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A HPLC Method for Determination of Total Plasma Homocysteine with Rapid Sample Preparation
Authors: Žáková Pavla | Kanďár Roman | Skalický Jiří | Čegan Alexander | Ventura Karel
Year: 2003
Type of publication: ostatní - přednáška nebo poster
Name of source: Clinical Chemistry and Laboratory Medicine Special Supplement
Publisher name: Walter de Gruyter GmbH & Co. KG
Place: Berlin
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Titles:
Language Name Abstract Keywords
cze HPLC metoda s rychlou přípravou vzorku pro stanovení celkového homocysteinu v plazmě homocysteine, HPLC, electrochemical detection, cardiovascular and vascular occlusive diseases, immunochemical method
eng A HPLC Method for Determination of Total Plasma Homocysteine with Rapid Sample Preparation Introduction. Several clinical studies have indicated that high plasma homocysteine concentration is an independent risk factor for cardiovascular and vascular occlusive diseases. In the clinical laboratory homocysteine is measured by a variety of techniques, including immunochemical and HPLC methods. Aim. To develop of HPLC method without pre- or postcolumn derivatization. Method. We reduced of EDTA plasma with 1,4-dithioerytritol containing internal standard penicillamine. After incubation, protein precipitation was achieved by addition of sulfosalicylic acid. Supernatant was injected onto 80 x 4.6 mm RP-C18 column (HR-80, particle size, 3 microm; ESA inc.). The mobile phase consisted of sodium dihydrogen phosphate adjusted to optimal pH with orthophosphoric acid, octane sulfonate and acetonitrile. We detected reduced homocysteine with ESA Coulochem II electrochemical detector, using coulometric cells. Results. Analytical performance of this method is satisfactory with intra-assay CV 2.8% and 97.0% recovery. The sensitivity of the method at 95% confidence limit is 0.5 microM. The linearity was excellent in the range 0.5 - 60.0 microM. Comparison of this method with immunoassay by linear regression shows that results obtained by HPLC-ED are higher: HPLC-ED = 0.9236 (immunoassay) + 0.97 microM, R2 = 0.9387. Conclusions. Our results suggest that this HPLC-ED method is inexpensive to immunochemical methods and suitable from the measurement of total homocysteine in clinical samples. This work was supported by grants GACR 203/02/0023 and MSMT 253 100 002. homocysteine, HPLC, electrochemical detection, cardiovascular and vascular occlusive diseases, immunochemical method

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