PART III: LABORATORY METHODS OF ANALYSIS
A. Sample preparation
139 Procedures
140 D. Chromatography of choline
1. UPLC Condition
a. Set column temperature at 40°C and sample temperature at 4°C
b. Prepare mobile phase A in the combination of acetonitrile/water/ethanol/1 M ammonium acetate/ glacial acetic acid (800/127/68/3/2, v/v)
c. Prepare mobile phase B in the combination of acetonitrile/water/ethanol/1 M ammonium acetate/ glacial acetic acid (500/500/85/27/18, v/v)
d. Program the gradient elution as follow for aqueous phase:
0.0 min 0% B 0.4 ml/min
3.0 min 0%B 0.4 ml/min
10.0 min 40% B 0.4 ml/min 14.0 min 45% B 0.3 ml/min 18.0 min 60% B 0.3 ml/min 19.0 min 100%B 0.5 ml/min 27.0 min 100%B 0.5ml/min
29.0 min 0%B 0.4ml/min
e. Program the gradient elution as follow for organic (chloroform) phase:
0.0 min 0% B 0.4 ml/min
3.0 min 0%B 0.4 ml/min
10.0 min 20% B 0.4 ml/min 14.0 min 60% B 0.3 ml/min 16.0 min 100% B 0.3 ml/min 17.0 min 100%B 0.5 ml/min 20.0 min 100%B 0.5ml/min
22.0 min 0%B 0.4ml/min
f. Sample injection volume is 10 µl, full-loop injection
g. Wash needle with 0.1% formic acid in water (weak wash); or 0.1% formic acid in methanol (strong wash)
h. For aqueous phase, allow LC effluent from 5-26 min into MS; for organic phase, allow effluent from 4-14 min into MS.
2. MS condition
a. Set 0.6 ml/min portion of column effluent into MS b. Acquire ESI-MS spectra in positive ion mode
c. Use nitrogen as both desolvation (350 L/hr) and cone (50 L/hr) gas.
d. Set 350 °C for desolvation temperature.
e. Set 105°C for source temperature.
141 f. Capillary voltage is 5 kV; cone voltage is 180 V
g. Use argon gas as collision gas at 3.5 X 10-3 mBar
h. For aqueous phase. Use m/z 104 (Cho), m/z 113 (Cho-d9), m/z 258 (GPCho), m/z 267 (GPCho-d9), m/z 184 (PCho) and m/z 193 (PCho-d9).
i. For organic phase. Use m/z 184 (PtdCho and SM); m/z 193 (PtdCho-d9) and m/z 187 (SM-d3).
3. Prepare calibration curve standard by mixing 20 nmol of deuterium-labeled internal standards with varying amount of analytes.
4. The calibration curves are constructed by relating the varying amounts of each analyte to their relative response factors (RRFs) as determined by the ratio of the peak area of the analyte to that of the corresponding deuterium-labeled internal standard.
5. Calculate total choline content (mg/100g food) in sample as the sum of Cho, GPCho, Pcho, PtdCho and SM. Report individual metabolites as mg choline moiety per 100g of food.
References
Koc, H., Mar, M-H., Ranasinghe, A., Swenberg, J.A. & Zeisel, S. H. 2002. Quantitation of choline and its metabolites in tissues and food by liquid chromatography/ electrospray ionization-isotope dilution mass spectrometry. Analytical Chemistry. 74: 4734-4740.
Zeisel, S.H., Mar, M-H, Howe, J-C, Hoilden, J.M. 2003. Concentrations of choline- containing compounds and betaine in common foods. Journal of Nutrition. 133: 1302- 1307.
142 3.3.4.9 DETERMINATION OF ASCORBIC ACID (VITAMIN C) IN FOOD
(HIGH PRESSURE LIQUID CHROMATOGRAPHY METHOD)
Principles
High pressure liquid chromatography analytical methods are now replacing the traditional titrimetric and colorimetric methods for more accurate determination of total vitamin C.
HPLC method is able to distinguish between L-ascorbic acid and isoascorbic acid and eliminate other deficiencies of traditional method. For example, colour interference by highly coloured extracts during the titration’s end point, and overestimation of total vitamin C content due to reduction of dye by other substances like ferrous, tannin and cuprous.
Ascorbic acid in sample is extracted in acidic environment and completed rapidly in subdued light. Metaphosphoric acid is chosen as extractant because it inhibits L-ascorbic acid oxidase and metal catalysis, precipitates proteins and compatible to LC system. Besides, tris (2- carboxyethyl)-phosphine (TCEP) is included in extractant to convert dehydroascorbic into its reduced form and to stabilize ascorbic acid.
Reverse-phase HPLC procedure is employed in this method and 0.05% of formic acid is used as mobile phase with a Phenomenex C18 column. The diode array detector is set at 255 nm for L-ascorbic acid determination.
Chemicals/ Reagents
1. Extraction buffer preparation
a. Prepare an extraction buffer containing 5 % of metaphosphoric acid (MPA), 1 mM of ethylenediaminetetraacetic acid (EDTA) and 5 mM of tris(2- carboxyethyl)phosphine (TCEP).
b. Prepare 5 % MPA by dissolving 50 g of MPA with deionized water and make to volume of 1 L.
c. Dissolve 150 mg of EDTA and 717 mg of TCEP with 5% of MPA, then top up to volume of 500 ml in volumetric flask. Refrigerate the extraction buffer until use.
2. Mobile phase preparation
a. Prepare 0.05% of formic acid by dissolving 0.5 g of formic acid with deionized water and top up to volume of 1 L in volumetric flask. Filter the mobile phase with 0.45 µm nylon membrane filter using solvent filtration apparatus. Prepare mobile phase freshly prior to every analysis.
3. Vitamin C standard solution
a. Use crystalline L-ascorbic acid (99% purity, e.g. from Sigma Chemical Co.) as standard for ascorbic acid determination.
b. Ascorbic acid standard stock solution. Weigh 10 mg of standard and make up to 100 ml with 5% MPA to give a concentration of 100 µg/ml. Prepare freshly prior to analysis
c. Ascorbic acid standard working solution. Pipette 10 ml of standard stock solution (b) in 100 ml volumetric flask and make up to volume with 5% MPA. The final concentration of working solution is 10 µg/ml.
Note: Preparation of all standards for ascorbic acid should be carried out in a room with subdued light. All sample treatment and analytical procedures were also carried out in this room.
143 Apparatus/Instruments
1. Analytical balance 2. Conical flask: 100 ml 3. Homogenizer
4. Centrifuge
5. Filtration system with 0.45 µm PVDF membrane filter 6. Volumetric flask: 50 ml
7. HPLC with UV detector.
8. Analytical column: RP C18, 150 mm x 4.6 mm I.D and particle size 5 µm 9. Guard column: length 20 mm, I.D 4.6 mm
HPLC conditions
1. Reverse phase C18 column: 150 mm x 4.6 mm I.D and particle size 5 µm (e.g.
Atlantis. Waters).
2. Guard column holder with a guard column: 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 diode arrays multiple wavelength detector (DAD), G1315 for ascorbic acid detection at the wavelength of 254 nm.
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 10 g (fresh sample) or 2 g (dried sample) of the foodstuff into 100 ml conical flask.
2. Add 15 ml of cooled extraction buffer into the flask and extract for 10 min with a homogenizer.
3. Repeat the extraction procedure in (2) for 3-4 times and combine the extract.
4. Centrifuge the extract at 10°C, 5000 rpm for 30 min.
5. Decant the supernatant and filter with 0.45 µm PVDF membrane filter.
6. Quantitatively transfer the filtered extract to a 50 ml volumetric flask and top up to volume with extraction buffer (V1).