Vitamin B1 HPLC

For Research Use Only. Not For Use In Diagnostic Procedures.

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This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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Catalog Number: Size: Version:

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30-2201 100 Determinations 21.09.2007 ALPCO 12/7/2007

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For the determination of Vitamin B1 (thiamin pyrophosphate) in EDTAwhole blood.

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1. INTENDED USE

This assay is intended for the quantitative determination of Vitamin B1 in EDTA-blood. This assay is designed for research use only.
2. SUMMARY AND EXPLANATION OF THE TEST

Vitamin B1 (thiamin) is a water-soluble Vitamin, which consists of a pyrimidine and thiazol ring linked via a methylene bridge. It is sensitive to alkaline solution, oxidation and reduction.

Vitamin B1 is produced by plants and microorganisms. It is found free, peptide bound and as phospho-esters (mono-, di- and triphosphoesters). In humans and other animals Vitamin B1 supplemented by food.The intake of thiamin in the gut is maintained by active transport and passive diffusion. The different phospho-esters are synthesized by phosphorylation and dephosphorylation. The active form in metabolism is thiamin pyrophos- phate and thiamin triphosphate in the brain. After dephosphorylation thiamin is secreted by the kidney. Thiamin pyrophosphate plays an important role as a co-enzyme in carbohydrate and amino acid metabolism. An important reaction is oxidative carboxylation. Thiamin itself is required for stimulating nerve cells. In addition, it stimulates the fatty acid and cholesterol-synthesis in nervous tissues.

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Beri Beri is a classic example of a disease caused by a lack of Vitamin B1. The disease has been traditionally seen in people in Asia who predominantly eat white rice. Symptoms are paralysis, drop in muscle mass, and heart failure. Other diseases are Wernicke's encephalopathy, Korsakow-syndrome and several forms of Landry's paralysis. In addition, many alcoholics are deficient in thiamin.

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2 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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3. PRINCIPLE OF THE TEST The first step in the determination of thiamin pyrophosphate includes the sample preparation with additional derivatization. During the precipitation higher molecular substances are removed. After centrifugation, the supernatant is used for the derivatization (10 min at 60C). Vitamin B1 will be transformed into a fluorescent product. The sample is cooled, centrifuged, and injected into the HPLC system.

The separation via HPLC follows an isocratic method at 30C using a "reversed phase" column; one run lasts 12 minutes. The quantification is performed with the delivered calibrator. The concentration is calculated via integration of the peak areas. Summary

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In addition to many other parameters, the HPLC method has the advantage of simultaneous handling of many analytes in a single test. The HPLC system enables even laboratories without experience in high performance liquid chromatography to use this technique quickly and precisely for routine determination. Unlike immunoassays with up to six calibrators per test, a one-point calibration is sufficient to calibrate the test system. It is possible to automate the sample application and calculation of the results so that even higher sample numbers can be handled.

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3 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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The HPLC technique provides an easy, fast, and precise method for quantitative determination of vitamin B1. The kit contains all reagents necessary for sample preparation and separation in ready to use form, with the exception of the column.

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4. MATERIAL SUPPLIED

Cat. No KC 2201lm KC 2201 ka KC 2201fr KC 220 1vl KC 2201 rb KC 2201dl KC 2201 lc KC 2201 ko

Content MOPHA CAL PREC DIL REABUF DER SOLC CTRL1, CTRL2

Kit Components Mobile phase

Calibrator, lyophilized Precipitating reagent Dilution solution Reaction buffer

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Solution C

Derivatization solution (lyoph.)

5. MATERIAL REQUIRED BUT NOT SUPPLIED

Centrifuge

HPLC with Fluorescence-detector Reversed phase C 1 8 -column

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6. PREPARATION AND STORAGE OF REAGENTS The calibrator(CAL), EDTA- whole blood with a defined concentration of thiamin pyrophosphate, should be resuspended with dilution solution (volume is given on the label), aliquoted and stored at -20 C. The concentration of Vitamin B1 is may vary slightly from lot to lot; the concentration is given on the label.

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Heatable shaker or waterbath Vortex mixer

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Various pipettes

4 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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1.5 ml reaction tubes (Eppendorf)

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Control 1 and 2; 250uL lyophilized

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Quantity 1 x 1000 ml 1 vial 5 ml 20 ml 5 ml 5.5 ml 5.5 ml 2x3 vials

Reconstitute the controls (CTRL1, CTRL2) in 250uL of dilution solution. Re-suspend the derivatization solution (DER) with 5.5 ml of solution C (SOLC). The dissolved derivatization solution is stable for 6 month at 2-8 C.

All other test components are ready to use.

Store test reagents at 2-8 C and the calibrator (CAL) and controls (CTRL1, CTRL2) at 20 C. They are stable up to the expiry date which is given on the label. 7. PRECAUTIONS

EDTA-whole-blood has to be used in this assay. Vitamin B1 is light and temperature sensitive. Therefore, protect samples from light and cool immediately after collection.

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The samples are stable in the dark at 2-8C for 1 day. For longer storage, samples should be frozen at -20C. Do not freeze the samples a second time.

9. ASSAY PROCEDURE
Procedural notes

Quality control guidelines should be observed. Incubation time, incubation temperature and pipetting volumes of the components are defined by the producer. Any variation of the test
5 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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8. SPECIMEN COLLECTION AND PREPARATION

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Reagents should not be used beyond the expiration date shown on the kit label.

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The supplied reagents contain reagents like acetonitrile (mobile phase) and acid (precipitating reagent). Even when diluted they should still must be handled with care. They can cause acid burns and should be handled with gloves, eye protection, and appropriate protective clothing. Any spills should be wiped up immediately with plenty of water. Avoid inhalation of any vapors.

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Human source materials used in kit components were tested and found to be negative for HIV, Hepatitis B and Hepatitis C. However, for safety reasons, all kit components should be treated as potentially infectious.

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procedure, that is not coordinated with the producer, may influence the test results. The assay should always be performed according to the enclosed manual.

Sample preparation

Pipette into 1.5 ml reaction tubes:

50 l sample (EDTA-whole blood), calibrator (CAL) or control (CTRL1, CTRL2) + 150 l dilution solution (DIL) + 50 l precipitating reagent (PREC)

Mix well. Leave the tubes for 10 minutes at 2-8C and centrifuge afterwards at 10,000 x g for 10 minutes. Take 150 l supernatant

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Incubate for 10 minutes at 60C on a shaker or in a water bath. Cool to 2-8C. Centrifuge at 10,000 x g for 5 minutes. (The prepared supernatant is stable for 3 days at 2-8 C.) Inject 50 l of the supernatant for chromatography into the HPLC-system.

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+ 50 l derivatization solution (DER)

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+ 50 l reaction buffer (REABUF)

6 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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Chromatographic conditions
Column material: Bischof Eurobond; 5 m;

Nucleodur Sphinx RP18; 5 m Column dimension: Flow rate: 125 x 4 mm 0.8 - 1.2 ml/min Emission: Temperature: Injection volume: Running time: 30 C 50 l

Fluorescence Detection: Excitation:

365 nm 440 nm

12 minutes

It is recommended that a guard column is used to extend column life.

11. RESULTS

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CALCULATION

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Peak height sample x conc. calibrator (ng/ml) = Peak height calibrator
B

B1 sample

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After the analysis, the column should be flushed with 30 ml distilled water (1 ml/min) and stored in 50% methanol in distilled water (ca. 30 ml, flow 0.7 ml/min). Before use, the system should be equilibrated with ca. 30 ml mobile phase (MOPHA).

7 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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10. TREATMENT OF THE COLUMN

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Lichrospher RP18; 5 m

Typical chromatogram

0,40

0,30

0,20

0,10

0,00

-0,10 0,0 1,0 2,0 3,0 4,0

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5,0 6,0 7,0 8,0

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We do not recommend measuring lipaemic samples. The measurement of serum and plasma samples is possible but not recommended, because the concentration is mostly below the detection limit.

13. QUALITY CONTROL

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Expected values

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12. LIMITATIONS

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32 95 ng/ml (mean 2SD)
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Vitamin B1 normal values:

It is recommended that each laboratory should establish its own normal range. Above mentioned values are only for orientation and may vary from other published data.

This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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9,0 10,0

0,50

Volt

Vitamin B1

Controls

14. PERFORMANCE CHARACTERISTICS Precision and reproducibility

4.3% Inter-Assay CV: 3.2% 4.7%

Linearity Detection limit

(59.0 ng/ml)[n=6]

(33.0 ng/ml)[n=12] (63.2 ng/ml)[n=12]

15. DISPOSAL

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The mobile phase (MOPHA) and derivatization solution (DER) must be disposed of as a non-halogenated solvent. The precipitation solution (PREC) can be neutralized with NaOH and disposed of as a salt solution. (Important: Reaction will produce heat, be careful.) Please refer to the appropriate national guidelines.

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This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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0.5 ng/ml

up to 250 ng/ml

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Intra-Assay CV:

3.3%

(31.2 ng/ml)[n=6]

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Control samples or EDTA-blood pools should be analyzed with each run of calibrators and samples. Results, generated from the analysis of the control samples, should be evaluated for acceptability using appropriate statistical methods. The results for the samples may not be valid if one or more values of the quality control samples within the same assay are outside the acceptable limits.

16. TROUBLESHOOTING

No peaks Double peaks Contaminating peaks

Broad peaks, tailing Variable retention times

System not equilibrated

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Baseline is drifting

Detector lamp did not reach working temperature yet Detector lap is too old

Baseline is not smooth

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17. REFERENCES 1. Tallaksen C.M.E., T. Bohmer, H. Bell (1991). Cocomitant determination of thiamin and its phosphate esters in human blood and serum by high performance liquid chromatography. J. Chromatogr, 564, 127-136.

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System not equilibrated

Imprecise pump delivery

Imprecise pump delivery Detector flow cell is dirty

This Protocol is for Reference Purposes Only. 10 DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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Air in system Auto sampler vials contaminated Precolumn/ column exhausted Drift in temperature Imprecise pump delivery

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Renew lamp Rinse system mobile phase for 15 min Check pump, degas the system Check pump, degas the system Clean flow cell

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Problem No signal

Possible Reason Defective or no connection to evaluation system Detector lamp defective Injector is congested Dead volume in fittings and/ or column Injector dirty Contamination at the head of the column

Solution Check signal cord and connection Change Lamp Check Injector Renew fittings and/or column Clean injector Change direction of the column and rinse for 30 min at low flow rat (0.2 ml/min) with mobile phase Degas pump Use new vial or clean them with methanol Use new precolumn/ column Use a column oven Check pump, degas the system Rinse system mobile phase for 15 min Wait

18. GENERAL NOTES ON THE TEST AND TEST PROCEDURE

This assay was produced and put on the market according to the IVD guidelines of 98/79/EC.

The test components contain organic solvents. Avoid contact with skin or mucous membranes. All reagents in the test package are for research use only.

Reagents should not be used beyond the expiration date shown on the kit label. Do not interchange different lot numbers of any kit component within the same assay. Quality control guidelines should be observed. Incubation time, incubation temperature and pipetting volumes of the components are defined by the producer. Any variations of the test procedure, that are not coordinated with the producer, may influence the results of the test.

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11 This Protocol is for Reference Purposes Only. DO NOT use this copy to run your assay; use the protocol included with the kit ONLY.

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2. Herbeth B., J. Zittoun, L. Miravet, M. Boureay-Causse, G. Carre-Guery, E. Delacoux, C. Le Devehat, A. Lemoine, J.P. Mareschi, J. Martin, G. Portier de Courcy and J. Sancho (1986). Reference intervals for vitamin B1, B2, E, D, retinol, and folate in blood: Usefulness of dietary selection criteria. Clin. Chem. 32/9, 1756-1759.