Determination of homocysteine in human saliva by liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry: Profiles in healthy adults

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. © 2013 bentham science publishers.