Homocysteine

KR120 HOMOCYSTEINE FOR HPLC

INTENDED USE

Reagent kit for the hplc determination of homocysteine in plasma/serum.

KIT SPECIFICATION

CONTENSTS FOR 100 ANALYSES:

INTRODUCTION

Cardiovascular diseases and stroke are the most common diseases resulting from atherosclerosis. the common risk factors are well knowm: overweight,blood high pressure, diabetes mellitus, smoking and disordes in fat metabolism or hingh blood cholesterol, respectively. however at each second heart attack patient these factors are in the normal range, and also at many patients that suffer from a stroke. Consequently there must be additional factors that cause atheroscleosis.

For a long time homocysteine was discussed to be the responsible substance for vascular diseases. Since that time a variety of epidemiologic studies were conducted to show the suitability of homocysteine as an idependent risk factor for ayherosclerosis. To day, an enhancedd homocysteine level in plasma (hyperhomocysteinemia)is related to an increased risk of atherosclerotic affections. Currently a plasma homocysteine can be decreased by of folic aci or vitamin b12. Reduction in homocysteine levels may be achieved by proper diet and vitamin supplementation (folic acid,vitamin b6,vitaminb12) therapy control of patients with hyperhomocyteinemia has to be perforned by measurement of homocysteine,and additionally,by monitoring of vitamin b6 and b12.

EQUIPMENT AND INSTRUMENTAL PARAMETERS

The analysis of homocysteine in serum/plasma requires  a hplc including isocratic system with hplc pump, autosampler, autoinjectorand fluorescence etector. The use of a solvent degasser prevents air bubbles and ensures a stable baseline.

Injection volüme : 25µl

Analytical run time : 6.5 min

Flow rate :  1.2 ml/min

Column temperature :   romm temperature(approx.25°c)

Fluorescence detector  : EX : 385 nm

                                 : EM :  515 NM

COLLECTION AND STORAGE OF PATIENT SPECIMENS 

Homocysteine is determined from plasma and serum. Storage life is up to 4 days at temperature,at+2to+8 oc up to 4 week and deep-freeze (-20 oc)for longer storage (up to a maximum 1 year.)

RECONSTITUTION OF THE SERUM CALIBRATOR

The prepared calibration standard is used to calibrate the system. To reconstıtute the lyophilized  serum calibration standadrt pıpette exactly 1.0 ml ultrapure water into the vial. Let the vial stand at room temperature for about 10-15 min shake until the vial contents are homogeneous. Before sampling allow the control to reach romm temperature (18 to 25 oc) and  swirl gently to ensure homogeneity return the control to storage at 2 to 8 oc ımmediately after use.

SAMPLE PREPARATION PROCEDURE 

-Pipette 100 µl serum/plasma (calıbrators, controls, specımen) 25µl internal standard and reagent a (reductıon reagent) a into a labelled light-protected reactıon vial mix briefly.

-Sample vortex-mix for 5 s.

-Add 100 µlreagent b (precipitatıon reagent )vortex-mix for 5s.

-Exactly ıncubate for 20 min at +60 oc.

-Refrigerate for 5 min +2-8 Oc.

-Inject 25 µl of the ınto the HPLC system.

RECOVERY

the analytical recovery was determined from the slope of the calibrations curves of spıked serum samples and diluted standard solutıons:

homocysteine:92%

LINEARITY

the method is line from the designated limit of quantification up to at least the stated upper limit.