Evaluation of five multisteroid LC‒MS/MS methods used for routine clinical analysis: comparable performance was obtained for nine analytes.

OBJECTIVES: A mass spectrometry (LC‒MS/MS)-based interlaboratory comparison study was performed for nine steroid analytes with five participating laboratories. The sample set contained 40 pooled samples of human serum generated from preanalyzed leftovers. To obtain a well-balanced distribution across reference intervals of each steroid, the leftovers first underwent a targeted mixing step. METHODS: All participants measured a sample set once using their own multianalyte protocols and calibrators. Four participants used in-house developed measurement platforms, including IVD-CE certified calibrators, which were used by three participants; the 5th lab used the whole LC‒MS kit from an IVD manufacturer. All labs reported results for 17-hydroxyprogesterone, androstenedione, cortisol, and testosterone, and four labs reported results for 11-deoxycortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEAS), and progesterone. RESULTS: Good or acceptable overall comparability was found in Bland‒Altman and Passing‒Bablok analyses. Mean bias against the overall mean remained less than ±10 % except for DHEAS, androstenedione, and progesterone at one site and for cortisol and corticosterone at two sites (max. -18.9 % for androstenedione). The main analytical problems unraveled by this study included a bias not previously identified in proficiency testing, operator errors, non-supported matrix types and higher inaccuracy and imprecision at lower ends of measuring intervals. CONCLUSIONS: This study shows that intermethod comparison is essential for monitoring the validity of an assay and should serve as an example of how external quality assessment could work in addition to organized proficiency testing schemes.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. METHODS: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. RESULTS: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels. CONCLUSIONS: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

Trimethylamine N-oxide (TMAO) in patients with subarachnoid hemorrhage: a prospective observational study.

BACKGROUND: It is suspected that microbiome-derived trimethylamine N-oxide (TMAO) may enhance platelet responsiveness and accordingly be thrombophilic. The purpose of this prospective observational study is to evaluate TMAO in patients with subarachnoid hemorrhage (SAH) and compare it with a control group. A secondary aim was to investigate TMAO in the cerebrospinal fluid (CSF) from SAH patients. This should provide a better understanding of the role of TMAO in the pathogenesis of SAH and its thrombotic complications. METHODS: The study included patients with diagnosed spontaneous SAH recruited after initial treatment on admission and patients with nerve, nerve root, or plexus disorders serving as controls. Blood samples were gathered from all patients at recruitment. Additionally, sampling of SAH patients in the intensive care unit continued daily for 14 days. The CSF was collected out of existing external ventricular drains whenever possible. RESULTS: Thirty-four patients diagnosed with SAH, and 108 control patients participated in this study. Plasma TMAO levels at baseline were significantly lower in the SAH group (1.7 µmol/L) compared to the control group (2.9 µmol/L). TMAO was detectable in the CSF (0.4 µmol/L) and significantly lower than in plasma samples of the SAH group at baseline. Plasma and CSF TMAO levels correlated positively. The TMAO levels did not differ significantly during the observation period of 15 days. CONCLUSIONS: Although we assumed that patients with higher TMAO levels were at higher risk for SAH a priori, plasma TMAO levels were lower in patients with SAH compared with control subjects with nerve, nerve root, or plexus disorders on admission to the hospital. A characteristic pattern of plasma TMAO levels in patients with SAH was not found.

Influence of Trimethylamine N-Oxide on Platelet Activation.

Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence platelet-derived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of platelet-derived eicosanoid release were analyzed. The method showed high reproducibility (CVs ≤ 5.3%), good recovery rates (96-98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.

Inclusion of 11-Oxygenated Androgens in a Clinical Routine LC-MS/MS Setup for Steroid Hormone Profiling.

11-Oxygenated androgens (11-OAs) are being discussed as potential biomarkers in diagnosis and therapy control of disorders with androgen excess such as congenital adrenal hyperplasia and polycystic ovary syndrome. However, quantification of 11-OAs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) still relies on extensive sample preparation including liquid-liquid extraction, derivatization and partial long runtimes, which is unsuitable for high-throughput analysis under routine laboratory settings. For the first time, an established online-solid-phase extraction-LC-MS/MS (online-SPE-LC-MS/MS) method for the quantitation of seven serum steroids in daily routine use was extended and validated to include 11-ketoandrostenedione, 11-ketotestosterone, 11ß-hydroxyandrostenedione and 11ß-hydroxytestosterone. Combining a simple protein precipitation step with fast chromatographic separation and ammonium fluoride-modified ionization resulted in a high-throughput method (6.6 min run time) featuring lower limits of quantification well below endogenous ranges (63-320 pmol/L) with recoveries between 85% and 117% (CVs ≤ 15%). Furthermore, the ability of this method to distinguish between adrenal and gonadal androgens was shown by comparing 11-OAs in patients with hyperandrogenemia to healthy controls. Due to the single shot multiplex design of the method, potential clinically relevant ratios of 11-OAs and corresponding androgens were readily available. The fully validated method covering endogenous concentration levels is ready to investigate the diagnostic values of 11-OAs in prospective studies and clinical applications.

Association of plasma trimethylamine N-oxide levels with atherosclerotic cardiovascular disease and factors of the metabolic syndrome.

BACKGROUND AND AIMS: The association of plasma trimethylamine N-oxide (TMAO) with atherosclerotic cardiovascular disease (ASCVD), diabetes mellitus (DM) and its determinants, as well as the role of TMAO as a predictor for short and long-term mortality, is still under discussion. We investigated associations between four plasma metabolites of the TMAO pathway and different clinical manifestations of atherosclerosis, diabetes determinants, and risk of short and long-term mortality in patients with stable ASCVD, acute myocardial infarction (AMI), cardiogenic shock (CS), and DM in three independent cohorts. METHODS: TMAO and its dietary precursors were simultaneously quantified by liquid chromatography-tandem mass spectrometry in a total of 2655 participants of the German Leipzig Research Center for Civilization Diseases (LIFE)-Heart study, LIFE-Adult study, and the European Culprit Lesion Only PCI versus Multivessel PCI in Cardiogenic Shock (CULPRIT-SHOCK) multicenter trial. Associations with ASCVD manifestations, metabolic syndrome, 30-day mortality of patients with AMI and CS, and long-term mortality of subjects with suspected coronary artery disease (CAD) were analyzed. RESULTS: TMAO plasma levels were not independently associated with stable ASCVD. Elevated TMAO plasma concentrations were independently associated with obesity (odds ratio, 1.23; p < 0.01) and DM (odds ratio, 1.37; p < 0.001) in LIFE-Heart. The latter association was confirmed in LIFE-Adult. We found no association of TMAO plasma levels with short-term mortality in patients with AMI and CS. However, TMAO plasma levels were independent predictors of long-term mortality in patients with suspected CAD (hazard ratio, 1.24; p < 0.05). CONCLUSIONS: Potential proatherogenic mechanisms of TMAO seem to have no short-term effect in AMI. Presented associations with diabetes mellitus and obesity suggest that TMAO might have a functional role in metabolic syndrome.

Hair Cortisol Concentration in Healthy Children and Adolescents Is Related to Puberty, Age, Gender, and Body Mass Index.

INTRODUCTION: Hair cortisol concentrations (HCC) have been found to be related to various common childhood diseases, like otitis media, conjunctivitis, respiratory viral infections, and asthma. However, the confounding effects of age, gender, body mass index (BMI), pubertal stage (Tanner stages), socioeconomic status (SES) as well as of some hair maintenance procedures on HCC are still not well examined. METHODS: A population-based cohort of 434 children aged between 5 and 18 years was examined for HCC between January 2012 and February 2015 in the context of the Leipzig Research Centre for Civilization Diseases (LIFE) Child study. Thereby, anthropometric data, gender, BMI, SES and pubertal status were assessed. HCC was measured by liquid chromatography mass spectrometry. RESULTS: In the total cohort, HCC levels ranged between 0.95 and 29.86 pg/mg. In prepuberty, boys showed significantly higher HCC than girls (6.54 vs. 3.73 pg/mg, p < 0.05). During puberty HCC values in both genders converged. Higher BMI was significantly associated with higher HCC in both genders. In girls, HCC did not differ depending on Tanner stages. In boys, HCC was significantly higher in Tanner stage 1 than in stages 2-5. CONCLUSION: Measuring cortisol concentration in hair gives information about long-term release of cortisol. We have found that puberty, gender, and BMI had a profound effect on HCC. As a result, further research should take into account the potentially confounding role of puberty, gender and BMI and may use the results of our study as a reference at determining values of HCC in healthy children.

Correction: Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

[This corrects the article DOI: 10.1371/journal.pgen.1008145.].

Advancement in steroid hormone analysis by LC-MS/MS in clinical routine diagnostics - A three year recap from serum cortisol to dried blood 17α-hydroxyprogesterone.

Steroid analysis by LC-MS/MS in daily clinical routine diagnostics requires high-throughput conditions including fast chromatographic separation. Hereby, signal interferences may occur due to limited specificity in complex biologic matrices. During the last three years of routine steroid analysis in our laboratory and roughly 50,000 measurements, about 1% was affected by interferences, mainly serum cortisol (>90%) and dried blood 17α-hydroxyprogesterone (17-OHP). To overcome specificity problems, enhanced chromatography, ionization polarity switching, and detection via two-stage fragmentation (MS3) using a quadrupole linear ion trap were investigated in our study. Signal interferences of serum cortisol were eliminated by applying a protocol for automated method switching without changing the basic high-throughput LC-MS/MS setup. This approach includes negative ionization and extended chromatography from 4 to 6.6 min using the fourfold column length. From 9 samples affected by cortisol interference using the high-throughput method, 8 could be reliably analyzed applying the method switching protocol. Moreover, the applicability of the high-throughput method as second tier analysis in congenital adrenal hyperplasia (CAH) diagnostics from dried blood was verified with 100% diagnostic specificity. In addition, the combination of fast LC and MS3 detection enables specific quantitation of 17-OHP from dried blood spots on a screening time scale. This approach may be an alternative to the newborn screening for CAH by immunoassay due to its higher specificity, reducing the number of false positive results by 90%. In this work we recap experiences from three years of clinical routine steroid analysis via LC-MS/MS and present a unique analytical setup that enables both high-throughput and enhanced resolution analysis of steroid hormones in serum and dried blood.

Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids (HCAR) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA1, HCA2) but humans and other hominids contain a third member (HCA3) in their genomes. A plausible hypothesis why HCA3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA3 in vitro and in vivo. The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA3. D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA3-dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.

Evaluation of hair cortisol and cortisone change during pregnancy and the association with self-reported depression, somatization, and stress symptoms.

Hair cortisol levels are used to measure long-term stress, while its inactive metabolite cortisone is often not assessed. We measured hair cortisol concentrations (HCC) and hair cortisone concentrations (HCNC) via liquid chromatography quadrupole linear ion trap mass spectrometry (LC-MS3) in 62 pregnant women who participated in the LIFE CHILD STUDY in their 2nd and 3rd trimester between 12/2011 and 11/2014. Sociodemographic factors, pregnancy-related factors, and hair characteristics were assessed. Degree of severity of depression, somatization, and stress were evaluated in both trimesters with a self-reported Patient Health Questionnaire (PHQ). Multivariate regression analyses were conducted between HCC and potential influencing factors, as well as with subscales of the PHQ, with HCNC and with the ratio of HCNC to HCC. Spearman correlation coefficients were calculated between steroid concentrations and subscale scores of the PHQ, as well as between the log2-fold change in HCC and HCNC and the change in PHQ subscale scores. HCC increased 1.3-fold and HCNC increased 1.5-fold by the 3rd trimester. HCNC was more than three times higher than HCC in both trimesters. We found significant associations of PHQ subscores with HCNC. The PHQ depression score was negatively correlated with HCNC in the 2nd trimester (p < .05). The PHQ stress score change was negatively correlated with the fold change of HCNC (p < .05) and with the change in the ratio of HCNC to HCC (p < .001). Our study suggests an association of cortisol/cortisone metabolism with self-reported stress in the 2nd and 3rd trimester of pregnancy. Since associations with PHQ subscores were only found with cortisone or the ratio of cortisone to cortisol, but not with cortisol alone, both cortisone and cortisol should be used as a marker for stress in pregnant woman.

Multicenter performance evaluation of a second generation cortisol assay.

BACKGROUND: Untreated disorders of the adrenocortical system, such as Cushing's or Addison's disease, can be fatal, and accurate quantification of a patient's cortisol levels is vital for diagnosis. The objective of this study was to assess the analytical performance of a new fully-automated Elecsys® Cortisol II assay (second generation) to measure cortisol levels in serum and saliva. METHODS: Four European investigational sites assessed the intermediate precision and reproducibility of the Cortisol II assay (Roche Diagnostics) under routine conditions. Method comparisons of the Cortisol II assay vs. liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard for cortisol measurement, were performed. Cortisol reference ranges from three US sites were determined using samples from self-reported healthy individuals. RESULTS: The coefficients of variation (CVs) for repeatability, intermediate precision, and reproducibility for serum samples were ≤2.6%, ≤5.8%, and ≤9.5%, respectively, and for saliva were ≤4.4% and ≤10.9%, and ≤11.4%, respectively. Agreement between the Cortisol II assay and LC-MS/MS in serum samples was close, with a slope of 1.02 and an intercept of 4.473 nmol/L. Reference range samples were collected from healthy individuals (n=300) and serum morning cortisol concentrations (5-95th percentile) were 166.1-507 nmol/L and afternoon concentrations were 73.8-291 nmol/L. Morning, afternoon, and midnight saliva concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively. CONCLUSIONS: The Cortisol II assay had good precision over the entire measuring range and had excellent agreement with LC-MS/MS. This test was found suitable for routine diagnostic application and will be valuable for the diagnosis of adrenocortical diseases.

Falsely elevated serum oestradiol due to exemestane therapy.

In this study, we present a case of falsely elevated oestradiol (E2) concentration, determined by two immunoassays, in a breast cancer patient receiving exemestane therapy. The positive bias of immunochemical measurements was revealed using liquid chromatography tandem mass spectrometry which showed undetectable E2 concentration. The discrepancy is expected to be a consequence of the structural resemblance of E2 and exemestane sharing the same steroidal backbone. Inaccurate laboratory findings in therapy monitoring, as in this case, may lead to unnecessary changes of therapy.

Liquid chromatography quadrupole linear ion trap mass spectrometry for quantitative steroid hormone analysis in plasma, urine, saliva and hair.

Steroid analysis is being conquered by liquid chromatography tandem mass spectrometry (LC-MS/MS) benefiting from higher standardization, selectivity and diversity. Regarding high throughput in routine diagnostics rapid chromatography is mandatory. Introducing MS(3) (MS/MS/MS), specificity of mass spectrometric detection can be enhanced without sacrificing analysis time. 100mL of human plasma/serum, saliva, urine and 10-20mg of hair are used for the simultaneous quantification of 17α-hydroxyprogesterone, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC-MS/MS or LC-MS(3). Steroids can be analyzed in 4min after a single manual dilution and protein precipitation step. In complex sample matrices like hair MS(3) detection was found to be appropriate for quantitation. Lower limits of quantitation ranged from 37pmol/L (estradiol) up to 3.1nmol/L (DHEAS). General accuracy was 89-107% with between-run imprecision ≤10%. Comparison to immunoassays revealed significant differences in quantitation for urinary cortisol (-71% mean), aldosterone (-40% mean) and plasma aldosterone (-45% mean). The comparison of MS(2) and MS(3) quantitation of hair cortisol also revealed significant differences. In general, quantitation via MS(3) was not applicable for a long time. But with the current generation of mass spectrometers quantitation via MS(3) can be superior to MS(2) regarding specificity and accuracy when dealing with matrix issues. However, drawbacks regarding flexibility and precision have to be taken into account. Concludingly, simple protein precipitation combined with rapid online SPE LC-MS/MS/MS allows us to quantify over broad, essential concentration ranges in human serum, saliva, urine and hair.

Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children?

BACKGROUND: Dysregulation of the adrenal cortex has been assessed with measurement of salivary cortisol. So far salivary cortisol is routinely measured with immunoassay (IA). However, liquid chromatography-tandem mass spectrometry (MS) is known to offer better specificity. We compared the concentrations of salivary cortisol measured by MS and IA at basal and stress induced conditions and evaluated reasons for the difference in method-dependent cortisol results. METHODS: Saliva samples (n=2703) were collected from 169 children (age range: 8-14 years; 81 healthy children; 55 with internalizing and 33 with externalizing disorders) under circadian conditions and during the Trier Social Stress Test for Children (TSST-C). Biochemical analyses were performed with MS for cortisol and cortisone, IA (IBL, RE62011) for cortisol, and enzyme kinetic assay for α-amylase. RESULTS: MS and IA showed mostly comparable results for circadian activity and TSST-C response with similar statistical power. However, IA measured cortisol concentrations about 2.39-fold higher than MS. We found that this difference in measured values between MS and IA was mainly due to different standardization of IA compared to MS. In addition, at cortisol IA concentration below 5 nmol/L, cross-reactivity with cortisone was found to contribute to the lower concordance between MS and IA. CONCLUSIONS: Immunoassay and LC-MS/MS were largely comparable in the interpretation of salivary cortisol dynamics in stress research. But the IA method revealed a restricted accuracy in the measuring range below 5 nmol/L.

The hyporeactivity of salivary cortisol at stress test (TSST-C) in children with internalizing or externalizing disorders is contrastively associated with α-amylase.

BACKGROUND: Stress biomarkers of the autonomic nervous system and hypothalamic-pituitary-adrenal axis (HPA-axis) can be measured via alpha-amylase (AA) and cortisol and cortisone in saliva. Objectives were to determine 1) the response patterns of cortisol, cortisone, and AA under both circadian conditions and the Trier Social Stress Test for Children (TSST-C), 2) which reactivity index is most suitable to differentiate internalizing or externalizing disorders from controls, and to explore 3) the interaction between AA and cortisol in the presence of internalizing or externalizing disorders. METHODS: Saliva samples (n = 2893) from children with internalizing (n = 55) or externalizing disorders (n = 33) and healthy children (n = 81) were analyzed for cortisol, cortisone, and AA under circadian conditions and TSST-C. RESULTS: Circadian rhythm of three biomarkers did not differ between diagnostic groups. Age and gender were significant predictors for cortisol and awakening time influenced all three biomarkers significantly. TSST-C responses appeared sequentially in the order of AA, cortisol, and cortisone. Trajectories of cortisol and cortisone responses, not in AA, were significantly lower in children with internalizing or externalizing disorders than in healthy children. Cortisol percentage increase appeared to be the most suitable reactivity index to detect the difference between the diagnostic groups. Internalizing disorders had a negative association between AA decrease and cortisol increase (ß = -.199, p < .05, R(2) = .304). Externalizing disorders had a positive association between AA baseline and cortisol increase (ß = .229, p < .05, R(2) = .304). CONCLUSION: An altered HPA-axis response during stress might result from chronic allostatic load in internalizing disorders and underaroused stress response system in externalizing disorders.

Multi-class method for biomonitoring of hair samples using gas chromatography-mass spectrometry.

Currently, non-invasive biomonitoring of human exposure to organic pollutants bases upon the analysis mainly of urine and human breast milk. While mostly persistent organic pollutants are the center of interest, the aim of our study was to develop a method for the determination of different chemical classes of emerging pollutants (organophosphorus flame retardants, plastic additives such as phthalates, bisphenol A, insecticides, antimicrobials, preservatives and musk fragrances) in hair by gas chromatography-mass spectrometry. The preferred sample preparation included hydrolysis of the hair with trifluoroacetic acid in methanol followed by a liquid-liquid extraction using hexane/ethyl acetate. The validated method is characterized by recoveries higher than 77 % for most analytes, relative standard deviations below 16 % and limits of detection between 2 pg mg(-1) (HHCB) and 292 pg mg(-1) (propylparaben) using 50 mg of dry hair. After respective blank corrections, bis-(2-ethylhexyl)phthalate (DEHP) and the musk fragrance HHCB were the predominant compounds determined in all hair samples at concentrations between 32 and 59 ng mg(-1) and 0.8-13 ng mg(-1), respectively. The bactericide triclosan and the insect repellent N,N-diethyl-3-methylbenzamide (DEET) were detected in selected hair samples at 2 and 0.8 ng mg(-1), respectively.