Higher paracetamol levels are associated with elevated glucocorticoid concentrations in hair: findings from a large cohort of young adults.

Paracetamol is one of the most commonly used over-the-counter medications. Experimental studies suggest a possible stress-suppressing effect of paracetamol in humans facing experimental stress-inducing paradigms. However, no study has investigated whether paracetamol and steroid hormones covary over longer time frames and under real-life conditions. This study addresses this gap by investigating associations between steroid hormones (cortisol, cortisone, and testosterone) and paracetamol concentrations measured in human hair, indexing a timeframe of approximately three months. The data came from a large community sample of young adults (N = 1002). Hair data were assayed using liquid chromatography-tandem mass spectrometry. Multiple regression models tested associations between paracetamol and  steroid hormones, while adjusting for a wide range of potential confounders, such as sex, stressful live events, psychoactive substance use, hair colour, and body mass index. Almost one in four young adults from the community had detectable paracetamol in their hair (23%). Higher paracetamol hair concentrations were robustly associated with more cortisol (ß = 0.13, ηp = 0.016, p < 0.001) and cortisone (ß = 0.16, ηp = 0.025, p < 0.001) in hair. Paracetamol and testosterone hair concentrations were not associated. Paracetamol use intensity positively correlated with corticosteroid functioning across several months. However, a potential corticosteroid-inducing effect of chronic paracetamol use has yet to be tested in future experimental designs.

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method including 25 novel synthetic opioids in hair and subsequent analysis of a Swiss opioid consumer cohort.

Major public health concern is raised by the evidence that common drugs like heroin are now frequently laced or replaced with highly potent novel synthetic opioids (NSOs). The objective of this study was to explore the prevalence and patterns of NSOs in a cohort of Swiss opioid users by hair analysis. Hair analysis is considered an ideal tool for retrospective consumption monitoring. Hair samples from 439 opioid users in Zurich were analyzed. Study inclusion required a previous positive hair test result for heroin metabolites, oxycodone, fentanyl, methadone, or tramadol. The samples were extracted with a two-step extraction procedure, followed by a targeted LC-MS/MS (QTRAP® 6500+) analysis in multiple reaction monitoring mode for a total of 25 NSOs. The method underwent full validation and demonstrated good selectivity and sensitivity with limits of detection (LOD) as low as 0.1 pg/mg. The analyzed sample cohort demonstrated a positivity rate for NSOs of 2.5%, including the following NSOs: butyrylfentanyl, acrylfentanyl, furanylfentanyl, methoxyacetylfentanyl, ocfentanil, U-47700, isobutyrylfentanyl and benzylfentanyl. Furthermore, we were able to identify specific consumption patterns among drug users. The results indicate that hair analysis is a valuable tool for investigating the prevalence of NSOs in drug-using populations, which seems to be low in the case of Swiss opioid users. Nevertheless, the results highlight the need for sensitive analytical detection methods in forensic toxicology to identify and monitor substance distribution in different populations.

Associations between hair-derived cannabinoid levels, self-reported use, and cannabis-related problems.

RATIONALE: As cannabis potency and cannabis use are increasing in newly legalized markets, it is increasingly important to measure and examine the effects of cannabinoid exposure. OBJECTIVES: The current study aims to assess how hair-derived cannabinoid concentrations - offering insight into three-month cumulative exposure - are associated with common self-report measures of cannabis use and cannabis use-related problems. METHODS: 74 near-daily dependent cannabis users self-reported their quantity of cannabis use, cannabis use-related problems, and estimated cannabis potency. Hair samples were provided to quantify Δ9-THC, CBD, and CBN using LC-MS/MS and THC-consumption was verified by analyzing THC-COOH in hair using GC-MS/MS. RESULTS: Cannabinoids were detectable in 95.95% of the hair samples from individuals who tested positive on a urine screen for cannabis. Δ9-THC concentrations were positively associated with measures of self-reported potency (relative potency, potency category, and perceived 'high'), but Δ9-THC, CBD, CBN concentrations and THC/CBD ratio were not associated with self-reported quantity of use. Self-reported potency, but not hair-derived concentrations, were associated with withdrawal and craving. Self-reported quantity of cannabis use, but not cannabinoid concentrations, were associated with cannabis use-related problems. CONCLUSIONS: The use of hair-derived cannabinoid quantification is supported for detecting cannabis use in near-daily users, but the lack of associations between hair-derived cannabinoid concentrations and self-report measures of use does not support the use of hair analyses alone for quantification of cannabinoid exposure. Further research comparing hair-derived cannabinoid concentrations with other biological matrices (e.g. plasma) and self-report is necessary to further evaluate the validity of hair analyses for this purpose.

Stress Markers, Executive Functioning, and Resilience Among Early Adolescents With Complex Congenital Heart Disease.

Importance: Infants with complex congenital heart disease (cCHD) may experience prolonged and severe stress when undergoing open heart surgery. However, little is known about long-term stress and its role in neurodevelopmental impairments in this population. Objective: To investigate potential differences between early adolescents aged 10 to 15 years with cCHD and healthy controls in physiological stress markers by hair analysis, executive function (EF) performance, and resilience. Design, Setting, and Participants: This single-center, population-based case-control study was conducted at the University Children's Hospital Zurich, Switzerland. Patients with different types of cCHD who underwent cardiopulmonary bypass surgery during the first year of life and who did not have a genetic disorder were included in a prospective cohort study between 2004 and 2012. A total of 178 patients were eligible for assessment at ages 10 to 15 years. A control group of healthy term-born individuals was cross-sectionally recruited. Data assessment was between 2019 and 2021. Statistical analysis was performed from January to April 2023. Exposure: Patients with cCHD who underwent infant open heart surgery. Main Outcomes and Measures: Physiological stress markers were quantified by summing cortisol and cortisone concentrations measured with liquid chromatography with tandem mass spectrometry in a 3-centimeter hair strand. EFs were assessed with a neuropsychological test battery to produce an age-adjusted EF summary score. Resilience was assessed with a standardized self-report questionnaire. Results: The study included 100 patients with cCHD and 104 controls between 10 and 15 years of age (mean [SD] age, 13.3 [1.3] years); 110 (53.9%) were male and 94 (46.1%) were female. When adjusting for age, sex, and parental education, patients had significantly higher sums of hair cortisol and cortisone concentrations (ß, 0.28 [95% CI, 0.12 to 0.43]; P < .001) and lower EF scores (ß, -0.36 [95% CI, -0.49 to -0.23]; P < .001) than controls. There was no group difference in self-reported resilience (ß, -0.04 [95% CI, -0.23 to 0.12]; P = .63). A significant interaction effect between stress markers and EFs was found, indicating a stronger negative association in patients than controls (ß, -0.65 [95% CI, -1.15 to -0.15]; P = .01). The contrast effects were not significant in patients (ß, -0.21 [95% CI, -0.43 to -0.00]; P = .06) and controls (ß, 0.09 [95% CI, -0.11 to 0.30]; P = .38). Conclusions and Relevance: This case-control study provides evidence for altered physiological stress levels in adolescents with cCHD and an association with poorer EF. These results suggest that future studies are needed to better understand the neurobiological mechanisms and timing of alterations in the stress system and its role in neurodevelopment.

Associations of psychoactive substances and steroid hormones in hair: Findings relevant to stress research from a large cohort of young adults.

OBJECTIVE: Epidemiological studies increasingly use hair samples to assess people's cumulative exposure to steroid hormones, but how the use of different psychoactive substances may affect steroid hormone levels in hair is, so far, largely unknown. The current study addresses this gap by establishing the substance exposure correlates of cortisol, cortisone, and testosterone in hair, while also accounting for a number of relevant covariates. METHOD: Data came from a large urban community-sample of young adults with a high prevalence of substance use (N = 1002, mean age=20.6 years, 50.2% female), who provided 3 cm of hair samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantified cortisol, cortisone, and testosterone, as well as delta-9-tetrahydrocannabinol (THC), 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), cocaine, several opioids, and their respective metabolites. Multiple linear regression models with covariates were used to predict steroid hormone levels from substance exposure in a four-step approach: In the full sample, low and high substance hair concentrations (median split) were first tested against no use for each substance individually (step 1) and for all substances together (step 2). Then, within the participants with any substance in hair only, the continuous hair concentration of each substance in pg/mg (step 3) and finally of all substances together, were regressed (step 4). RESULTS: Low, high, and continuous levels of THC in hair were robustly associated with higher levels of cortisol (sig. in step 1 low THC: ß = 0.29, p = .021; high THC: ß = 0.42, p = .001; step 2: low THC: ß = 0.27, p = 0.036, and high THC: ß = 0.40, p = .004, and step 4: ß = 0.12, p = .041). Participants with high MDMA levels had higher levels of cortisone without adjusting for other substances (step 1: ß = 0.34, p = .026), but this effect was not significant in the other models. While high THC levels were associated with lower levels of testosterone in step 2 (ß = -0.35, p = .018), MDMA concentration was positively related to testosterone concentration with and without adjusting for other substances (step 3: ß = 0.24, p = .041; step 4: ß = 0.17, 95%, p = .015) in male participants. CONCLUSION: The use of psychoactive substances, especially of cannabis and ecstasy, should be considered in studies investigating steroid hormones in hair.

Advances in testing for sample manipulation in clinical and forensic toxicology-part B: hair samples.

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology.

Associations of different hormonal contraceptive methods with hair concentrations of cortisol, cortisone, and testosterone in young women.

Hair concentrations of cortisol, cortisone, and testosterone are non-invasive measures of cumulative steroid hormone levels. Use of contraceptives co-varies with levels of cortisol and cortisone in women's hair. It is unclear, however, how different contraceptive methods (i.e., that differ in their steroid hormone composition) affect corticosteroid and testosterone hair levels. The current study examines associations of contraceptives with hair steroid hormone concentrations in females from the community (N = 464, M = 20.6 years old, age range = 19-22). Self-reported contraceptives were first categorized as combined estrogen-progestin or progestin-only, and then analyzed individually in follow-up analyses. Multiple regressions adjusting for body mass index (BMI) and hair characteristics revealed that levels of hair cortisol, cortisone, and testosterone were significantly lower in women who used combined estrogen-progestin methods than in women who did not use hormonal contraception (ßcortisol(log) = -0.29; ßcortisone(log) = -0.28; ßtestosterone(log) = -0.36), showing moderate to large effect sizes (d = 0.64, d = 0.71, and d = 0.81, respectively). Concentrations of hair cortisol were lower in women who used progestin-only contraceptives (ß = -0.49) compared to no contraceptive use, with a large effect size (d = 1.67). Follow-up analyses revealed that the association of the three steroid hormones with estrogen-progestin methods was strongest for the combined oral "micro-pill." Future studies of hair steroid hormones should take into account the specific type of contraceptive used, as this may affect study results.

Single sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids in hair.

Recently, we published a multi-analyte method for the simultaneous analysis of 116 drugs and pharmaceuticals including different substance groups like opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics based on a single sample workup followed by a single analytical measurement with LC-MS/MS. However, in some cases, additional analysis of further substance groups, such as cannabinoids and endogenous steroids, is required, which are analyzed in our laboratory using separate sample preparation and separate analytical methods. The goal of this study was to use the knowledge from the different sample preparations and combine them into a single sample preparation and extraction workflow for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids, and endogenous steroids to be analyzed with the appropriate analytical methods. A partial validation of selected parameters such as selectivity, linearity, limit of quantification (LOQ), accuracy, precision and robustness for the different analytical methods was carried out and revalidated. In addition, comparative measurements of quality controls and authentic pools were performed and statistically evaluated using the unpaired t-test or the non-parametric Mann-Whitney test. The results using the newly established sample preparation and extraction were in good agreement with the original data. In conclusion, the newly established sample preparation is suitable for the combined extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids, and gives reliable results for quantification of various substances.

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

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.

Alterations of Stress-Related Glucocorticoids and Endocannabinoids in Hair of Chronic Cocaine Users.

BACKGROUND: Previous research in animals and humans has demonstrated a potential role of stress regulatory systems, such as the hypothalamic-pituitary-adrenal (HPA) axis and the endocannabinoid (eCB) system, in the development of substance use disorders. We thus investigated alterations of HPA and eCB markers in individuals with chronic cocaine use disorder by using an advanced hair analysis technique. METHODS: We compared hair concentrations of glucocorticoids (cortisone, cortisol) and the eCBs 2-arachidonylglycerol, anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA) between 48 recreational cocaine users (RCU), 25 dependent cocaine users (DCU), and 67 stimulant-naïve controls. Self-reported substance use and hair concentrations of substances were also assessed. RESULTS: Significantly higher concentrations of hair cortisone were found in RCU and DCU compared with controls. Hair concentrations of OEA and PEA were significantly lower in DCU compared with RCU and controls. Additionally, within cocaine users, elevated cocaine hair concentration was a significant predictor for increased glucocorticoid and decreased OEA hair levels. Moreover, higher 3,4-methyl​enedioxymethamphetamine hair concentration was correlated with elevated cortisone and AEA, OEA, and PEA levels in hair within cocaine users, whereas more self-reported cannabis use was associated with lower eCBs levels in hair across the total sample. CONCLUSION: Our findings support the hypothesis that the HPA axis and eCB system might be important regulators for substance use disorders. The mechanistic understanding of changes in glucocorticoid and eCB levels in future research might be a promising pharmacological target to reduce stress-induced craving and relapse specifically in cocaine use disorder.

Systemic detectability of dexamethasone and prednisolone after eye drop application in horses.

BACKGROUND: Equine sport agencies list steroids as prohibited substances for competing horses. OBJECTIVES: The objective of this study was to investigate if the controlled substances dexamethasone and prednisolone are detectable in equine serum and urine samples during and after treatment with eye drops and if this can generate a positive doping test. STUDY DESIGN: Prospective cohort study. METHODS: The study cohort included 11 horses. One eye of the horses was treated with either dexamethasone (Maxitrol® 0.1%, n = 5 eyes) or prednisolone (Pred forte® 1%, n = 6 eyes) eye drops 3 times daily for 14 days. Dexamethasone and prednisolone concentrations were determined in serum and urine at day 0 (negative control), 1, 7, 14, 15, 17 and 21 using liquid chromatography-tandem mass spectrometry. Blood samples were collected within 2 hours post application. Urine samples were collected during spontaneous urination. RESULTS: All serum samples (range: 0.7-43 ng/mL, mean 2.1 ng/mL) and urine samples (range 1.2-5 ng/mL, mean 0.8 ng/mL) showed measurable amounts of dexamethasone during the course of treatment. Concentrations in both serum and urine samples were below limit of detection (LOD) 24 hours after the last dexamethasone treatment (day 15). All serum samples (range 1.1-32.5 ng/mL, mean 6.4 ng/mL) and urine samples (range 3.7-19 ng/mL, mean 4.6 ng/mL) were positive for prednisolone during treatment. Urine samples were below LOD on day 15; serum samples on day 21. CONCLUSIONS: Dexamethasone and prednisolone eye drops can induce detectable drug levels in serum and urine samples of horses after a 14-day treatment plan. This can lead to a positive doping result. All samples tested negative (below LOD of the analytical method) for dexamethasone one day and for prednisolone one week after treatment cessation.

Quantification of Classic, Prescription and Synthetic Opioids in Hair by LC-MS-MS.

The current use and misuse of synthetic and prescription opioids in the USA has reached epidemic status. According to the US Department of Health and Human Services, every day more than 130 people in the USA die after overdosing on opioids, and 2.1 million had an opioid use disorder in 2018. Hair is becoming an alternative matrix of increasing interest in forensic toxicology to investigate drug use and abuse patterns due to its long window of detection. The focus of this project was to develop and validate a method that simultaneously detects and quantifies 27 classic, prescription and synthetic opioids in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized in a bead mill. Twenty-five milligrams of hair powder were incubated in a buffer overnight. Mixed mode cation exchange solid phase extraction was carried out before undergoing reversed-phase chromatographic separation, successfully resolving isobaric opioids. We used two multiple reaction monitoring transitions in positive mode to identify each analyte. The linearity range was 1-500 pg/mg for fentanyl and synthetic opioids and 10-500 pg/mg for prescription and classic opioids. Imprecision was <17.5% and bias ranged from -13.6 to 12.0%. Majority of compounds showed extraction efficiency >50%, and ion suppression from -89.2 to -26.6% (CV < 19%, n = 10). This method was applied to 64 authentic cases, identifying 13 compounds from our panel. A sensitive and specific method was developed for the identification and quantification of 27 classic, prescription and synthetic opioids in hair by LC-MS-MS.

Towards Best Practice in Hair Metabolomic Studies: Systematic Investigation on the Impact of Hair Length and Color.

Untargeted metabolomic studies are used for large-scale analysis of endogenous compounds. Due to exceptional long detection windows of incorporated substances in hair, analysis of hair samples for retrospective monitoring of metabolome changes has recently been introduced. However, information on the general behavior of metabolites in hair samples is scarce, hampering correct data interpretation so far. The presented study aimed to investigate endogenous metabolites depending on hair color and along the hair strand and to propose recommendations for best practice in hair metabolomic studies. A metabolite selection was analyzed using untargeted data acquisition in genuine hair samples from different hair colors and after segmentation in 3 cm segments. Significant differences in metabolites among hair colors and segments were found. In conclusion, consideration of hair color and hair segments is necessary for hair metabolomic studies and, subsequently, recommendations for best practice in hair metabolomic studies were proposed.

Cheating on forensic hair testing? Detection of potential biomarkers for cosmetically altered hair samples using untargeted hair metabolomics.

Hair analysis has become an integral part in forensic toxicological laboratories for e.g. assessment of drug or alcohol abstinence. However, hair samples can be manipulated by cosmetic treatments, altering drug concentrations which eventually leads to false negative hair test results. In particular oxidative bleaching of hair samples under alkaline conditions significantly affects incorporated drug concentrations. To date, current techniques to detect cosmetic hair adulterations bear limitations such as the implementation of cut-off values or the requirement of specialized instrumentations. As a new approach, untargeted hair metabolomics analysis was applied to detect altered, endogenous biomolecules that could be used as biomarkers for oxidative cosmetic hair treatments. For this, genuine hair samples were treated in vitro with 9% hydrogen peroxide (H2O2) for 30 minutes. Untreated and treated hair samples were analyzed using liquid-chromatography high-resolution time-of-flight mass spectrometry. In total, 69 metabolites could be identified as significantly altered after hair bleaching. The majority of metabolites decreased after bleaching, yet totally degraded metabolites were most promising as suitable biomarkers. The formation of biomarker ratios of metabolites decreasing and increasing in concentrations improved the discrimination of untreated and treated hair samples. With the results of this study, the high variety of identified biomarkers now offers the possibility to include single biomarkers or biomarker selections into routine screening methods for improved data interpretation of hair test results.

Hair cortisol measurement in older adults: Influence of demographic and physiological factors and correlation with perceived stress.

AIMS: This study aimed to investigate correlation between hair cortisol levels and perceived stress scale (PSS) in addition to a range of demographic and physiological factors in a sample of older adults. EXPERIMENTAL: Hair cortisol concentrations were established from 42 older adults aged between 60 and 80 years old. Age, sex, hair colour, smoking status, employment status, daytime sleeping, medication, waist to hip ratio (WHR) and PSS scores were assessed through a questionnaire. Hair cortisol concentration was assessed through liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS). RESULTS: Amongst the older adult group there was no statistically significant correlation between hair cortisol concentration and age, employment status, daytime sleep duration, WHR or PSS. When compared to previous data assessing hair cortisol in toddlers (7 months to 3 years old), adolescents (12-17 years old) and adults (18-60 years old) it is observed that there is a trend for higher hair cortisol in older adults (60-80 years old). Hair cortisol concentrations were significantly higher in males (n = 20) than in females (n = 22) and in participants with dark brown hair (n = 8). No relationship was investigated between hair cortisol concentration and smoking status or medication intake. CONCLUSIONS: The results confirm that hair samples are a useful alternative to the current mediums that are used to analyse biological cortisol. The results also validate the use of LC-MS/MS as an effective analytical method for the quantitation of hair cortisol concentrations.

Endogenous steroid hormones in hair: Investigations on different hair types, pigmentation effects and correlation to nails.

Steroid hormone analysis is widely used in health- and stress-related research to get insights into various diseases and the adaption to stress. Hair analysis has been used as a tool for the long-term monitoring of these steroid hormones. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous identification and quantification of seven steroid hormones (cortisone, cortisol, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, progesterone) in hair. Cortisol, cortisone, androstenedione, testosterone and progesterone were detected and quantified in authentic hair samples of different individuals. Significantly higher concentrations for body hair were found for cortisone and testosterone compared to scalp hair. Furthermore, weak correlations for the majority of steroids between scalp and body hair indicate that body hair is not really suitable as alternative when scalp hair is not available. The influence of hair pigmentation was analyzed by comparing pigmented to non-pigmented hair of grey-haired individuals. The results showed no differences for cortisol, cortisone, androstenedione, testosterone and progesterone concentrations (p > 0.05) implying that hair pigmentation has not a strong effect on steroid hormone concentrations. Correlations between hair and nail steroid levels were also studied. Higher concentrations of cortisol and cortisone in hair were found compared to nails (p < 0.0001). No significant correlation for cortisone, cortisol, androstenedione, testosterone and progesterone concentrations were found between hair and nails. These results demonstrate that matrix-dependent value ranges for hair and nail steroid levels should be established and applied for interpretation.

A possible new oxidation marker for hair adulteration: Detection of PTeCA (1H-pyrrole-2,3,4,5-tetracarboxylic acid) in bleached hair.

Hair analysis has become a valuable tool in forensic toxicology to assess drug or alcohol abstinence. Yet, hair adulteration by cosmetic products presents a major challenge for forensic hair analysis. Oxidative treatments, e.g. bleaching, may lead to analyte loss and thereby to false negative results. Currently, the eumelanin degradation product 1H-pyrrole-2,3,5-tricarboxylic acid (PTCA) serves as a marker for oxidative hair treatment, but requires the definition of cut-off values. To investigate further eumelanin degradation products as markers for oxidative hair treatment, hair samples with and without in vitro bleaching (hydrogen peroxide (H2 O2 ) concentrations 1.9% up to 12%; incubation times 15 min, 30 min, 60 min) were analyzed by liquid chromatography coupled to high-resolution time of flight mass spectrometry (HPLC-HRMS). The distribution of eumelanin degradation products along the hair shaft was investigated for routine applicability after segmentation of cosmetically untreated hair samples and authentically treated hair samples. The signals of the eumelanin degradation products PTCA, 1H-pyrrole-2,3,4-tricarboxylic acid (isoPTCA), and 1H-pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) were found to be significantly elevated after in vitro bleaching already with low H2 O2 concentrations and after short incubation times. In contrast to PTCA and isoPTCA, PTeCA was not detectable in cosmetically untreated segments up to 12 cm from hair root and was only formed through the oxidation process. The results of the study show that the detection of PTeCA within the proximal 3 to 6 cm segment can be applied to reliably detect hair adulteration attempts through hair bleaching.

(Un)targeted hair metabolomics: first considerations and systematic evaluation on the impact of sample preparation.

The interest in metabolomic studies has rapidly increased over the past few years. Changes of endogenous compounds are typically detected in plasma or urine. However, the use of hair allows for long-term monitoring of metabolomic changes and has recently started being applied to metabolomic studies. Within the proposed study, we aimed for a systematical investigation of different pre-analytical parameters on detected metabolites from different chemical classes in hair. For this purpose, three different parameters were varied: (1) multi-step decontamination (dichloromethane (DCM), acetone, H2O, acetone; H2O, acetone, DCM, acetone; and H2O, methanol/acetone), (2) homogenization (pulverization vs. cutting into snippets), and (3) extraction (acetonitrile (ACN)/buffer pH 4 vs. ACN/H2O vs. ACN/buffer pH 8.5). To include as many metabolites as possible, samples were analyzed by high-resolution time of flight mass spectrometry coupled to liquid chromatography (HPLC-HRMS) and additionally by gas chromatography high-resolution mass spectrometry (GC-HRMS) followed by untargeted-like data processing, respectively. The application of different decontamination procedures yielded similar results, although pointing to a trend towards increased washing-out effects if protic solvents were used as a first washing step. Pulverization of hair samples was favorable in terms of detected and tentatively identified metabolites. Evaluation of extraction solvents showed differences in extraction yield for the majority of investigated metabolites, yet, a prediction of metabolite extraction according to their pKa values was not possible. Overall, successive decontamination with DCM, acetone, H2O, and acetone; homogenization by pulverization; and extraction with ACN/H2O produced reliable results. Graphical abstract.

Non-medical prescription opioid users exhibit dysfunctional physiological stress responses to social rejection.

Non-medical prescription opioid use (NMPOU) recently increased dramatically, especially in the U.S. Although chronic opioid use is commonly accompanied by deficits in social functioning and dysregulation of the hypothalamic-pituitary adrenergic (HPA) stress axis, little is known about the impact of NMPOU on psychosocial stress responses. Therefore, we measured physiological responses of the autonomic nervous system and the HPA axis to social rejection using the Cyberball paradigm. We compared 23 individuals with NMPOU, objectively confirmed by hair and urine analyses, with 29 opioid-naïve, healthy controls. As expected, heart rate variability (HRV), an index of parasympathetic activity, increased significantly during exclusion within controls, while in the NMPOU group only a trend in the same direction was found. However, increased HRV was robustly moderated by opioid craving indicating worse emotion regulation to social exclusion specifically in individuals with high opioid craving. Greater levels of the adrenocorticotropic hormone and cortisol responses to social rejection were found in the NMPOU group indicating hyperreactivity of the HPA axis to social exclusion. Self-ratings suggest that opioid users were aware of rejection, but less emotionally affected by exclusion. Furthermore, controls showed greater negative mood after the Cyberball confirming the task's validity. Moreover, NMPOU individuals reported a smaller social network size compared to controls. Present findings suggest that chronic NMPOU is associated with dysfunctional physiological responses to psychosocial stressors such as social rejection. In sum, NMPOU was associated with poorer regulation of the parasympathetic nervous system, especially under opioid craving highlighting its potential importance in relapse prevention.

Systematic investigations of endogenous cortisol and cortisone in nails by LC-MS/MS and correlation to hair.

Hair samples are increasingly used for measuring the long-term stress mediator cortisol. However, hair is not always available and nails (finger or toe), as a keratinized matrix, may be an alternative to hair. In order to measure cortisol and cortisone in the nail matrix, an LC-MS/MS method has been developed and validated using 13C3-labeled surrogate analytes. Both analytes were measured in ESI negative mode as formic acid adducts. Different sample preparation techniques were assessed, and single-step extraction in methanol was established for determination of cortisone and cortisol in the nail matrix. The method was successfully validated with limits of detection (LOD) and limits of quantification (LOQ) of 0.5 and 1.0 pg/mg for cortisol and cortisone, respectively. The calibration curve was linear up to a concentration of 500 pg/mg. Recovery was good for both analytes and showed values over 50%. Matrix effects with ion suppression occurred for both substances but could be corrected by the use of internal standard. Accuracy and precision were in the accepted range of ± 20% for both substances. The method was successfully applied to determine cortisol and cortisone concentrations in authentic nail samples. Cortisol and cortisone concentrations varied significantly among different fingernails, being highest in the little fingernails and lowest in the thumbnails. It could be shown that even in only 1 mg nail sample cortisol and cortisone can be reliably quantified. No correlation between hair and nail cortisol and cortisone concentrations could be found. Furthermore, cortisol and cortisone concentrations were significantly higher in hair. Graphical abstract.