25-Hydroxyvitamin D2-D3 Serum VD-200 UHPLC Analysis Kit User Manual

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1 Page 1 / Hydroxyvitamin D2-D3 Serum VD-200 UHPLC Analysis Kit User Manual ZV C

2 Page 2 / 12 Table of Contents 1. INTENDED USE SUMMARY AND EXPLANATION IVD SYMBOLS TEST PRINCIPLE WARNING AND PRECAUTIONS STORAGE AND STABILITY MATERIALS SUPPLIED MATERIALS REQUIRED BUT NOT SUPPLIED PROCEDURE NOTES PRE-SET UP INSTRUCTIONS LIMITATIONS OF THE PROCEDURE SAMPLE PRETREATMENT QUALITY CONTROL CALCULATION OF RESULTS INTERPRETATION OF RESULTS EXPECTED VALUES HPLC-UV PARAMETERS ANALYTICAL PERFORMANCE SAMPLE CHROMATOGRAM REFERENCES... 12

3 Page 3 / INTENDED USE Quantitative HPLC analysis kit for 25-hydroxy metabolites of Vitamin D2 and D3 in human serum samples. 2. SUMMARY AND EXPLANATION Vitamin D, commonly known as the sunshine vitamin, is essential for maintaining calcium metabolism and bone health. The major physiologic function of Vitamin D is to maintain the blood calcium and phosphorus levels within the normal range in order to maintain metabolic functions that are essential for most life processes, Vitamin D3 and Vitamin D2 are metabolized to 25-OH- Vitamin D3 and 25-OH-Vitamin D2 in the liver by the 25-OHase enzyme. The quantitation of these metabolites is widely used for determining of vitamin D status in human. The Vitamin D status has a clinical significance in a variety of disorders which related to hormones. The measurements of the serum concentration of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 have been used for differential diagnosis of hypocalcemic and hypercalcemic metabolic bone disorders. The analysis of these metabolites is considered to be difficult. Some reasons of the difficulties are below. The low concentrations of 25-OH Vitamin D metabolites in serum (level of μg/l). The low stability in the presence of light and heat. The presence of other metabolites which have chemical similarity. The large amount of other related neutral lipids (such as cholesterol) 25-OH Vitamin D2-D3 VD-200 UHPLC Analysis Kit was developed for rapid, sensitive and reliable quantitative detection of the 25-OH Vitamin D2-D3 concentrations in human serum samples and it gives results in 6 minutes with minimal sample preparation. Main methods and procedures that have been selected are based on EN ISO and 98/79/EC.

4 Page 4 / IVD SYMBOLS Conformite European Manufacturer In vitro diagnostic Lot number Order number Sufficient for <N> Tests Temperature limits 4. TEST PRINCIPLE The 25-hydroxyvitamin D metabolites are extracted from human serum samples using an organic extraction solution after deproteinization step. Separation was made with an isocratic elution on Zivak 25-OH Vitamin D HPLC column and measured by Zivak VD-200 UHPLC system. Accuracy and reliability of the analysis could be verified by the serum controls which are supplied with the kit. 5. 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. 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.

5 Page 5 / STORAGE AND STABILITY This analysis kit can be shipped at room temperature. 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. 7. MATERIALS SUPPLIED Order No Volume Symbol Component ZV R1-10 ZV R2-10 ZV MP-10 ZV MT-10 1 x 350 ml 1 x 300 ml 2 x 1.5 L 2 x 1.5 L R1 R2 MP MT Reagent 1, Contains aqueous buffer Reagent 2, Contains Internal Standard Mobile Phase A, Contains organic solvent SPE Buffer, Contains organic solvent ZV W x 2 L WB1 Wash Solution 1 ZV W x 2 L WB2 Wash Solution 2 ZS-2ML x 100 pcs 2 ml glass vial ZS x 100 pcs 2 ml glass vial caps pre-slit ZV-4027-KK-10 1 x 1 pc KK User Guide

6 Page 6 / MATERIALS REQUIRED BUT NOT SUPPLIED Order No Volume Symbol Component ZV S1-10 Calibrator Level 1 Calibrator Level-1 ZV S2-10 Calibrator Level 2 Calibrator Level-2 ZV S3-10 Calibrator Level 3 Calibrator Level-3 ZV S4-10 Calibrator Level 4 Calibrator Level-4 ZV S5-10 Calibrator Level 5 Calibrator Level-5 ZV S6-10 Calibrator Level 6 Calibrator Level-6 ZV K1-10 Control Level 1 Control Level-1 ZV K2-10 ZV C x 1 pc Control Level 2 Control Level-2 25-Hydroxyvitamin D2-D3 Serum VD-200 UHPLC Column ZS µl pipette ZS µl pipette tips Bidistilled or deionized water 9. PROCEDURE NOTES Any inappropriate handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pre-treatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only.

7 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. Page 7 / PRE-SET UP INSTRUCTIONS Instrument Setup Purge UHPLC pumps with 5000 µl/min flow rate after connecting mobile phase A and SPE buffer. Purge pumps until there are no air bubbles inside the tubing lines. Connect wash solution into system and purge syringe pumps until there are no air bubbles in tubing lines. Make sure the bottle of mobile phase is closed well, otherwise components of the mobile phase could evaporate; this alters the retention times. System will be stabilized automatically when analysis started from the Zivak VD-200 Full Automated Vitamin D2/D3 UHPLC Analyzer. Valve Connections Injection Valve Switching Valve

8 Page 8 / 12 Preparation of Calibrators Add 3 ml of deionized water to each serum calibrator and dissolve. After preparation, they must be aliquoted into 400 µl volumes and stored at -20 C. Preparation of Controls Add 3 ml of deionized water to each serum control and dissolve. After preparation, they must be aliquoted into 400 µl volumes and stored at -20 C. 11. 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. 12. SAMPLE PRETREATMENT Sample Pretreatment (Automated) If Calibrator/Control will be analyzed, take 400 µl of calibrator/control sample into glass vial and put vials into related tray on instrument. Place serum samples into system tray. Analysis will automatically start after clicking Start button on software. Injections, calculations and reporting done automatically by instrument. Prepared samples are stable for 24 hours at 2-4 C temperature.

9 Page 9 / 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 (Quality Control) Certificate. If the criteria 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. 14. CALCULATION OF RESULTS The obtained ratios of the analytes area to IS Area is used for calculation of Vitamin D2 and D3 results. A linear calibration curve will created with these calculated ratios. Calculation formula is given below. D3 Area Ratio = D2 Area Ratio = D3 Area IS Area D2 Area IS Area D3 Amount Ratio = D2 Amount Ratio = D3 Amount IS Amount D2 Amount IS Amount Sample signals above the highest control level have to be confirmed by a reference method. 15. INTERPRETATION OF RESULTS 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 coordinated with the recommendations of the responsible society of this geographic region. The results themselves should not be the only reason for any therapeutic consequences. They have to be correlated to other clinical observations and diagnostic tests.

10 Page 10 / EXPECTED VALUES These reference ranges represent clinical decision values, based on the 2011 Institute of Medicine report, that apply to males and females of all ages, rather than population-based reference values. Population reference ranges for 25-OH-VitD vary widely depending on ethnic background, age, geographic location of the studied populations, and the sampling season. <10 ng/ml ng/ml ng/ml ng/ml >80 ng/ml Severe deficiency Mild to moderate deficiency Optimum levels Increased risk of hypercalciuria Toxicity possible Could be associated with osteomalacia or rickets May be associated with increased risk of osteoporosis or secondary hyperparathyroidism Optimum levels in the healthy population; patients with bone disease may benefit from higher levels within this range Sustained levels >50 ng/ml 25OH-Vitamin D along with prolonged calcium supplementation may lead to hypercalciuria and decreased renal function 80 ng/ml is the lowest reported level associated with toxicity in patients without primary hyperparathyroidism who have normal renal function. Most patients with toxicity have levels >150 ng/ml. Patients with renal failure can have very high 25-OH-Vitamin D levels without any signs of toxicity, as renal conversion to the active hormone 1.25-OH-Vitamin D is impaired or absent It is recommended that each laboratory establishes its own range of normal values. 17. HPLC-UV PARAMETERS Instrument Column SPE Detector Wavelength Zivak VD-200 Full Automated Vitamin D2/D3 UHPLC Analyzer Zivak 25-Hidroxyvitamin D2-D3 Serum VD-200 UHPLC Analytical Column Zivak 25-Hidroxyvitamin D2-D3 Serum VD-200 UHPLC Trap Column Cartridge UV/VIS 264 nm Injection Volume 20 µl Column Oven Temperature 35 C

11 Page 11 / ANALYTICAL PERFORMANCE No Analyte Accuracy (%) Intra-assay Precision (%CV) Inter-assay Precision (%CV) OH Vitamin D3 25-OH Vitamin D2 Low level High level Low level High level Low level High level No 1 2 Analyte 25-OH Vitamin D3 25-OH Vitamin D2 LOD (µg/l) LOQ (µg/l) Linearity (R 2 ) 1. Day 2. Day 3. Day 0,9989 0,9979 0, ,9984 0,9951 0,9974 Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical substances tested. 19. SAMPLE CHROMATOGRAM

12 Page 12 / REFERENCES Alshahrani F., Aljohani N., Viatmin D: Deficiency, Sufficiency and Toxicity, Nutrients, 5 (9): , Khan J. Q., Fabian C. J., How I Treat Vitamin D Deficiency, Journal of Oncology Practice 6 No:2, , Aw T. C., Yap C., Vitamin D Measurements-Facts and Fancies, Department of Laboratory Medicine, Changi General Hospital, Proceedings of Singapore Healthcare, Volume 22, Number 3, 2013.

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