Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva.

A growing body of literature describes the potential effects of circadian disruption on human health. Indeed, psychiatric diseases, metabolic syndrome, and cancers may be linked to disturbance of the circadian rhythm. Currently, the best practice to assess circadian rhythm is the measurement of melatonin levels. Our goal was thus to develop and validate a highly sensitive LC-MS/MS method to follow salivary melatonin levels throughout the day and night. Our method reached a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. To our knowledge, it is the most sensitive method allowing quantitation of melatonin in saliva. Saliva, obtained from passive drooling or salivette, was extracted by an efficient and quick liquid-liquid extraction with no further cleanup needed. The method was validated according to the European Medicines Agency (EMA) guidelines and provided excellent results regarding accuracy, precision, linearity, selectivity, and specificity. Comparison between radioimmunoassay and our method was performed and showed differences at low levels, most likely due to cross-reactivity with other indols. To assess daytime melatonin levels in humans, salivary melatonin levels of ten volunteers were monitored throughout the day and showed lower daytime levels than reported in previous studies.

Daytime rest: Association with 24-h rest-activity cycles, circadian timing and cognition in older adults.

Growing epidemiological evidence points toward an association between fragmented 24-h rest-activity cycles and cognition in the aged. Alterations in the circadian timing system might at least partially account for these observations. Here, we tested whether daytime rest (DTR) is associated with changes in concomitant 24-h rest probability profiles, circadian timing and neurobehavioural outcomes in healthy older adults. Sixty-three individuals (59-82 years) underwent field actigraphy monitoring, in-lab dim light melatonin onset assessment and an extensive cognitive test battery. Actimetry recordings were used to measure DTR frequency, duration and timing and to extract 24-h rest probability profiles. As expected, increasing DTR frequency was associated not only with higher rest probabilities during the day, but also with lower rest probabilities during the night, suggesting more fragmented night-time rest. Higher DTR frequency was also associated with lower episodic memory performance. Moreover, later DTR timing went along with an advanced circadian phase as well as with an altered phase angle of entrainment between the rest-activity cycle and circadian phase. Our results suggest that different DTR characteristics, as reflective indices of wake fragmentation, are not only underlined by functional consequences on cognition, but also by circadian alteration in the aged.

Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples.

OBJECTIVES: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. METHODS: 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2). RESULTS: In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. CONCLUSIONS: This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Development and validation of a fast and reliable method for the quantification of glucagon by liquid chromatography and tandem mass spectrometry.

INTRODUCTION: The quantitation of glucagon remains a challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work. METHODS: Glucagon was extracted from plasma employing a mixed-mode anion exchange solid-phase extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories. RESULTS: Assay imprecision was below 4%. Recoveries were within 95-103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4 °C for less than 4 h. Diasource® RIA disagreed with our method. Mercodia® ELISA provided a closer agreement, also proven by external quality control samples. CONCLUSIONS: A rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon.

Evaluation of hazardous chemicals in edible insects and insect-based food intended for human consumption.

Due to the rapid increase in world population, the waste of food and resources, and non-sustainable food production practices, the use of alternative food sources is currently strongly promoted. In this perspective, insects may represent a valuable alternative to main animal food sources due to their nutritional value and sustainable production. However, edible insects may be perceived as an unappealing food source and are indeed rarely consumed in developed countries. The food safety of edible insects can thus contribute to the process of acceptance of insects as an alternative food source, changing the perception of developed countries regarding entomophagy. In the present study, the levels of organic contaminants (i.e. flame retardants, PCBs, DDT, dioxin compounds, pesticides) and metals (As, Cd, Co, Cr, Cu, Ni, Pb, Sn, Zn) were investigated in composite samples of several species of edible insects (greater wax moth, migratory locust, mealworm beetle, buffalo worm) and four insect-based food items currently commercialized in Belgium. The organic chemical mass fractions were relatively low (PCBs: 27-2065 pg/g ww; OCPs: 46-368 pg/g ww; BFRs: up to 36 pg/g ww; PFRs 783-23800 pg/g ww; dioxin compounds: up to 0.25 pg WHO-TEQ/g ww) and were generally lower than those measured in common animal products. The untargeted screening analysis revealed the presence of vinyltoluene, tributylphosphate (present in 75% of the samples), and pirimiphos-methyl (identified in 50% of the samples). The levels of Cu and Zn in insects were similar to those measured in meat and fish in other studies, whereas As, Co, Cr, Pb, Sn levels were relatively low in all samples (<0.03 mg/kg ww). Our results support the possibility to consume these insect species with no additional hazards in comparison to the more commonly consumed animal products.

Ultra-trace measurement of Dechloranes to investigate food as a route of human exposure.

Dechloranes, including Dechlorane Plus (syn- and anti-isomers), Dechlorane 602, Dechlorane 603, Dechlorane 604, Chlordene Plus, and Mirex are used as flame-retardants and were recently found in human serum of the European population. In order to investigate if food consumption would possibly be a significant route of exposure, we developed a method for the measurement of Dechloranes in food and feed. We showed that it was possible to extend the scope of the regular polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), dioxin like (DL-), and non-dioxin like (NDL-) regulated PCBs clean-up and fractionation procedure to Dechloranes and that no compound degradation occurred during the strong acidic treatments used for lipid digestion. Dechloranes were measured by gas chromatography coupled to triple quadrupole mass spectrometry (GC-QQQMS/MS). We optimized injection parameters by face centered experimental design (FCD). The electron ionization fragmentation was investigated to set appropriate multiple reaction monitoring (MRM) transitions. Instrumental and method limits of quantitation (iLOQs and mLOQs) were determined following EU guidelines for dioxin analyses in food. A total of 88 samples were analyzed to assess the prevalence of this route of exposure to humans. Average levels of the sum of Dechloranes ranged from 10 to 31pg/g fat, with the exception of fish, feed additives, and corn that were reported in pg/g wet weight at average levels of 9, 12, and 2pg/g ww. Based on Belgian food habits, a dietary intake was estimated to be 136pg/day. The relatively low reported levels indicate that other routes of human exposure should be considered.