D1.3.16 M37 (by 1/2/08) - Report on evaluation of analytical … · 2018-02-02 · 1 Project no.FP6...

1

Project no.FP6 - 513944

EuroFIR

EUROPEAN FOOD INFORMATION RESOURCE NETWORK

Instrument: Network of Excellence

Thematic Priority: 5 – Food Quality and Safety

D1.3.16 – M37 (by 1/2/08) - Report on evaluation of analytical methods

according with compilers needs 5/5 (Vitamins)

Due date of milestone or deliverable: 1/2/08

Actual submission date: 18/03/08*(available at Technical webside)

Start Date: January 01, 2005 Duration: 5 years

Organisation name of lead contractor for this deliverable or milestone –

INSA – Partner 24

Project co-funded by the European Commission within the Sixth Framework Programme

(2002-2006) Dissemination Level (please check appropriate box)

PU Public

PP Restricted to other programme participants (including the Commission Services)

RE Restricted to a group specified by the consortium (including the Commission Services)

CO Confidential, only for members of the consortium (including the Commission Services) x

EuroFIR guidelines for assessment of Methods of Analysis

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Methods of analysis

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Index

Vitamins 3

(i) Vitamin A Beta-carotene .................................................................... 4

(i) Vitamin A all-trans-retinol and 13-cis-retinol .................................... 9

(i) Vitamin B1 Thiamin ......................................................................... 14

(i) Vitamin B2 Riboflavin .................................................................... 19

(i) Vitamin C (L(+) ascorbic acid and dehydro L(+) ascorbic acid) ..... 24

(i) Vitamin D3, Vitamin D2 ................................................................... 28

(ii) .......................................................................................................... 28

(iii) Vitamin K1 and Vitamin K2 .............................................................. 33

(i) Folic acid and Folates ....................................................................... 38

(i) Vitamin B12 - Pantothenic acid ....................................................... 43

(i)


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Methods of analysis

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Vitamins


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Methods of analysis

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(ii) Vitamin A Beta-carotene

Figure 1- Structures of the main vitamin A –active carotenoids (reprint from Southgate & Greenfield, 2002)

Golden Standard

EN 12823-2:2000 Foodstuffs - Determination of vitamin A by high performance liquid

chromatography - Part 2: Measurements of Beta-carotene

Method Indicator

Name

Code

Scope

the determination of total- beta carotene in foodstuffs by high performance liquid

chromatography (HPLC).


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Methods of analysis

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Principle

Determination of the sum of beta _-carotene isomers in an appropriate sample solution by HPLC and

spectrometric detection in the visible range. The extract obtained after saponification as described in

EN 12823 -1 may be used for quantification. Identification on the basis of the retention times, and

determination by the external standard method using peak areas or peak heights, Internal standard

methods may also be used if the corresponding recovery

Key steps

Saponification

o by refluxing preferably under nitrogen using suitable amounts alcohol, antioxidant

and potassium hydroxide

Extraction

o Extraction the beta -carotene from the saponified sample solution by means of a

suitable solvent or solvent mixture (ex: n-hexane, dichloromethane),

Evaporation and dilution

o Evaporate the extract using a rotary evaporator under partial vacuum and re-

dissolve with the mixture compatible with mobile phase

Separation

o Stationary phase C18 reversed phase,

o Mobile phase (indicative) acetonitrile + methanolic ammonium acetatesolution+

dichloromethane + butylated hydroxy+ triethylamine

Identification

o by comparison of the retention time of the individual peaks in the chromatograms

obtained with the sample test solution and with the standard solution of beta

carotene

o Peak identification can also be performed by adding small amounts of the

appropriate standard solution to the sample test solution.

Detection

o UV/VIS 450 nm

o Diodo Array


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Methods of analysis

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Quantification

o Determination by the external standard method, integrating the peak areas or

determine the peak heights obtained for sample test solutions compare the

results with the corresponding values, with:

the standard substance with similar retention time

or construct a calibration curve.

o Check the linearity of the calibration function using a minimum three points of

beta carotene standard solution

Criteria for analytical performance and Analytical Quality

control

Method Performance

Table 1. Relevant parameters obtained during EN 12823-2 inter laboratory studies

Matrix

Parameter

Margarine

Vitamin

Drink

Pudding

Powder

Mixed

Vegetables

Repeatability

RSDr %

4,5

2,9

5,6

3,9

Reproducibility

RSD R%

9,7

6,5

9,3

15

Certified Reference Materials/Standard Reference Material

o BCR – 485- Mixed Vegetables Trans alfa carotene ; Trans beta carotene; Total

alfa carotene; Trans beta carotene

o NIST 2383 Baby Food

o NIST 2385 Slurried Spinach

Proficiency Testing Schemes (www.eptis.bam.de)

o Non register pt schemes for beta carotene

Other methods available

AOAC 975.23 Carotenoids in Eggs Colorimetric Method

http://www.eptis.bam.de/


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Methods of analysis

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AOAC 938.04 Carotenoids in Macaroni Products Colorimetric Method

Table 2. Codex CodexStan 234-1999)-Recommended methods of analysis

Nutrient Matrix

Principle

Vitamin A in foods in which carotenes have been added as

a source of vitamin A

Special

foods

Spectrophotometry

Remarks

The conditions applied to saponification are critical and need to be carefully controlled

using standard mixtures

Old Data

o Colorimetry can measure only total carotenoids .

Compatibility of old data

o Values from old data based on Carr& Price method give incompatible results

Recent Reviews on subject

o Christopher John Blake (2007) Status of Methodology for the Determination of

Fat-Soluble Vitamins in Foods, Dietary Supplements, and Vitamin Premixes.

JAOAC International 90, 897-910

o Meléndez-Martínez A J., Vicario I. M. and Heredia F J. (2007) Review: Analysis

of carotenoids in orange juice Journal of Food Composition and Analysis, 20,

638-649

o -Bernaldo de Quirós A.R. and Costa H. S. (2006) Analysis of carotenoids in

vegetable and plasma samples: A review Journal of Food Composition and

Analysis, 16, 97-111


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Methods of analysis

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References

Deharveng G., Charrondière UR, Slimani N., Southgate DAT., Riboli E. (1999)

Comparison of nutrients in food composition tables available in the nine European

countries participating in EPIC. European Journal of Clinical Nutrition 53, 60-79

Carr FH and Price EA (1926) Colour reactions attributed to vitamin A. . Biochem J 20,

497-501.

Greenfield H, Southgate DAT (2002) Food Composition Data – Production,

Management and Use. Elsevier Applied Science, London, UK

Finglas, P.M., Scott, K.J., Wilthöft, C. M., van der Berg, H. & de Froidmont – Görtz, I.,

1999. The certification of the mass fractions of vitamins in four reference materials:

wholemeal flour (CRM 121), milk powder (CRM421), lyophilized mixed vegetables

(CRM 485) and lyophilized pig’s liver (CRM 487). EUR-Report DOC/BCR/01/98.

Commission of the European Union, Luxembourg.

Neil, C.A., Schwartz, S.J. , Catignani, G.L.: Comparison of Liquid Chromatographic

Methods for Determination of Cis-Trans Isomers of beta -Carotene, J. Assoc. Off.

Anal. Chem. 74, 1991, 36-42

Saleh, H.M., Tan, B.: Separation and Identification of Cis/Trans Carotenoid Isomers, J.

Agric. Food Chem., 39, 1991, 1438-1443

[

EuroFIR assistance to this method/guidelines


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Methods of analysis

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(iii) Vitamin A all-trans-retinol and 13-cis-retinol

Vitamin A is a generic term that includes retinol, its esters and some isomers

Figure 2- Structures of the main vitamin A –active retinoids (reprint from Southgate & Greenfield, 2002)

Golden Standard

EN 12823-1:2000 Determination of vitamin A by HPLC - Part 1: Measurements of all-trans-

retinol and 13-cis-retinol

Method Indicator

Name

Code


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Methods of analysis

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Scope

The determination of vitamin A in foodstuffs by High performance liquid chromatography , The

determination of vitamin A content is carried out by measurement of all trans –retinol, 13 cis

retinol and β carotene. This chapter do not cover β carotene

Principle

Retinol is saponified by using methanolic or ethanolic potassium hydroxide solution and

extracted by na appropriate solvent, The determination is carried out by HPLC with either

fluorometric detection (F) or ultra-violet (UV) detection. The substances are identified on the

basis of the retention times and determined

Key steps

Homogeneity

o Homogeneity is necessary , Caution to avoid expose the samples to high

temperatures.

Saponification

o Food samples are saponified in alcoholic potassium hydroxide with addition of

an antioxidant, ascorbic acid, butylated hydroxytoluene(BHT) or pyrogallol

Extraction

o In suitable organic solvent

Identification

o Stationary phase normal phase column, with baseline resolution for all –trans

retinol and 13-cis-retinol

o Mobile phase n-hexane + n butanol (98+2) (volume parts) (indicative)

Detection

o Fluorescence, excitation: 325nm, emission: 475

o UV 325 nm

Quantification

o At least two independent determinations should be carried out


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Methods of analysis

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o Determination is usually carried out by external standard method by

comparision of peak heights or areas of sample solution with standard solution

with similar retention time or against calibration curve, and check the linearity of

the calibration curve


control

Method Performance

Table 3. Results obtained during Certification campaign of the Reference materials

Matrix

Parameter

Margarine

CRM 122

Milk Powder

CRM 421

All trans

retinol

13-cis

retinol

All trans

retinol

13-cis

retinol

Repeatability

RSDr

3.8 % 7.7 % 2.1 % 5.0 %

Reproducibility

RSDR

10.0 % 30.8 % 3.4 % 24.0 %


o CRM 122 Margarine

o CRM 421 Milk Powder



o Provider AarhusKarlshamn Sweden AB

Matrix ( testing method) Oil, vegetable

Testing method HPLC


AOAC 2001.13 Determination of Vitamin A (Retinol) in Foods Liquid Chromatography/

0.15 µg/g to 1 g/g



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Methods of analysis

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Table 4. Recommended methods of analysis (CodexStan 234-1999)-

Nutrient Matrix

Method

Reference

Principle

Vitamin A Infant formula and follow-up formula AOAC 974.29 Colorimetry

Vitamin A Margarine AOAC 960.45 Spectrophotometry

Vitamin A Minarine AOAC 960.45 Spectrophotometry

Vitamin A Special foods AOAC 974.29 Colorimetry

Retinol

Isomers

Infant formula and follow-up formula AOAC 992.04 Liquid

chromatography

Retinol Infant formula and follow-up formula AOAC 992.06 Liquid

chromatography

Remarks

Vitamin A is sensitive to UV radiation and to oxidizing agents. Vitamin A is very

sensitive to light and all preparations must be carried out in subdued lighting, preferably

gold lighting. The handling of food is extremely important

To compare the values obtained by HPLC. the definition of retinol should taken into

account

Old Data

o Colorimetric Carr & Price

.


o The colorimetric Carr-Price reaction of separation on ion-exchange columns

prone to interference.

Latest review paper on subject

Christopher John Blake (2007) Status of Methodology for the Determination of Fat-

Soluble Vitamins in Foods, Dietary Supplements, and Vitamin Premixes. JAOAC

International 90, 897-910


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Methods of analysis

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References




Brubacher, G., Muller-Mulot, W.; Southgate, D.A.T.., eds. 1985. Methods for the

determination of vitamins in foods. London, Elsevier Applied Science Publishers.



DeVries J W. Silvera KR. 2002 Determination of Vitamins A (Retinol) and E (alpha-

Tocopherol) in Foods by Liquid Chromatography: Collaborative Study JAOAC

International 85 , 424-434



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Methods of analysis

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(iv) Vitamin B1 Thiamin

Figure 3- Structure of Vitamin B1 (reprint from Southgate & Greenfield, 2002)

Golden Standard

EN 14122:2003 Foodstuffs- Determination of vitamin B1 by HPLC

Method Indicator

Name

Code

Scope

Determination of Vitamin B1in foodstuffs by HPLC . Vitamin B1 is the mass fraction of total

thiamin including the phosphorylated derivatives

Principle

Thiamin is extracted from food after acid hydrolysis followed by dephosphorylation using an

enzymatic treatment and quantified by HPLC with pré-or-post-column derivatisation to

thiochrome

Key steps

Extraction

o Acid hydrolysis is carried out by hydrochloric acid or sulphuric acid

Enzyme treatment

o The dephosphorylation can depend on sample matrix and on the enzyme

used. Taka diastase is used currently.


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Methods of analysis

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o thiamin pyrophosphate or thiamin monophosphate is added to test sample to

check the enzyme activity .

Identification

o Pre-column oxidation

Polyphenols are presented in same foods and can inhibited the

oxidative conversion of thiamin to thiochrome. The recovery of the

method should be checked. L

Stationary phase reverse phase

Mobile phase methanol and acetate buffer

Retention time of sample solution versus retention time of standard test

solution

o Pos-column oxidation

Stationary phase normal phase

Methanol and phosphate buffer containing tetraethlyammonium-

chloride and sodium heptanesulfonate

Detection

o Pre -column oxidation

Fluorometric excitation: 366nm and emission; 435 nm

o Pos-column oxidation

Fluorometric excitation: 368nm and emission; 440 nm

Quantification

o External calibration using a calibration curve of thiamine chloride

hydrochloride.


control

Method Performance*

Table 5. Results of inter laboratory studies

Matrix

Parameter

Tube

Feeding

Baby food Powdered

milk

Meal with

fruits

yeasts

Repeatability 7 % 8 % 7 % 7 % 9 %


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Methods of analysis

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RSDr

Reproducibility

RSDR

32% 21 % 16 % 19 % 13 %

*Results obtained in 1995

Table 6. Results of inter laboratory studies

Matrix

Parameter

Cereal Cereal Chocolate

powder

Food

supplement

Repeatability

RSDr

4 % 6 % 8 % 8 %

Reproducibility

RSDR

19 % 14 % 19 % 15 %

*Results obtained in 1995


o BCR –121; wholemeal flour

o BCR-421; milk powder

o BCR-485; mixed vegetables

o BCR-487 pig’s liver

o NIST-2387 Peanut butter

o NIST 1546 – Meat Homogenate

o NIST 2383 – Baby Food

o VMA 399 – Cereal

o NIST 1846- Infant formula powdered

o NIST 8435 Whole-milk powder

o NIST 3244 Protein powder


o WEPAL;

o Inter2000


Codex-Adopted-AOAC 942.23 Thiamine (Vitamin B1) in Human and Pet Foods

Spectroscopy/Fluorometer, Chromatography



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Methods of analysis

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AOAC 953.17 Thiamine (Vitamin B1) in Grain Products Fluorometric (Rapid) Method

AOAC 957.17 Thiamine (Vitamin B1) in Bread Fluorometric Method

AOAC 986.27 Thiamine (Vitamin B1) in Milk-Based Infant Formula

Spectroscopy/Fluorometer

Remarks

Thiamin is sensitive to heat and alkaline conditions. Appropriate precautions must be

undertaken during its analysis

Alternative HPLC conditions are described

Thiamin is present in foods combined with phosphate and must be hydrolysed and

treated with phosphate before analysis(2) + Diverse laboratories practices did not affect

overall performance of HPLC method

Old Data

o Microbiological,

o fluorimetry

o HPLC


o Three methods give similar results (Deharveng, 1999)

Latest review

o Blake, C.J.,(2007). Analytical procedures for water-soluble vitamins in foods

and dietary supplements:a review. Analytical Bioanalytical Chemistry,389, 63-

76

Chen P, Wolf WR. 2007LC/UV/MS-MRM for the simultaneous determination

of water-soluble vitamins in multi-vitamin dietary supplements. Anal Bioanal

Chem387(7):2441-8


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Methods of analysis

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References




Bell, J.G. 1974. Microbiological assay of vitamins of the B-group in foodstuffs.

Laboratory Practice, 23:235-242,252

Van den Berg, H., van Schaik, F.Finglas, P.M.,& de Froidmont,I. 1996. Third EUMAT

intercomparison on methods for the determination of vitamins B1,B2 and B6 in food.

Food Chem., 57:101-108



Wimalasiri, P.& Wills, R.B.H. 1985. Simultaneous analysis of thiamin and riboflavin in

foods by high-performance liquid chromatography J. Chromatogr., 318: 412-416



·Konings. Erik J.M (2006) Water-Soluble Vitamins. Journal of AOAC INTERNATIONAL

89 285-288



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Methods of analysis

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(v) Vitamin B2 Riboflavin

Figure 4- Structure of Riboflavin (reprint from Southgate & Greenfield, 2002)

Golden Standard

EN 14152:2003- Foodstuffs – Determination of Vitamin B2 by HPLC

i. Method Indicator

Name

Code

Scope

Determination of the vitamin B2 in foodstuffs by high performance liquid chromatography

(HPLC). The determination of vitamin B2. The determination content is carried out by

measurement of riboflavin,

Principle

Riboflavin in an apprpriate sample solution is determined after acid hydrolysis followed by

dephosphorylation using na enzymatic treatment by HPLC separation with fluorometric

detection


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Methods of analysis

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Key steps

Extraction

o Extraction is carried out by hydrochloric acid or sulphuric

Enzyme treatment

o The dephosphorylation is checked with vitamin B2 phosphates or riboflavin-5’-

phosphate.

o The enzyme used for the dephosphorylation ,is taka-diastase, and may

contain riboflavin. The amount of riboflavin in enzyme has to be consider in the

calculation of result

Identification

o Stationary phase- Normal phase ( Column Supelco)

o Mobile phase -Methanol: phosphate buffer containing tetraethyl

ammoniumchloride and sodium heptanesulfonate

o The riboflavin in the sample can be identified by comparison of the retention

time of the peak in the chromatograms obtained with the sample solution and

standard solution ( external standard)

Detection

o Fluorometric detector : excitation 468nm; emission 520 nm

Quantification

o Calibration curve.

o Comparison of the peak areas or peak heights of standard substance versus

sample solution


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Methods of analysis

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control

Method Performance

Table 7. Results of certification campaign of Reference Materials carried out by IRMM

Matrix

Parameter

CRM 421

milk powder

CRM 487 Pig liver

Repeatability

RSDr

3.17 % 1.71%

Reproducibility

RSDR

7,31 % 7.89 %


o BCR421 – Milk powder

o BCR 487- Pig liver

o NIST 2384 Baking Chocolate

o NIST 1546 Meat Homogenate


o VMA 399 Cereal

o NIST 1846 Infant formula powder

o NIST 8435 whole –milk powder

o NIST 2385 Slurried spinach



o FAPAS

o WEPAL


AACC-AOAC 981.15 Riboflavin in Foods and Vitamin Preparations Fluorometric

Method, Automated Method applicable to products which contain >=0.1 µg/g



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Methods of analysis

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AOAC 985.31 Riboflavin in Ready-To-Feed Milk-Based Infant Formula Fluorometric

Method

Table 8. (CodexStan 234-1999)-Recommended methods of analysis

Matrix

Method Reference Principle Type

Special foods AOAC 970.65 Fluorometry II

Remarks

Old Data

o HPLC

o Fluorimetry

o Microbiological


o Similar results are reported using microbilogical, fluorimetry and HPLC

methods (Van den Berg , 1996)

Latest review on methods of analysis

Chen P, Wolf WR. 2007LC/UV/MS-MRM for the simultaneous determination

of water-soluble vitamins in multi-vitamin dietary supplements. Anal Bioanal

Chem387(7):2441-8

Blake, C.J.,(2007). Analytical procedures for water-soluble vitamins in foods

and dietary supplements:a review. Analytical Bioanalytical Chemistry,389, 63-

76

References





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Methods of analysis

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Van den Berg, H., van Schaik, F.Finglas, P.M.,& de Froidmont,I. 1996. Third EUMAT

intercomparison on methods for the determination of vitamins B1,B2 and B6 in food.

Food Chem., 57:101-108



Fellman,J.K., Artz , W.E.,Tassinari,P.D., Cole C.I. & Augustin,J. 1982. Simultaneous

determination of thiamin and riboflavin in selected foods by high -performance liquid

chromatography. J. Food Sci. , 47:2048-2050,2067

Bell, J.G. 1974. Microbiological assay of vitamins of the B-group in foodstuffs.

Laboratory Practice, 23:235-242,252



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Methods of analysis

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(vi) Vitamin C (L(+) ascorbic acid and dehydro L(+)

ascorbic acid)

Figure 5- Structures of the common compounds with vitamin C activity (reprint from Southgate and

Greenfield, 2002)

Golden Standard

EN 14130:2003 Foodstuffs - determination of vitamin C by HPLC

Method Indicator

Name

Code

Scope

HPLC - method for determination of Vitamin C in foodstuffs. Vitamin C is the sum of L(+)

ascorbic acid and dehydro L(+) ascorbic acid

Principle

Vitamin C is extracted from the sample to be analysed using metaphosphoric acid solution. A

reducing solution is used to transform dehydro L(+) ascorbic acid. Total L (+) ascorbic acid

content is determined by HPLC with UV

Key steps

Extraction

o Vitamin C is extracted from the sample to be analysed using metaphosphoric

acid solution


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Methods of analysis

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Separation

o Vitamin C is separated from matrix through several steps including the addition

of L-Cysteine to sample extract solution and decreasing the pH (value

between 2,5 to 2,8) by adding metaphosphoric acid solution. The final solution

is filter and used for chromatography

Identification

o L ascorbic acid by comparison of the retention time of individual peaks in the

chromatograms of test sample with standard or by adding standard to the

sample

o Stationary phase: Reverse Phase

o Mobile phase potassium dihydrogen phosphate and methanol

Detection

o UV 265 265 nm : retention time 11,953 min (in the above conditions)

Quantification

o Using a calibration graph and check the linearity of the calibration graph


Control

Method Performance

Table 9. Results of inter laboratory studies EN 14130:

Matrix

Parameter

Orange

Juice

Liquid

Soup

Powdered

Milk

Freeze-

dried

soup

Breakfast

Cereal

Fruits

baby

food

Repeatability

RSDr

≤ 4.2 % ≤ 3.6 % ≤6.3% ≤8.8% ≤9.9% ≤5.3%

Reproducibility

RSDR

≤19.7% ≤ 21,6% ≤ 11.4% ≤ 15.5% ≤19.3% ≤18.0%


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Methods of analysis

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o BCR-431 BRUSSELS SPROUTS

o VMA 399 Cereal

o BCR 421 Milk powder

o NIST 1846 Infant formula powder

o NIST 3244 protein powder


o muva kempten matrix Food (infant) on milk basis Vitamin C

o Determination of Total Vitamin C in Fruit Juices and Related Products by Liquid

Chromatography: Interlaboratory Study (Brause,2003)


AOAC 967.21 Ascorbic Acid in Vitamin Preparations and Juices2,6-Dichloroindophenol

Titrimetric Method

AOAC 984.26 Vitamin C (Total) in Food Semiautomated Fluorometric Method

AOAC 985.53 (1998) Vitamin C (reduced ascorbic acid) in ready- to-feed milk-based

infant formula

Table 10. CodexStan 234-1999)-Recommended methods of analysis

Matrix


Special foods AOAC 967.21 Colorimetry

(dichloroindophenol)

II

Special foods AOAC 967.22 Microfluorometry II

Remarks

Old Data

o All analytical methods to measure total vitamin C, are broadly comparable

according with Deharveng & Southgate 1999.



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Methods of analysis

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o Titrimetry measures gives low results because only measure Ascorbic Acid

(Greenfield & Southgate)

Comparability of Old Data

Caution to preparation of food samples vitamin C content decrease with storage

time (Southgate 1985)

Variability due to region and season for potatoes is described by Nordbotten, 2000

References




Nordbotten A, Loken EB, Rimestad AH (2000) J. Food Comp. Anal. 13:369-377



Deutsch, M.J. & Weeks, C.E. (1965). Microfluorimetric assay for vitamin C. J. Assoc.

Off. Agric. Chem., 48: 1249-1256

Schlack, J.E. (1974). Quantitative determination of L-ascorbic acid by gas-liquid

chromatography. J. Assoc. Off. Anal. Chem., 57: 1346-1348

Speek,AJ. Schrijver, J. & Schreurs, W.H.P. (1984). Fluorometric determination of total

vitamin C and total isovitamin C in foodstuffs and beverages by high-performance liquid

chromatography with precolumn derivatization. J. Agric. Food. Chem., 32:352-355

Brause A R. Woollard D C Indyk H E. 2003 Determination of Total Vitamin C in Fruit

Juices and Related Products by Liquid Chromatography: Interlaboratory Study JAOAC

International 86, 367-374


http://www.sciencedirect.com/science?_ob=IssueURL&_tockey=%23TOC%236879%232002%23999849995%23341462%23FLP%23&_auth=y&view=c&_acct=C000057403&_version=1&_urlVersion=0&_userid=2460193&md5=026e7b5945fc48c8c44a1488c1bf9392


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Methods of analysis

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(vii) Vitamin D3, Vitamin D2

(viii)

Includes two forms of vitamin D, cholecalciferol (D3) and ergocalciferol (D2), both used in

fortification. Foods of animal origin also contain 25-hydroxy-cholecalciferol.

Figure 6- Structures of the main compounds in foods with vitamin D activity (reprint from Southgate and

Greenfield, 2002)

Golden Standard

Foodstuffs - Determination of vitamin D by high performance liquid chromatography -

Measurement of cholecalciferol (D3) and ergocalciferol (D2). CEN Standard: EN 12821: 2000.

AOAC Method 2002.05 Cholecalciferol in Selected Foods

Method Indicator

Name

Code

Scope

Vitamin D determination in foods by HPLC. Vitamin D is naturally present in the majority of

foods as Vitamin D3 (cholecalciferol) and this is the form determined. Vitamin D2 (ergocalciferol)

is sometimes present in fortified foods and can also be determined using this method. Some

foods contain both vitamins D3 and D2 and this method is not applicable to those samples.


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Methods of analysis

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Principle

Vitamin D3 (cholecalciferol) and D2 (ergocalciferol) are saponified using alcoholic potassium

hydroxide solution and are extracted by a solvent. The determination of vitamin D3 or D2 in

sample extract solution is by semi-preparative normal phase HPLC followed by reversed-phase

analytical HPLC. If vitamin D3 is to be determined, then vitamin D2 is used as an internal

standard. If vitamin D2 is to be determined, then vitamin D3 is used as an internal standard.

Vitamin D is detected by ultraviolet (UV) spectrometry and peaks are identified based on

retention times and additionally by UV spectral profile if diode array detection is used.

Key steps

Saponification

D3 is saponified using alcoholic potassium hydroxide solution in a reflux

apparatus at boiling in a water bath

Extraction

o The Vitamin is extracted with n-heptane after vigorously shake (solid foods and

oils margarine, oils, infant formula, milk powder are wash with KOH).

Separation

o The fraction that contains Vitamins is separated by semipreparative cleanup

using normal-phase liquid chromatography. After evaporation and dilution in

acetonitrile-methanol, vitamin D3 is determined by reverse-phase LC with UV

detection at 265 nm.

Identification

o The identification of D2/D3 in semipreparative cleanup process is based on

retention time (17 min).

o The determination of Vitamin D3 should be free from interfering components as

determined by either a wavelength ratio (265:289 nm) or spectral monitoring

and peaks should be separated with a resolution better than 1.5

Detection

o Vitamin D is detected by ultraviolet (UV) spectrometry 265nm


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Methods of analysis

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Quantification.

o Quantification is based on the internal standard procedure using peak areas or

peak heights


control

Method Performance

Table 11. Results of inter laboratory studies of AOAC 2002.05

Matrix

Parameter

Mean g/100g (RSDr) (RSDR) Recovery

Milk

0,418

4.6 9.1 ------

Gruel 1.38 5.9 12.1 -----

Cooking oil 4.61 7.4 24.1 102

Margarine 8.39 6.5 6.8 ----

Infant formula 10.1 2.4 7.1 93.9

Fish Oil 11.6 2.2 17.7 92.9


o BCR –CRM 421 Vitamin D (Cholecalciferol)

o BCR - CRM 122 Vitamin D (Cholecalciferol)

o NIST 1846 Infant formula


o DGF;

o WEPAL

o AOCS


Bioassay



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Table 12. Codex stand 234-1999 Recommended methods of analysis

Nutrient Matrix


Vitamin D

Margarine AOAC 936.14 Bioassay II

Vitamin D

Minarine AOAC 936.14 Bioassay II

Vitamin D

Special foods AOAC 936.14 Rat bioassay IV

Vitamin D (D3,

milk based

infantformula)

Special foods AOAC 992.26 Liquid

chromatography

II

Remarks

Old Data

o Biological assay

o Colorimetry

o GC

o HPLC

o Rádio-immunoassay

Compatibility old data

o Older methods give unreliable data (Deharveng,1999)

Review paper on the subject

o Christopher John Blake (2007) Status of Methodology for the Determination

of Fat-Soluble Vitamins in Foods, Dietary Supplements, and Vitamin Premixes.

JAOAC International 90, 897-910

Others

o The best method should separate the three forms. Vitamin D is found at very

low concentrations.(Southgate, 2002)

o Vitamin D is found at very low concentrations (Southgate,2002)


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References






EuroFIR assistance to this method/guideline


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Methods of analysis

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(ix) Vitamin K1 and Vitamin K2

Vitamin K activity is phylloquinone(K1)+ menaquinones(K2)+menadione (synthetic K3)

Figure 7- Structures of the main natural compounds with vitamin K activity (reprint from Greenfield and

Southgate, 2002)

Golden Standard

EN 14148:2003 Foodstuffs - Determination of vitamin K1 by HPLC

Method Indicator

Name

Code

Scope

the determination of vitamin K1 in foodstuffs by high performance liquid chromatography

(HPLC). The determination of Vitamin K1 content is carried out by measurement of reduced

phylloquinone.

Principle

After enzymatic removal of fat from the sample vitamin K1 is determined in an appropriate

sample solution by high performance liquid chromatographic separation coupled with post-

column reduction and subsequent fluorometric detection. Vitamin K1 isomers are quantified as a

single unresolved peak with a C18 column


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Key steps

Extraction

the presence of lipid, which must be removed by digestion with lipase before extraction with

hexane. The solvent is evaporate under a stream nitrogen and residue dissolve in

methanol, which is applied to reverse-phase HPLC column. The eluated is reduced post-

columnwith zinc

Separation

o Stationary phase reverse phase column

o Mobile phase dichloromethane + methanol + zinc chlorideacetate solution

(indicative)

o Post-column reductor A stainless steel or glass column placed between

analytical column and fluorescence detector

Identification

by comparison of the retention time of the peak in the chromatograms obtained with the

sample test solution and with the standard solution.

Peak identification can also be performed by adding small amounts of the appropriate

standard solutions to the sample test solution.

Detection

Fluorometric, Excitation: 243 nm; Emission: 430 nm

Quantification

By external calibration, integrate the peak areas or determine the peak heights of the

sample and compare the results with the corresponding values for the standard substance.

The calculation is based on calibration curve prepared by standard solutions.

.


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Methods of analysis

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Criteria for analytical performance and Analytical

Quality control

Method Performance

Table 13. Results of inter laboratory studies carried out during preparation of EN 14148:2003

Matrix

Parameter

UHT

whole

liquid

milk,

non-

fortified;

Goat

whole

milk

powder,

non-

fortified;

Milk-

base

infant

formula,

oil-

filled,

fortified;

Whey-

based

infant

formula,

partially

oil-

filled,

fortified;

Soy-

based

infant

formula,

oil-

filled,

fortified;

Whey-

based

infant

formula,

oil-

filled,

fortified;

Whey-

based

infant

formula,

partially

oil-filled,

fortified;

NIST

SRM

1846, dry

blended

infant

formula

Repeatability

RSDr%

9,03

3,23

4,24

4,77

2,59

5,11

4,44

5,68

Reproducibility

RSD R%

10,94

5,81

5,50

6,63

4,33

7,66

4,56

6,78



o NIST 1846 Infant formula


o Non registry in the database


AOAC 999.15 Vitamin K in Milk and Infant Formulas Chromatography/Liquid

Chromatography >1 µg vitamin K1/100 g solids



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Nutrient Matrix


trans-Vitamin

K1

(Phylloquinone)

Ready-To-Feed

Milk-Based Infant

Formula

Codex-Adopted-AOAC

992.27

Liquid

Chromatography (75-

130 µg/L trans-vitamin

K1)

II

Old Data

o Colorimetry .

o Column chromatography

o CG

o HPLC


o No information is given either Southgate book or Deharveng paper on

compatibility

Latest review paper on subject

Christopher John Blake (2007) Status of Methodology for the Determination of Fat-Soluble

Vitamins in Foods, Dietary Supplements, and Vitamin Premixes. JAOAC International 90,

897-910

Remarks

Vitamin K is sensitive to alkali and UV radiation, appropriate precautions need to be

taken during analytical operations.

Most authors comment on great variability of the values and emphasize the need for

proper repeat sampling and replication of analysis

Vitamin K1 standard substance (Phyllochinone, 3-Phythylmenadione) can be obtained

from various suppliers. The purity of the phylloquinone standard may vary. It is therefore

necessary to determine the concentration of the calibration solution by UV-spectrometry

The vitamin K1 concentration in the final sample solution is very low. It is therefore

necessary to perform all operations with clean glassware to avoid contamination


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Methods of analysis

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References






Woolard, D.C., Indyk H.E., Bertram, Y.F and Cook, K.K.: Determination of Vitamin K 1

Isomers in Food by liquid Chromatography with C 30 Bonded-Phase Column, J. AOAC

intern. 85, 2002, 682-691

Indyk, H.E., and Woollard, D.C.: Vitamin K in Milk and Infant Formulas: Determination of

Phylloquinone and Menaquinone-4. Analyst 122, 1997, 465-469.



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Methods of analysis

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(x) Folic acid and Folates

Comprise a group of compounds related to folic acid. Folic acid does not occur naturally in

foods. Naturally folates are conjugates with 2 or more gama glutamyl residues and need to be

deconjugated to be absorbed and metabolically.

Figure 8- Structures of folacin (folates) (reprint from Southgate and Greenfield, 2002)

Golden Standard

EN


_________________________________________________________________________________

Methods of analysis

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Scope

A microbiological method for the determination of total folate content of foods by turbidimetric

detection of the growth of the micro-organism Lactobacillus casei, subp rhamnosus (ATCC

7469). The method allows for the determination of folates in foodstuffs, including naturally-

occurring folates and added folic acid (pteroylmnoglutamic acid).

Principle

Samples suspended in phosphate buffer are heated to enable extraction of folates. Protease

and -amylase treatment may be used to further digest the food matrix. Naturally occurring

folypolyglutamates are hydrolysed with -glutamyl hydrolyase (EC 3.4.19.9) to folylmono- or

folydi-glutamates. Extracted folates are diluted with basal medium containing all required

growth nutrients except folate. The growth response of Lactobacillus casei, subsp. rhamnosus

(ATCC 7469) to extracted folates is followed turbidimetrically and is compared to the growth

response to standard solutions of folic acid with known concentration. The method allows for the

optional use of a semi-automated liquid-handling system and a microplate or test tubes for

incubation of the micro-organism.

Key steps

Extraction

o Use of -glutamyl hydrolyse from fresh hog kidneys is recommended but

other hydrolyses (e.g. chicken pancreas, human/rat blood plasma) can be

used provided enzyme activity is verified;

Detection

o Use of absorbance at 282nm to measure folic acid concentrations of

stock standard solutions.

Quantification

o External procedure

Criteria for acceptance of data may be assay-format dependent,

and shall be established in each laboratory. As a minimum, the

following parameters shall be considered:


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Methods of analysis

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Maximum turbidity of inoculated blanks;

minimum turbidity of the most concentrated calibration

solution;

variation between replicate absorbance values obtained at

each calibration level;

variation of calculated folate amounts between all

dilutions of test sample solution;


control

Method Performance

Criteria for acceptance of data may include the checking of results with 5-

formyltetrahdrofolic acid as a standard substance. Results should be equivalent with

those produced with folic acid as a standard substance.

o Repeatability relative standard deviation (RSDr)<15%;

o Reproducibility relative standard deviation (RSDR) <25%.


o BCR 121- Wholemeal flour (total folate)

o BCR -421- Milk powder (total folate)

o BCR- 485 Mixed Vegetables (total folate)

o BCR -487 Pig’s liver (total folate)

o VMA-399 Cereal (Folic acid)

o NIST 1846 Infant formula powder (Folic acid)

o NIST 3244 Protein powder (Folic acid)


o FAPAS



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AOAC 2004.05A Total Folates in Cereals and Cereal Foods Microbiological,

Spectroscopy .Folate (Folic Acid) or naturally occuring folates of 7.6 µg/100g to 100%

folate. Uses 3-enzyme extraction procedure to free bound folates

The use of stable isotope dilution LC/MS/MS assays was proposed as reference

method (Blake,2007)

LC methods are currently published in literature the following key steps are

(Blake,2007)

o Liberation of folates from the food matrix

o Deconjugation from polyglutamates to the mmonoglutamate and diglutamate

forms

o Purification of folates from matrix

o Determination of folates and or folic acid by LC

Latest review on Folic acid and folates analysis in foods

o Arcot J, Shrestha A (2005) Folate: methods of analysis.Trends Food Sci Technol

16:253–266

o Quinlivan EP, Hanson AD, Gregory JF (2006). The analysis of folate and its metabolic

precursors in biological samples. Anal Biochim348:163–184

Remarks

Old Data (before 1999)

o Radio –immune assay

o HPLC

o AOAC using Streptococus faecalis

Compatibility of old data (before 1999)

o The performance of microbiological assay depends on bacteria used (

o HPLC methods have possibility to measure different fractions, although

performance in collaborative studies has not satisfactory


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o Radio-immune assay no results available for different folate forms

o Analytical methods are neither inter-compatible nor consistent , resulting in

incomparable values.

References






Blake, C.J.,(2007). Analytical procedures for water-soluble vitamins in foods and

dietary supplements:a review. Analytical Bioanalytical Chemistry,389, 63-76


[email protected]

mailto:[email protected]


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Methods of analysis

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(xi) Vitamin B12 - Pantothenic acid

(CAS 79-83-4). It is present bound to proteins or in the form of salts. Only the dextro-form is

active

Figure 9- Structure of pantothenic acid (reprint from Southgate and Greenfield, 2002)

Reference Standard

AOAC 992.07 (Codex recommended method) Pantothenic acid in milk –based infant formula

(xii) Method Indicator

Name

Code

Scope

Determination of pantothenic acid in milk based infant formula

Principle

Bound pantothenic are released to free form, and assay by turbidimetric microbiological growth

response using Lactobacillus plantarum as the test microorganism.

Key steps

Extraction

o the food is extracted with water and where the food is rich in fats these are best

removed before analysis. The aqueous extract is usually autoclaved and the

pH adjusted with acid and alkali to around pH 6.8


_________________________________________________________________________________

Methods of analysis

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Identification

o The mixture of test sample with lactobacillus plantarum after incubation

overnight is heat treated and growth of lactobacillus is measured

turbidometrically

Detection in a calibrate photometer

o Draw a calibration curve best representing 3 or more individual curves. Using

inoculum solution content (pantothenic acid and lacto bacillus plantarum) and

standard stock solution (pantothenic acid standard solution) and measure

transmittance at any specific wave length between 540 nm and 660nm

Quantification

o For each level of test solution used determine the amount of vitamin by

interpolation of standard concentration response curve by plotting % T reading

of each level against cell content (mg dry weight) of respective tube


control

The following parameters need to be consider

The minimum Transmittance of tubes containing highest level of standard inoculated

solutions

The maximum Transmittance of uninoculated blank level

variation between replicate of transmittance values obtained at each calibration level;

variation of calculated amounts between all dilutions of test sample solution

Method Performance

Table 15. Results of inter laboratory studies obtained during AOAC 992.07 method validation

Matrix

Parameter

Milk Based Infant

Formula

Repeatability

RSD r %

4.59%

Reproducibility

RSDR%

10.23%


_________________________________________________________________________________

Methods of analysis

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o NIST 2387 Peanut butter

o NIST 1546 Meat Homogenate


o VMA 399 Cereal

o NIST 1846 Infant formula powdered

o NIST 8435 Whole milk powder



o In the database non registry for PT schemes on panthothenic acid

i. Other methods available

o A general procedure by HPLC with fluorescence detection for free and total

pantothenic acid with fluorescence detection developed by Pakin, (Pakin,2004)

is being evaluated by CEN TC275

Extraction with pepsine, pantetheinase and alkaline phosphatase

Purification on strong anion-exchanhe solid-phase extraction cartridges

Separation on a C18 column

Postcolumn reaction involving alkaline hydrolysis of pantothenic acid to

-alanine. Reaction of -alanine with o-phthaldialdehyde in the

presence of 3-mercaptopropionic acid to form fluorescent 1-alkylthio-2-

alkylisoindole


Matrix

Method Reference Principle

Infant formula and

follow-up formula

The Analyst 89 (1964)(1) 3-6,

232

US Dept Agr., Agr. Handbook

97 (1965)

Microbioassay

Enriched foods

Special foods

AOAC 945.74 Microbioassay

non-enriched foods

Special foods

The Analyst 89 (1964):1, 3-6,

ibid. 232

US Dept Agr., Agr. Handbook

97 (1965)

Microbioassay



_________________________________________________________________________________

Methods of analysis

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Remarks

Pantothenic acid in free form is unstable and extremely hygroscopic.

The classical method is microbilogical using lactobacillus plantarum . the food is

extracted with water and where the food is rich in fats these are best removed before

analysis

Old Data

o Non information available


o Non information available

Latest review on subject

Chen P, Wolf WR. 2007LC/UV/MS-MRM for the simultaneous determination of

water-soluble vitamins in multi-vitamin dietary supplements. Anal Bioanal

Chem387(7):2441-8

Blake, C.J.,(2007). Analytical procedures for water-soluble vitamins in foods and

dietary supplements:a review. Analytical Bioanalytical Chemistry,389, 63-76

References






Pakin C, Bergaentzlé M, Hubscher V, Aoudé-Werner D, Hasselmann C (2004) J

Chromatogr A 1035:87–95

Mittermayr R, Kalman A, Trisconi MJ, Heudi O (2004) J Chromatogr A 1032:1–6

Rychlik M (2003) Analyst 128(7):832–837


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Methods of analysis

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Rychlik M, Roth-Maier D (2005) Int J Vit Nutr Res 75:218–223

Sadecka J, Karasova G, Polonsky J (2003) Eur Food Res Technol 216:440–444

Woollard DC, Indyk HE, Christiansen SK (2000) Food Chem 69:201–208


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