Determination of Homocysteine in Human Saliva by Liquid Chromatography and Electrospray Ionization Quadrupole Time-of-flight Mass Spectrometry: Profiles in Healthy Adults
Daniel A. Abaye,
Joshua S. Boateng.
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.
Keywords: Acetonitrile precipitation, healthy adults, Homocysteine, liquid chromatography mass spectrometry, quadrupole
time-of- flight, saliva.
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