Effects of saliva collection using cotton swabs on melatonin enzyme immunoassay

Nine healthy males were recruited and each provided four saliva samples on a single day to yield a total of 36 samples. Saliva samples were directly collected in plastic tubes using plastic straws, and subsequently pipetted onto cotton swabs (cotton saliva collection) and into clear sterile tubes (passive saliva collection). The melatonin levels were analyzed in duplicate using commercially available ELISA kits.The mean melatonin concentration in cotton saliva collection samples was significantly lower than that in passive saliva collection samples at higher melatonin level (>6 pg/mL). The Bland-Altman plot indicated that cotton swabs causes relative and proportional biases in the assay results. For lower melatonin level (<6 pg/mL), although the BA plots didn't show proportional and relative biases, there was no significant correlation between passive and cotton saliva collection samples.Our findings indicate an interference effect of cotton swabs on the assay result of salivary melatonin at lower melatonin level. Cotton-based collection devices might, thus, not be suitable for assessment of DLMO.Melatonin, produced by the pineal gland [1,2], has often been assessed for determination of human circadian phase as dim light melatonin onset (DLMO) [3-6]. Assessing salivary melatonin has recently been used as an alternative method for blood analysis because the level of salivary melatonin is correlated with that of blood melatonin [7]. Furthermore, collecting salivary samples is less intrusive and easier for participants than collecting urine and blood samples.Although various acceptable and easy-to-use devices have been used for saliva collection, cotton swabs are among the most common. However, previous studies have reported that cotton swabs yield low levels of melatonin [8,9]. In these studies, saliva samples were collected by spitting into a clear bottle (passive saliva collection), and exogenous melatonin was artificially added to the samples and loaded on cotton s