A highly sensitive assay of melatonin at the femtogram level in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry

Abstract

A highly sensitive and specific assay was developed for routine analysis of melatonin at the femotogram level in human plasma. Melatonin and the deuterated internal standard [(2H4)melatonin] were measured by gas chromatography/negative ion chemical ionization mass spectrometry with methane as the reagent gas. A simple liquid-liquid extraction procedure was used to isolate the two compounds of interest from the complex biological matrix. Melatonin was converted to the fluorinated derivative with pentafluoropropionic anhydride. The mass spectrometer was tuned to monitor the very intense and stable ions at m/z 320 and 323 which were generated into the ion source by a dissociative capture process. This assay was conducted with 1 ml of plasma and the quantification limit of the method was statistically calculated as 0.5 pg ml-1. The very low relative standard deviations and mean percentages of error calculated during the within-day or between-day repeatability assays have clearly demonstrated the ruggedness of the technique for the routine quantitative measurement of melatonin in plasma. Some results on the melatonin circadian rhythm are presented to illustrate the applicability of this powerful gas chromatographic/mass spectrometric method.

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

“…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.…”

“…However, enzymatic immunoassays are widely known to show crossreactions or non-specific bindings, [17][18][19][20][21]28 especially for the determination of trace amounts of an analyte like melatonin. 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 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.…”

Quantitative determination of melatonin in milk by LC-MS/MS

“…There are many methods reported for quantification of melatonin with different kind of matrix like plasma, saliva, pineal gland and biological samples by GC-MS Chemical ionisation (Fourtillan et al 1994), LC-MS/MS (Eriksson et al 2003;Cao et al 2006), GC-ECD (Greiner and Chan 1978), HPLC-FLD (Rizzo et al 2002;Vitale et al 1996;Iinuma et al 1999;Munoz et al 2009;Lizuka et al 2007;Itoh et al 1997), HPLC-Electrochemical detector (Chanut et al 1998) and HPLC chemiluminesence (Lu et al 2002). Melatonin in human milk was reported by RIA and the method was validated by GC-MS (Illnerova et al 1993).…”