Endocannabinoid and steroid analysis in infant and adult nails by LC–MS/MS

Abstract

A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC–MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5–20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC–MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults. Graphical abstract

Peripubertal Testosterone, 17β-Estradiol and Progesterone Concentrations in Hair and Nails in Dobermann Dogs

“…It is not surprising that the highest P4 concentrations were observed at the second postpubertal sampling time, as both biological specimens had been exposed to accumulating circulating hormones for a longer duration. Compared to P4 concentrations in claws from calves and pregnant cows [ 25 ], P4 concentrations in nails in our study were very similar to the ~20 pg/mg reported for calves and fell within the wide range of 14.32–103.66 pg/mg reported for pregnant cows, but were more than twofold higher than the range of 0.7–12.8 pg/mg reported in women using a different laboratory technique [ 39 ]. Because the laboratory technique employed by Comin et al [ 25 ] was similar to ours, it can be assumed that, although species-related differences must be considered, sexual steroid concentrations in nails/claws are strongly influenced by the method of analysis.…”

“…Nevertheless, the similar profiles of T concentrations in both hair and nails suggest that both biological specimens can serve as alternatives to assess changes in T concentration in dogs. The concentrations of E2 and P4 in males did not exhibit significant changes over the study period in either biological specimen, with similar profiles and low concentrations, albeit about tenfold higher than the P4 concentrations reported in nails in men (1.0–2.1 pg/mg) [ 39 ].…”

“…Focusing on peripubertal hormone changes in male dogs, our results showed a significant approximately twofold increase in testosterone (T) accumulation in both biological specimens immediately before puberty, which remained high until the end of the study, indicating that T secretion by the testes precedes the onset of puberty. The average >2.6 pg/mg concentrations of T in the hair were higher than the range of 0.3–2.7 pg/mg reported in men’s hair [ 27 ], and the average T concentrations in nails were threefold higher than the 0.4–0.8 pg/mg reported in human nails [ 39 ]. It should be noted, however, that different laboratory techniques were employed in those studies compared to our study, potentially accounting for the divergent concentrations found.…”

Tacrolimus monitoring in hair samples of kidney transplant recipients

“…Drugs and metabolites can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) from biological matrices, including blood ( 15 ), hair ( 16 ), and nails ( 17 ). In forensic toxicology, retrospective quantification of chemicals in hair samples has gained widespread acceptance.…”