25-OH-Vitamin D3/D2

25-OH-VİTAMİN D3/D2 FOR HPLC

INTENDED USE

KR102 reagent kit for the hplc detemination of 25-oh-vitamin3/d2 in plasma/serum.

KIT SPECIFICATION

CONTENTS FOR 100 ANALYSES:

INTRODUCTION

the term “vitamin d” means a group of steroids that possess antırachitic activity. Clinically relevant members of this group are vitamin d2 (ergocalciferol)and vitamin d3(cholecalciferol). At sufficient sun light (uv-b),vitamin d3 can be produced by the skin and therefore does not meet the classical definition of a vitamin. Vitamin d plays an important role for the incorporationof calcium into the bone matrix. Vitamin d deficiencies (hypovitaminosisd) therefore interfere with bone mineralisation. The deficiency leads to rickets in children and osteomalacıa in adults. The cutaneous bıosynthesis of vitamin d3 is the most important source of vitamin d.dietary intake becomes critical when exposure to sunlight is limited,due to the reason that only a few foods naturally contain adequate or high amounts on vitamin d,for example sea fish and cod liver oil.

Vitamin D obtained by nutrition (D2 and D3) or endogenously (d3) formed, is metabolised in the liver into 25-oh-vitamin d. By further hydroxylatıon ın the kıdney, the bıologically active form of the vitamin ,1,36-(oh)2 vitamin d, is formed for the assessment of the vitamin d status however, the concentration of the primary metabolites, 25-oh-vitamin d,is used. The main reason for this is a long metabolite half-life (3 weeks),that gives an indication of vitamin d stores over long periods. Also, the plasma/serum sonsentrations of 25-oh-vitamin d are approximately 1000-fold larger than for 1,25-(oh)2 vitamin d.

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EQUIPMENT AND PARAMETERS

the analysis of 25-oh-vitamin d3/d2 in serum/plasma requires a hplc including isocratic system with hplc pump, autosampler, autoinjector and uv detector. the use of a solvent degasser prevents air bubles and ensures a stable baseline.

Injection volume:25 µl

Analytical run tıme:12 min

Flow rate:0.7 ml/min

Column temperature:ambident (approx.25 °c)

uv detector:265 nm

COLLECTION AND STORAGE OF PATIENT SPECIMENS

25-Oh-Vitamin D İs Determined From Plasma And Serum. Storage Life İs Up To 3 Days At Ambident Temperature,At +2to+8°C Up To 1 Week And Deep-Freeze (-18 °C) For Longer Storage (Up To A Maximum 1 Year)

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RECONSTITUTION OF THE SERUM CALIBRATOR

The Prepared Calibratıon Standard İs Used To Calibrate The System. To Reconstitute The Lyophilized Serum Calibration Standard,Pipette Exactly 2.0 Ml Ultrapure Water İnto The Vial.Let The Vial Stand At Room Temperature For About 10-15 Min Shake Until The Vial Contents Are Homogeneous.

Storage Life Of The Reconstituted Calibration Standard:

the reconstituted calibration standard is stable 2 weeks when kept light protected and stored at 2-8 °c.lf the calibrators cannot be analyzed within this period, they should be aliquoted and stored frozen below -18°c at least 3 months.

SAMPLE PREPARATION PROCEDURE

• pipette 500 µl serum/plasma(calibrators,controls,specimen)and 50µl internal standard into a labelled ,light-protected reation vial,mix briefly.
• add 500µl precıpıtatıon reagent vortex-mix for 20 s.
• incubate for 10 min at +4 °c.
• centrifuge 5 min at 13000 rpm.
• ımmediately apply the complete supernatant to a labelled sample clean up column and draw through by centrifugation 1500 rpm about 2 min,discard effluent.
• draw 2x 1 ml wash buffer ı through column by centrifugation 1500 rpm about 2 min,discard effluent.
• draw 75 µl wash buffer ıı through column by centrifugation 1500 rpm about 2 min ,discard effluent.
• change collection vial,apply 200 µl elution buffer to the columni draw through completely by centrifugation 1500 rpm about 2 min the eluates must be collected into glass vials.
• dilute the eluate with 20 µl ultrapure water and mix.
• inject 25 µl of the eluate into the hplc system.
• the eluates/prepared samples can be kep for 1 week at room temperature or refrigerated (+2-8°c) for approx.2 weeks (in glass vials and light protected)for longer periods of storage deep-freeze (-18°c) the sample.

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RECOVERY

The analytical recovery was determined from the slope of the calibrations curves of spiked serum samples and diluted standard solutions:

25-OH-VİTAMİN D3 :88%

2-OH-VİTAMİN D2:89%

INTERNAL STANDARD:86&