Tina M. Binz

The influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair.

Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE), non-volatile, direct metabolites of ethanol have been shown to be suitable markers for the evaluation of social and chronic excessive alcohol consumption. Previous investigations have shown that the regular use of hair-care products with high alcohol content lead to an increase of FAEE concentration and consequently gave false-positive results for the determination of FAEE in hair. In this study we investigated the influence of a long-term hair treatment with EtOH containing lotion, on the EtG concentrations in hair. In this study 7 volunteer subjects (classified as either rare, social or heavy drinkers) treated the right side of their scalp every day during a one or two month period with a commercial hair tonic (Seborin), which contains 44.0% ethanol (vol%). Collection of hair specimens from both sides of the scalp was done one day before hair treatment, one week and one month after treatment (for 5 subjects also after two months of treatment). A hair segment of 3 centimeters (cm) was cut and then washed with water and acetone, and then pulverized. EtG was quantified by GC/MS after pulverization and 2h of ultrasonication in water, extraction by solid phase extraction using Oasis MAX columns and derivatization with HFBA. Measurements were done in negative chemical ionization mode using EtG-D5 as internal standard. Comparison of EtG concentration in the treated and in the non-treated hair specimens did not show any increase at the different dates of collection for the 7 subjects. In conclusion, these results show that there is no indication for an increase of EtG after use of ethanol containing hair cosmetics.

Systematic investigation of the incorporation mechanisms of zolpidem in fingernails

Nails are attracting increasing interest in forensic toxicology as an alternative to hair. The goal of this study was to systematically investigate the incorporation of drugs in fingernails after single drug dose, exemplified for zolpidem. Fingernail samples from ring fingers were collected one week before, and then 24 h and weekly after intake for a period of three to five months. Hair samples were taken six weeks after intake. Nail specimens were pulverized and extracted with methanol (internal standard: zolpidem-D6 ) under sonication. Extracts were analyzed by a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method, which was developed and validated for this study. The lower limit of quantification (LLOQ) for a 5-mg sample was 0.1 pg/mg nail. Zolpidem was detected continuously in fingernail clippings. The mean window of detection of zolpidem in fingernail clippings was 3.5 months. Unwashed nail specimens taken 24 h after intake showed the highest zolpidem concentrations indicating external contamination by sweat. External contamination experiments revealed that zolpidem could be incorporated in fingernails by sweat to such an extent that it remained irremovable by daily hygiene. Averagely 3 months after intake a concentration peak was reached, suggesting outgrowth of the nail part which had been formed while the drug circulated in blood. Hair concentrations were higher than the maximum nail concentrations. Pigmented hair contained more zolpidem than non-pigmented hair from the same strand. From all these results it can be concluded, that fingernail clippings may represent a useful alternative and/or complementary matrix in cases of, for example, drug-facilitated sexual assault or monitoring of constant consumption behavior.

Rapid extraction, identification and quantification of oral hypoglycaemic drugs in serum and hair using LC-MS/MS

A sensitive and accurate LC-MS/MS method for the identification and quantification of 5 oral anti-diabetics (glipizide, glibenclamide, gliclazide, gliquidone and metformin) in serum and hair was developed using glibornuride as the internal standard. We have developed a rapid and robust extraction procedure by using acetonitrile for serum protein precipitation and methanol for the extraction of anti-diabetics from hair. Anti-diabetics (ADs) were separated by UPLC over a C18 column and detection was performed on a Waters Xevo TQ MS mass spectrometer in positive ionization mode using electrospray ionization. Each AD was identified by three specific ion transitions in multiple reaction monitoring (MRM) mode. The method was validated according to international guidelines. For all compounds the variation coefficient (CV) was <20%, and accuracies ranged from 85 to 115% in serum and hair. The limits of detection (LODs) were <1.5 ng/mL for all ADs in serum and <3.59 pg/mg in hair. Recoveries varied from 56.41% (gliclazide) to 67.58% (glipizide) in serum and from 68% (gliclazide) to 91.2% (metformin) in hair. The method was successfully applied to quantify ADs in serum of 33 patients and in hair of 15 patients.

The influence of cleansing shampoos on ethyl glucuronide concentration in hair analyzed with an optimized and validated LC–MS/MS method

Ethyl glucuronide (EtG) is widely used as a marker for assessment of alcohol consumption behavior. In this study the influence of special cleansing shampoos on ethyl glucuronide concentrations in hair was investigated. For that purpose an optimized LC-MS/MS method was developed using a Hypercarb™ porous graphitic carbon (PGC) column and validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Twenty-five hair samples of persons with known alcohol consumption behavior were investigated (21 positive samples and 4 blank samples). The hair samples were divided into two strands of hair and were analyzed after treatment with one out of four cleansing shampoos and without shampoo treatment. EtG concentrations in hair did not show any significant differences after a single application of the different cleansing shampoos. EtG was still detectable in all the positive hair samples without significant concentration change. These results clearly demonstrated that a single application of the tested cleansing shampoos did not remove EtG from hair and therefore had no influence on EtG concentration in analytical hair analysis.

Time resolved analysis of quetiapine and 7-OH-quetiapine in hair using LC/MS-MS.

Hair analysis is a powerful tool for retrospective drug analysis and has a wide application window. This article describes the simultaneous determination and quantification of the short-acting atypical antipsychotic drug quetiapine and its main metabolite 7-OH quetiapine in hair. A sensitive and accurate method for the determination of these two compounds was developed using high-performance liquid chromatography coupled to tandem mass spectrometry detection (LC-MS/MS). The method was applied to 10 real case samples. For five patients, a time resolved hair analysis was done. Results varied from 0.35 ng/mg to 10.21 ng/mg hair for quetiapine and from 0.02 ng/mg to 3.19 ng/mg hair for 7-OH-quetiapine.

Endogenous cortisol in keratinized matrices: systematic determination of baseline cortisol levels in hair and the influence of sex, age and hair color

The measurement of hair cortisol is increasingly used to measure long-term cumulative cortisol levels and investigate its role as an important stress mediator. In this study a comparative statistical analysis of five independent studies (all analyzed in our laboratory) was performed to investigate baseline ranges of cortisol values in hair and evaluate potential influences of sex, age and hair color. Cortisol concentrations in hair of 554 subjects were measured and a comparative statistical analysis was performed. The analysis showed that cortisol levels significantly differ depending on age. The toddler group (7 months (0.6 years) to 3 years) showed significantly higher values (median 10pg/mg, p-value<0.0001, d=0.78) than the adolescent group. The adolescent groups showed significantly lower (p-value<0.0001, d=0.58 and p<0.0001, d=0.13) values (median 2.4pg/mg and 2.8pg/mg) than the adult group (median 5.8pg/mg). Furthermore, in the adult group men showed significantly higher cortisol values than women (p-value<0.05, d=0.17). This effect could not be seen in the adolescent group. Black hair showed higher cortisol concentrations than blond hair (p-value<0.0001, d=1.3). In addition, two rounds of interlaboratory comparisons for hair cortisol samples between four laboratories revealed very consistent results. Our results demonstrate that baseline cortisol levels are generally low in hair thus making a standardized and well-elaborated analytical method indispensable for accurate determination. Age-dependent normative baseline cortisol levels (toddlers, adolescents and adults) are highly recommended based on the comparative analysis comprising five independent studies.

Hair Cortisol in Twins: Heritability and Genetic Overlap with Psychological Variables and Stress-System Genes

Hair cortisol concentration (HCC) is a promising measure of long-term hypothalamus-pituitary-adrenal (HPA) axis activity. Previous research has suggested an association between HCC and psychological variables, and initial studies of inter-individual variance in HCC have implicated genetic factors. However, whether HCC and psychological variables share genetic risk factors remains unclear. The aims of the present twin study were to: (i) assess the heritability of HCC; (ii) estimate the phenotypic and genetic correlation between HPA axis activity and the psychological variables perceived stress, depressive symptoms, and neuroticism; using formal genetic twin models and molecular genetic methods, i.e. polygenic risk scores (PRS). HCC was measured in 671 adolescents and young adults. These included 115 monozygotic and 183 dizygotic twin-pairs. For 432 subjects PRS scores for plasma cortisol, major depression, and neuroticism were calculated using data from large genome wide association studies. The twin model revealed a heritability for HCC of 72%. No significant phenotypic or genetic correlation was found between HCC and the three psychological variables of interest. PRS did not explain variance in HCC. The present data suggest that HCC is highly heritable. However, the data do not support a strong biological link between HCC and any of the investigated psychological variables.

Contact to Nature Benefits Health: Mixed Effectiveness of Different Mechanisms

How can urban nature contribute to the reduction of chronic stress? We twice measured the concentration of the “stress hormone” cortisol in the hair of 85 volunteer gardeners (six months apart), relating cortisol level change to (self-reported) characteristics of their recreational activities. Both time spent in nature and physical activity led to decreases in cortisol, while time spent being idle led to an increase. At high levels of present stressors, however, the relationship for time spent in nature and for idleness was reversed. Time spent with social interaction had no effect on cortisol levels. Our results indicate that physical activity is an effective means of mitigating the negative effects of chronic stress. The results regarding the time spent in nature and time spent being idle are less conclusive, suggesting the need for more research. We conclude that if chronic stress cannot be abolished by eradicating its sources, public health may take to measures to reduce it—providing urban nature being one effective possibility.

Steroid profiling in nails using liquid chromatography-tandem mass spectrometry

HIGHLIGHTSAn LC‐MS/MS method was developed for the quantification of 12 steroid hormones in nails.3 steroid hormones were detected in nails for the first time.Six steroid hormones were detected and quantified in human fingernails of mothers and their infants.No significant differences could be found between right and left hand steroid levels. ABSTRACT The retrospective analysis of endogenous steroid hormones in nails can be used to elucidate endocrine diseases and thus help with their diagnosis and treatment. A liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) based method was developed for the simultaneous identification and quantification of 12 steroid hormones (aldosterone, cortisone, cortisol, corticosterone, 11‐deoxycortisol, androstenedione, 11‐deoxycorticosterone, testosterone, dehydroepiandrosterone (DHEA), 17&agr;‐hydroxyprogesterone (17‐OHP), dihydrotestosterone (DHT) and progesterone) in human fingernails. Steroid hormones were extracted from 0.5mg to 10mg pulverized nail clippings by methanolic extraction, followed by a liquid‐liquid extraction. The analysis was conducted with LC‐MS/MS in electrospray ionization positive mode. The method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, recovery and robustness. It was successfully applied for steroid profiling in nails of mothers and their infants where cortisol, cortisone, testosterone, progesterone, androstenedione and 11‐deoxycorticosterone could be detected. Furthermore, it could be shown that there is no significant difference in concentrations between left and right hand for cortisol, cortisone and progesterone. A positive linear correlation between cortisol and cortisone in nails was found. In conclusion, it could be shown that nails are a suitable matrix for the retrospective monitoring of cumulative steroid hormone levels.

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 tasks 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.