values were 3.7 pg/mg for cortisone, 1.7 pg/mg for cortisol, 2.6 pg/mg for androstenedione, 1.1 pg/mg for testosterone and 6.2 pg/mg for progesterone. Testosterone and progesterone levels were significantly and positively correlated between hair and nails ( r = 0.49 and r = 0.38, respectively), whereas cortisol ( r = −0.03), cortisone ( r = −0.07) and androstenedione ( r = −0.05) showed no significant correlation. Higher concentrations of cortisol and cortisone in hair were found compared to nails ( P -value < 0.05). Investigations in nails of mothers and their newborn infants showed no significant difference in left and right hand concentrations of cortisol, cortisone, androstenedione, testosterone and progesterone ( P -value > 0.05). Regarding the hair pigmentation, there were no significant differences in pigmented and non-pigmented hair for cortisol, cortisone, androstenedione, testosterone and progesterone ( P -value > 0.05). The results showed that steroid profiling in hair and nails is a promising tool for various research and clinical applications. Higher steroid concentrations in hair might be attributed to affinity of cortisol and cortisone to bind to melanin. However, there were no significant differences in pigmented and non-pigmented hair, suggesting that the binding to melanin is not very strong and weaker interactions can play a role in the incorporation of steroid hormones into the hair matrix. Right and left hand values showed no significant differences, which indicated that the sample collection can be done on either hand on the same fingernail. We developed a highly sensitive and selective LC-MS/MS method for steroid profiling in hair and nails. With help of surrogate analytes, low endogenous amounts of steroid hormones were quantified. Nails and hair are both suitable matrices for steroid profiling with different baseline values.