PART III: LABORATORY METHODS OF ANALYSIS
C. Chromatography of Pantothenic Acid
1. Identify pantothenic acid in sample by comparing their retention time with the retention time of standards.
2. Obtain a standard curve by using the peak area given by each standard solution (0.5 – 2 µg/ml)
3. Quantify pantothenic acid of sample by comparing integrated chromatographic peak areas from the test samples to peak areas of known amounts of standards.
4. The total amount of pantothenic acid in sample is expressed in mg/100g of sample.
Reference
Pakin, C., Bergaentzlé, M., Hubscher, V., Aoudé-Werner, D., Hasselmann, C. 2004.
Fluorimetric determination of pantothenic acid in food by liquid chromatography with post column derivatization. Journal of Chromatography A. 1035, 97-95.
119 3.3.4.4 DETERMINATION OF PYRIDOXINE (VITAMIN B6) IN FOOD
(HIGH PRESSURE LIQUID CHROMATOGRAPHY METHOD) Principles
Vitamin B6 in food is dephosphorylated by enzyme hydrolysis and detected using fluorescence as it provides a high sensitive and specific detection mode for the analysis of all vitamin B6 forms commonly encountered in foods. Pyridoxine hydrochloride (PN), pyridoxal hydrochloride (PL) and pyridoxamine dihydrochloride (PM) is detected and total vitamin B6 is calculated with the equation PN = PN + (1.01PL) + (0.79PM).
Chemicals/ Reagents
1. Acetonitrile, HPLC grade.
2. HCl, 0.01M 3. HCl, 0.1M 4. HCL, 1M
5. Sodium acetate, 2M
6. Potassium hydroxide, 3.5M
7. Acid phosphatase, 25 unit/ml, prepare freshly prior to analysis.
8. β-glucosidase, 45 unit/ml, prepare freshly prior to analysis.
9. Triethylamine, 99%, 4 mM
10. Potassium dihydrogen phosphate, 81 mM 11. Ortho-phosphoric acid, 85%, 19 mM 12. 1-octan-sulfonic acid, 2.2 mM 13. Sodium monohydrate, 99%
14. Mobile phase preparation.
a. HPLC eluent-buffer. Prepare buffer (1L) consist of 2.2 mM 1-octan-sulfonic acid in 81 mM potassium dihydrogen phosphate and 19 mM 85% phosphoric acid and 4.0 mM triethylamine. Adjust pH to 2.75 with 3.5 M potassium hydroxide.
b. Prepare mobile phase that contain 93% HPLC eluent-buffer and 7%
acetonitrile.
c. Filter mobile phase with 0.45 µm nylon membrane filter using solvent filtration apparatus daily prior to analysis.
15. Vitamin B6 standard solutions
a. Use pyridoxine hydrochloride (PN), pyridoxal hydrochloride (PL) and
pyridoxamine dihydrochloride (PM) (e.g. from Sigma Chemical Co) as standards for vitamin B6 determination.
b. Standard stock solution. Weigh 10 mg of standards and make up to 100 ml with 0.1 M HCl to give a concentration of 100 µg/ml. Prepare freshly prior to analysis.
c. Standard working solution. Pipette 1 ml of standard stock solution (b) in 100 ml volumetric flask and make up to volume with 0.1 M HCl. The final concentration of working solution is 1 µg/ml.
120 Apparatus/Instruments
1. Ultrafiltration equipment: 0.45 µm, 47 mm diameter membrane filter 2. Volumetric flask: 100 ml, 1L
3. Balance: analytical sensitivity ± 0.1 mg 4. Conical flask: 50 ml
5. Beaker: 25 ml, 50 ml 6. Mechanical shaker 7. Autoclave
8. Centrifuge
9. Incubator: at 45°C
10. Filter paper, Whatman No.1 11. HPLC sample vials
12. Syringe filter: 0.45 µm PVDF
13. Analytical column: C18, particle size 3 µm, 150 mm x 4.6 mm I.D 14. HPLC with fluorescence detector
HPLC conditions
1. Reverse phase C18 column of 150 mm x 4.6 mm I.D and particle size 3 µm (e.g.
Hypersil, Phenomenex).
2. Guard column holder with a gurd column with an I.D of 4.6 mm and length of 20 mm packed with the same material as that of the analytical column.
3. Agilent 1200 series standard and preparative autosampler, G1239.
4. Agilent 1200 series binary pump, G1312 to deliver the mobile phase.
5. Agilent 1200 series micro vacuum degasser, G1379.
6. Agilent 1200 series fluorescence detector, G1321 7. Solvent cabinet to keep the mobile phases.
8. A computer with an Agilent ChemStation control software.
9. A HP LaserJet 5L printer to print the results and chromatograms.
Procedures
A. Sample extraction
1. Weigh accurately 5g of the foodstuff into a 100 ml extraction flask.
2. Add 50 ml of 0.1 m HCl; homogenize the extract for 10 min with a orbital shaker.
3. Autoclave extracts at 121°C for 30 min (animal origin sample); 5 min (vegetable origin sample).
4. Cool the extract to room temperature immediately on ice.
5. Adjust the cooled extract to pH 4.5 with 2 M sodium acetate.
6. Dilute the extract and make up to volume of 100 ml with deionized water.
7. Centrifuge the extract at 8500 g, 5°C for 10 min.
8. Decant the supernatant and filter with filter paper.
9. For animal origin sample, take 15 ml of the filtered supernatant and mix with 1 ml of acid phosphatase solution in 30 ml measurement flask.
10. For vegetable origin sample, take 15 ml of the filtered supernatant and then mix with 1 ml of acid phosphatase and 3 ml of β-glucosidase in 30 ml measurement flask.
11. Incubate the mixture at 45°C for 18 hours.
121 12. Cool the mixture to room temperature and add 5 ml of cool 1 M HCl. Then, fill up the
flask with 0.01 M HCl until 30 ml (V1).
13. Filter the extract with 0.45 µm PVDF syringe filter into a HPLC vial. (For turbid sample, centrifuge it at 14,000 g for 10 min at 5°C prior to filtration.)
C. Instrument set up procedures
1. Before turning on the HPLC, check the column, reservoir and mobile phase to ensure that the correct column and mobile phase is used.
2. Turn on the Agilent Chemstation control software and HPLC system, then, warm up instrument for at least 30 min before starting.
3. Open the purge valve on the pump module by turning the knob counter clockwise several turns so that later priming solvent will directly deliver to waste container.
4. Set the pump 100% at flow rate of 5 ml/min to primp every channel in solvent delivery system with respective mobile phase for several minutes.
5. Turn off the pump and close the purge valve. Set the required mobile phase composition (isocratic) and flow rate of 1 ml/min for vitamin B6 analysis.
6. Turn on the pump again and allow the column to equilibrate with mobile phase which usually need 1 hour.
7. Set fluorescence detector excitation at 333 nm and emission at 375 nm.
8. To check whether column is equilibrated, inject in above standard mixture for 2 runs and if the elution time of the standards is the same for the 2 runs then the column is ready for sample injection.
9. Key in the sample identity and build the sequence in SAMPLE SEQUENCE for autosampler.
10. Inject 50 µl of extract for HPLC analysis.