title: Determination of homocysteine in human saliva by liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry: profiles in healthy adults.
abstract: Homocysteine (Hcys) is a non-essential amino acid associated with a range of diseased and abnormal metabolic conditions. Hcys concentration in saliva is routinely determined by enzyme assays, which are broadly specific, but can be expensive and suffer from cross-reactivity. Total Hcys (tHcys) concentrations in eight healthy adults were determined to establish the inter-day variation during resting, normal and intensive physical activity, using the more sensitive analytical techniques of liquid chromatography and tandem mass spectrometry without prior derivatization. Saliva (~ 1.5 mL) was collected over four days; early morning (EA), normal activity (NA) and during physical activity (PA). Samples were processed by disulphide reduction, acetonitrile precipitation and then centrifugation-filtration. Extracts were chromatographically resolved and analysed on a quadrupole time- of-flight (QToF) mass spectrometer. The protonated [M+H]+, m/z 136.101 and product ([M+H]+- HCOOH) m/z 90.103 ions were then monitored against an internal standard (13CHcys) and external set of calibration standards. Mean tHcys concentration for the whole group, including exercise was 6.6 ± 8.0 (range 0.2 - 29.6 nmol/mL). Overall, concentration of tHcys was greater in males than the females but not significantly (p > 0.05). The mean EA concentration was significantly (p < 0.05) greater than NA for both males (p = 0340) and females (p = 0.0045). There were large within-subject variations (coefficient of variation; CV%; 24% to 103%). The limits of detection (LOD) and quantification (LOQ) were 0.07 and 0.22 nmol/mL, respectively. The procedure potentially provides a convenient means of analyzing salivary Hcys as a diagnostic disease marker.
authors.affiliation: Department of Pharmaceutical, Chemical and Environmental Sciences, Faculty of Engineering and Science, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, UK. J.S.Boateng@gre.ac.uk.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Abaye
authors.forename: Birthe
authors.initials: B
authors.lastname: Nielsen
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Boateng
mesh_terms: D000097:Acetonitriles; D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006710:Homocysteine / Q000032:analysis*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D055815:Young Adult
chemical_list: D000097:Acetonitriles; D006710:Homocysteine; C032159:acetonitrile