Vitamin A-E Serum HPLC Analysis Kit

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1 Vitamin A-E Serum HPLC Analysis Kit ZV C ZIVAK TEKNOLOJI SANAYI VE TICARET LIMITED SIRKETI Gursel Mh. Eski Besiktas Cd. No:44/1-2 KAGITHANE-ISTANBUL-TURKEY (212) FAX: +90 (212)

2 INDEX Table of Contents Page 1. INTENDED USE SUMMARY AND EXPLANATION TEST PRINCIPLE WARNING AND PRECAUTIONS STORAGE AND STABILITY MATERIALS SUPPLIED: MATERIALS REQUIRED BUT NOT SUPPLIED PROCEDURE NOTES LIMITATIONS OF THE PROCEDURE PRE-TEST SET UP INSTRUCTIONS TEST PROCEDURE QUALITY CONTROL CALCULATION OF RESULTS INTERPRETATION OF RESULTS EXPECTED VALUES HPLC-ECD PARAMETERS ANALYTICAL PERFORMANCE SAMPLE CHROMATOGRAMS... 8 Page 2 / 8

3 1. INTENDED USE: Quantitative HPLC analysis kit for Vitamin A and Vitamin E in human serum &plasma samples. 2. SUMMARY AND EXPLANATION: The level of vitamin A in the plasma or serum is a reflection of the quantities of vitamin A and carotene ingested and absorbed by the intestine. Vitamin A plays an essential role in the function of the retina (adaptation to dim light), is necessary for growth and differentiation of epithelial tissue, and is required for growth of bone, reproduction, and embryonic development. Together with certain carotenoids, vitamin A enhances immune function, reducing the consequences of some infectious diseases. Vitamin A in excess can be toxic. Vitamin E contributes to the normal maintenance of biomembranes, the vascular system, and the nervous systems; and provides antioxidant protection for vitamin A. Deficiency of vitamin E in children leads to reversible motor and sensory neuropathies; this problem also has been suspected in adults. Deficiencies of vitamins A and E may arise from poor nutrition or from intestinal malabsorption. People, especially children, at risk include those with bowel disease, pancreatic disease, chronic cholestasis, celiac disease, cystic fibrosis, and intestinal lymphangiectasia. Vitamin A-E HPLC Analysis Kit was developed for rapid, sensitive and reliable quantitative detection of the Vitamin A-E concentrations in human serum samples and it gives results in 8 minutes with minimal sample preparation. Main methods and procedures that we selected are based on EN ISO and 98/79/EC. 3. TEST PRINCIPLE Vitamin A and vitamin E are extracted from human serum samples using an organic extraction solution after deproteinization step. Separation was made with a isocratic elution on Zivak Vitamin A-E HPLC column and measured by HPLC-UV system. Accuracy and reliability of the analysis could be checked by the serum controls which are supplied with the kit. 4. WARNING AND PRECAUTIONS For in-vitro diagnostic use only. For professional use only. Read the instructions carefully,before you start. In case of damage of the kit package, please contact Zivak or your supplier. Do not use expired kits and components. Please check the batch no and expiry date before start. Although the concentration of all toxic components is low, for safety reasons the sample preparation station should be placed in an exhaust hood. Protective gloves and goggles should be worn. Please take any necessary precautions to prevent infection with blood borne pathogens while working with biological fluids. Appropriate bio-safety precautions and disposal of bio-hazardous wastes should be followed. Please check the labels on reagent bottles. Reagents of this kit contain hazardous material may cause eye and skin irritations. Page 3 / 8

4 5. STORAGE AND STABILITY This analysis kit can be shipped at room temperature. Reagent 1, MP, WB should be stored at room temperature. Calibrator and controls should be stored at 2-8 C. After arrival, reagent 2, Calibrator and controls (after reconstitution) must be stored at -20 C. All the other components of the kit should be stored at room temperature. Please make sure that you keep reagent 1 at room temperature otherwise it will become unusable. All components are guaranteed until expiry date when stored at recommended temperatures and used as described in these instructions. 6. MATERIALS SUPPLIED ZV Volume Symbol Component ZV S1-10 1x 5ml Calibrator Level 1 Lyophilized, Serum Calibrator Level-1 ZV K1-10 1x 5ml Control Level 1 Lyophilized, Serum Control Level-1 ZV K2-10 1x 5ml Control Level 2 Lyophilized, Serum Control Level-2 ZV R1-10 1x 50ml R1 Reagent 1, Contains aqueous buffer ZV R2-10 1x 40ml R2 Reagent 2, Contains deuterated internal standart ZV WB-10 1x 0,5 L WB Washing Solution ZV MP-10 1x 2,5 L MP Mobile Phase, Contains organic solvent ZV-4013-KK-Eng 1x 1pc User Guide 7. MATERIALS REQUIRED BUT NOT SUPPLIED Zivak Vitamin A-E Serum HPLC Analytical Column µl pipette µl pipette Pipette tips Vortex mixer 2,0 ml eppendorf tube Eppendorf tube centrifuge Bidistilled or deionised water 0,5 ml conical autosampler vial or vial insert Page 4 / 8

5 8. PROCEDURE NOTES Any inappopriate handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Leave aside all reagents and specimens to reach room temperature (18-25 C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each reagent, standard or specimen. Do not interchange the vial caps. Always keep vials closed when not been used. Do not re-use wells/tubes or reagents. Incubation time affects results. All tubes or wells should be handled in the same order and time sequences. 9. LIMITATIONS OF THE PROCEDURE Specimen collection and storage have a significant effect on the test results. Serum samples from earlier days must be kept frozen before the analysis. 10. PRE-TEST SETUP INSTRUCTIONS Preparation of Calibrators: Add 5 ml deionized water to serum calibrator and dissolve. After preparation, calibrator must be aliquoted into 250 µl volumes and stored at -20 C. Preparation of Control Level I: Add 5 ml deionized water to Serum Control Level I and dissolve. After preparation, Control Level I must be aliquoted into 250 µl volumes and stored at -20 C. Preparation of Control Level II: Add 5 ml deionized water to Serum Control Level II and dissolve. After preparation, Control Level II must be aliquoted into 250 µl volumes and stored at -20 C. Page 5 / 8

6 11. TEST PROCEDURE 1. Take 200 L of patient sample (or calibrators and controls) into the sample preparation tube. 2. Add 250 L Reagent 1 and vortex for 10 seconds. 3. Add 200 L Reagent 2 and vortex for 10 seconds. 4. Centrifuge at x rpm for 3 minutes. 5. Take the upper phase into the autosampler vial. 6. Inject 20 L to the HPLC system. The prepared sample is stable at 2-4 C for 24 hours. 12. QUALITY CONTROL The test results are only valid if the test has been performed by following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All standards and kit controls must be found within the acceptable ranges as stated on the QC Certificate. If the criterias are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. In case of any deviation the following technical issues should be proven: Expiry dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. It is recommended to participate at appropriate quality assessment trials. 13. CALCULATION OF RESULTS The obtained area of the standard is plotted against their concentration. The standard curve is calculated by a linear regression or a weighted linear regression function. Using computer programs, the curve is best described by a 1-point linear regression fit with linear axes. For the calculation of the regression curve, apply each signal of the standards (one obvious outlier of duplicates might be omitted and the more plausible single value might be used). The concentration of the samples can be read directly from the regression function. Sample signals above the highest standard have to be confirmed by a reference method. Page 6 / 8

7 14. INTERPRETATION OF RESULTS Vitamin A: The World Health Organization recommendations supplementation when vitamin A levels fall below 20.0 mcg/dl. Severe deficiency is indicated at levels <10.0 mcg/dl. Vitamin A values >120.0 mcg/dl suggest hypervitaminosis A and associated toxicity. Vitamin E (alpha-tocopherol): -Values that indicate need for supplementation: -Premature: <2.0 mg/l -Neonate: <2.0 mg/l -Child (3 months): <3.0 mg/l -Child (2 years): <3.0 mg/l -Adults: <3.0 mg/l -Values that indicate significant excess: -Adults: >40.0 mg/l Various societies recommend different Cut-Off values for repetition of the measurement and the application of confirmatory assays. Depending on the application of patient samples from different populations, it is highly recommended that each laboratory establishes its own range of normal values and that this distribution of values is co-ordinated with the recommendations of the responsible society of this geographic region. The results themselves should not be the only reason for any therapeutical consequences. They have to be correlated to other clinical observations and diagnostic tests. 15. EXPECTED VALUES VITAMIN A (RETINOL) 0-6 years: mcg/dl 7-12 years: mcg/dl years: mcg/dl > or =18 years: mcg/dl VITAMIN E (ALPHA-TOCOPHEROL) 0-17 years: mg/l > or =18 years: mg/l Significant deficiency: <3.0 mg/l Significant excess: >40 mg/l It is recommended that each laboratory establishes its own range of normal values. Page 7 / 8

8 16. HPLC-UV PARAMETERS Device Zivak ASP-100 HPLC-UV System Column Zivak Vitamin A-E Serum HPLC Analytical Column Injection volume 20 µl Gradient min 100% A 08:00 min 100% A Flow 1,20 ml/min Column Oven 30 C Detector UV/VIS Wavelength min 325nm 02:00-08,00 min 295nm 17. ANALYTICAL PERFORMANCE No Analyte LOD (mg/l) LOQ (mg/l) Accuracy (%) Precision (%CV) Linearity (R 2 ) 1 Vitamin A ,999 2 Vitamin E ,998 Analytical Specificity (Cross Reactivity): No cross-reactivities were found with the typical substances tested. 18. SAMPLE CHROMATOGRAM Page 8 / 8

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