Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Abstract

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability, and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1×100 mm, 3.5 μm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 μm, 125 Å). Quantitation was done using the SRM transitions m/z 233→174 and m/z 219→160 for MEL and NAS, respectively. The analytical response ratio vs. concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL).

Development of a Fully-automated On-line Oxidation Column-switching HPLC System for the Determination of Endogenous Melatonin in Human Clinical Samples

“…In these methods, a high concentration of organic solvents, such as 20 -50% of MeOH or MeCN, was required as the mobile phase. [25][26][27]29,30 However, melatonin could not be oxidized in these mobile phases containing a high concentration of organic solvents.…”

“…Until now, various analytical methods, such as enzyme immunoassay, [18][19][20][21]28 GC-MS [22][23][24] and HPLC methods, [25][26][27]29,30 have been reported for the determination of melatonin. Enzymatic methods (both RIA and ELISA) are frequently used, and various assay kits are commercially available.…”

“…GC-MS methods with N-trimethylsilyl or pentafluoropropionic derivatization are sensitive, [22][23][24] however, both derivatization procedures require skillful techniques. Concerning HPLC, various methods combining an electrochemical detector, 25 a fluorescence detector 26 or MS 27,29,30 have been reported. However, the limit of detection of melatonin using these analytical methods are around 5 fmol/injection, and a 1 -5 mL blood sample is thus needed for the determination of melatonin.…”

“…However, the amount of endogenous melatonin is extremely low, and a 1 -5 mL blood sample is needed for the determination of melatonin by the methods already reported. [17][18][19][20][21][22][23][24][25][26][27] To obtain 1 -5 mL of blood, blood sampling from the cubital vein is normally required, and it is practically a severe task for patients to measure the circadian rhythm of melatonin (blood should be obtained 6 -8 times within one day). Therefore, a highly sensitive and selective analytical method, which can determine endogenous melatonin using 10 -100 μL of plasma (100 μL of plasma can be obtained by a less invasive method) is required.…”

“…Radioimmunoassay (RIA), [18][19][20] enzyme-linked immunosorbent assay (ELISA), 21,28 gas chromatography (GC) [22][23][24] and high-performance liquid chromatography (HPLC) [25][26][27]29,30 are frequently used. The melatonin rhythm in human plasma/serum and saliva has also been studied using these methods, and the amounts of melatonin are reported to be 3 to 10 pM in the diurnal period.…”