BACKGROUND: Determination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine. METHODS: As a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP(18) cartridge; mobile phase: 0.1 mol/l KH(2)PO(4) with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min. RESULTS: Homocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 micro mol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants. CONCLUSIONS: The described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier.
BACKGROUND: Determination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine. METHODS: As a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP(18) cartridge; mobile phase: 0.1 mol/l KH(2)PO(4) with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min. RESULTS:Homocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 micro mol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants. CONCLUSIONS: The described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier.
Authors: Iori Ueki; Heather B Roman; Lawrence L Hirschberger; Carolyn Junior; Martha H Stipanuk Journal: Am J Physiol Endocrinol Metab Date: 2012-03-13 Impact factor: 4.310
Authors: Jodi L Brewster; Petr Pachl; James L O McKellar; Maria Selmer; Christopher J Squire; Wayne M Patrick Journal: J Biol Chem Date: 2021-05-18 Impact factor: 5.157
Authors: Marcus J Miller; Adam D Kennedy; Andrea D Eckhart; Lindsay C Burrage; Jacob E Wulff; Luke A D Miller; Michael V Milburn; John A Ryals; Arthur L Beaudet; Qin Sun; V Reid Sutton; Sarah H Elsea Journal: J Inherit Metab Dis Date: 2015-04-15 Impact factor: 4.982