“Melatonin” (N-acetyl-methoxytryptamine) is secreted from a part of the brain called suprakiasmic nuclui. This hormone has the highest level of secretion at night and the lowest during the day. The biological clock of the body or the circadian rhythm is regulated through the secretion of hormones such as melatonin and cortisol. Disturbance in circadian rhythm is specifically connected with shift working jobs. Melatonin is known as a good bio-marker to show circadian rhythm irregularities. So, in most studies, it is used to determine the degree of disturbance in circadian rhythm due to the shift working jobs in occupational health. Therefore, trace analysis of such bio-marker in shift workers is highly important. Through this study, the dispersive liquid–liquid micro-extraction (DLLME) was optimised for the analysis of human salivary melatonin. Different variables including extracting solvent, dispersing solvent, volume of extracting solvent, volume of dispersing solvent, sample pH, sample ionic strength, extraction time, and centrifugation time were screened using one factor at a time design (OFAT) and then, the significant variables were selected as optimum values. Optimum conditions were: extracting solvent (CCl4) 200 ml, dispersing solvent (AcN) 2 ml, sample pH 7.00, sample ionic strength 5%, extraction time 3 min, and centrifugation time at 4500 rpm 6 min. To validate achieved procedure, accuracy and precision of the optimised method was evaluated at three concentrations of 50, 100, and 250 pg.ml-1. Based on this evaluation, CV% of 6.29, 2.39, and 1.82 were achieved respectively for day-to-day reproducibility as well as CV% of 4.49, 2.68, and 1.83 at the same three concentrations respectively for within-day reproducibility. Considering the appropriate results of the study, dispersing liquid-liquid micro-extraction procedure (DLLME) can be used for preparation of trace melatonin hormone in saliva samples as a bio-marker of circadian rhythm of shift workers in field of occupational health.
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