Analytical Procedures for the determination of Vitamins B1 & B2 in foods, feeds and supplements

Part 1: Overview of methods Part 2: An evaluation of selected HPLC Procedures.

A report published as part of the Government Chemist Programme Project CB1.4 - The determination of BB-Vitamins

Analytical Procedures for the determination of Vitamins B1 & B2 in foods, feeds and supplements

Part 1: Overview of methods Part 2: An evaluation of selected HPLC Procedures.

27/09/2007 Report number: LGC/GC/2007/019

Author: Paul Lawrance; LGC. Approved by : Michael Walker

A report published as part of the Government Chemist Programme Project CB1.4 - The determination of BB-Vitamins LGC Limited 2007

Contents
Introduction........................................................................................................................................1 Legislation .........................................................................................................................................1 Occurrence and properties .................................................................................................................2 Vitamin B1 - Thiamin .....................................................................................................................2 Vitamin B2 - Riboflavin..................................................................................................................2 Methods of Analysis ..........................................................................................................................3 Supplements ...................................................................................................................................3 Foods ..............................................................................................................................................4 Part II : Evaluation of methods for the determination of thiamin and riboflavin in foods by HPLC ...........................................................................................................................................................7 Sample extraction (Joint extraction for riboflavin and thiamin) ...................................................7 Determination of Riboflavin...........................................................................................................8 Standards ........................................................................................................................................8 HPLC ..............................................................................................................................................9 Determination of thiamin..............................................................................................................10 Standards ......................................................................................................................................10 Derivatisation ...............................................................................................................................10 Pre-column derivatisation.............................................................................................................10 HPLC ............................................................................................................................................10 Post column derivatisation ...........................................................................................................11 Pre-column vs. post-column derivatisation ..................................................................................12 Method Performance ....................................................................................................................12 Thiochrome formation..................................................................................................................14 Iso-butanol extraction ...................................................................................................................15 SPE Clean-up................................................................................................................................15 Enzymes for enzymatic hydrolysis...............................................................................................16 Summary..........................................................................................................................................17 Determination of Thiamin and Riboflavin in foods and feeds (Protocol) ......................................18 References........................................................................................................................................22

Introduction
Vitamins B1 & B2 are two of the B-group vitamins required by humans for maintenance of health and the prevention of disease. They are present in many foodstuffs and are also added to fortified foods and dietary supplements. It is important to have reliable methods of analysis for these vitamins both for nutritional reasons and also for official control purposes when these vitamins are added to foods and supplements. Manufacturers must be able to demonstrate that label declarations or nutritional claims are accurate and and official control analysts must be able to verify this by analysis.

Legislation
Thiamin and riboflavin are natural components of food. It is generally accepted that ingested thiamin and riboflavin have very low toxicity in humans therefore there are few specific concerns about excess intakes of these vitamins. Deficiency is rare in industrialised countries. Beriberi and other deficiency symptoms are still a concern in developing countries with a staple diet of polished rice but with a more varied diet, intakes are adequate. Mandatory restoration of processed flour with thiamin is required by the Bread and Flour Regulations 1998 and there are compositional standards for both vitamins (inter alia) in the Infant Formula and Follow-on Formula Regulations 1995, the Processed Cereal-based Foods and Baby Foods for Infants and Young Children Regulations 1997 as amended , and in the Foods Intended for Use in Energy Resticted Diets for Weight Reduction Regulations 1997. Riboflavin is also controlled by the Miscellaneous Additives in Food Regulations 1995 in respect of its use as a colorant rather than as a vitamin. Fortification of other foods is on a voluntary basis and there are no specific statutory requirements other than contained in the Food Labelling Regulations 1996 and the Food Safety Act 1990. Schedules 6 & 7 of the Food Labelling Regulations cover nutrition claims and labelling in respect of vitamins and other nutrients and claims in relation to the description of a food as a source of specified nutrients. In dietary supplements, vitamin additives are controlled by the Food Supplements Directive 2002/46/EC which was implemented in England by the Food Supplements (England) Regulations 2003. These regulations specify the vitamins which can be used, and their chemical forms, and have provision for the future establishment of minimum and maximum amounts that may be added, although these are still under evaluation. Manufacturers are required to show the amount of vitamins present in a product and must declare the amount on the label of pre-packed products both in numerical terms and for supplements, as a percentage of the Recommended Daily Amount (RDA) for the vitamin(s) concerned. The RDAs are given in Schedule 7 of the Food Labelling Regulations 1996 and in the Nutrition Labelling for Foodstuffs Directive, 90/496/EEC. In feeds, the provisions of the Feeding Stuffs (England) Regulations 2005 (& equivalent legislation) and the Feeding Stuffs (Sampling and Analysis) Regulations 1999 as amended, apply
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although there are no specific requirements for thiamin and riboflavin. Additives in feed are subject to Regulation (EC) No 1831/2003 of the European Parliament and of the Council on additives for use in animal nutrition but again there are no specific requirements for these vitamins.

Occurrence and properties

Vitamin B1 - Thiamin Vitamin B1 is the name given to thiamin and related compounds that have similar biological activity.
Chemical name: Molecular formula: Molar mass: 2-[3-[(4-amino-2-methyl- pyrimidin-5-yl) methyl]- 4-methyl-thiazol-5-yl] ethanol C12H17N4OS+ 265.36

Thiamin occurs in food as free thiamin, thiamin monophosphate (TMP), thiamin diphosphate (TDP or TPP), thiamin triphosphate (TTP), and also as adenosine triphosphate (ATP) or hydroxyl ethyl thiamin (HET). Supplements are generally fortified with thiamin hydrochloride or thiamin mononitrate. The USP standard is thiamin hydrochloride.( C12H18Cl2N4OS: Molar mass 337.3 g/mol) Thiamin is most stable in acid solutions at pH 2 - 4. Stability decreases as pH increases and thiamin is highly unstable at alkaline pH. Thermal degradation also occurs therefore prolonged heating should be avoided. Vitamin B2 - Riboflavin Vitamin B2 is the name given to riboflavin and related compounds with similar biological activity.
Chemical name: Molecular formula Molar mass
7,8-dimethyl-10-(D-ribo-2,3,4,5-tetrahydroxypentyl)benzo[g]pteridine-2,4(3H,10H)-dione

C17H20N4O6 376.364

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Riboflavin occurs in food as free riboflavin, as riboflavin-5-phosphate (flavin mononucleotide FMN) and as flavin dinucleotide (FAD). Supplements are fortified with riboflavin or, in the case of liquids, with riboflavin-5 phosphate. The USP standard is riboflavin. (C17H20N4O6 : MW 376.36) It is stable in acid solution, even when heated provided it is protected from light It deteriorates on exposure to light, especially in alkaline solution.

Methods of Analysis Analysis

Thiamin and riboflavin can be determined using chemical, microbiological or chromatographic procedures. The choice of method depends on the sample matrix, the vitamin concentration and the forms of the vitamins being extracted. Supplements In the case of supplements, the vitamins are added as stable, water soluble forms which are readily extractable in a acidic medium with or without the use of methanol. EDTA should be used for samples containing minerals as iron or copper can degrade thiamin during extraction. The extracted vitamins are analysed simultaneously by reverse phase or ion-pair HPLC. Since the levels are high, UV detection can be used. The HPLC system must be optimised to resolve the vitamins from other matrix components which may interfere. Note that it is possible to determine liquid supplement drinks by direct injection onto the HPLC. In this case however, it is important to realise that vitamin B2 may have been added as riboflavin 5- phosphate (FMN) because of its greater water solubility. The HPLC must be capable of resolving FMN which must then be quantified separately. The activity of FMN is equivalent to that of riboflavin on the basis that 1.37mg of sodium riboflavin-5-phosphate is equivalent to 1mg of riboflavin. Methods for the analysis of dietary supplements can be found in the United States Pharmacopoeia National Formulary (USP-NF)1. Two or three alternative procedures are given for the determination of thiamin and riboflavin in tablets, capsules and oral solutions with and without minerals. A method for the simultaneous determination of thiamin, riboflavin, niacin, vitamin B6 and calcium pantothenate in food supplements has been developed at LGC with funding from the Food Standards Agency and has been collaboratively tested on a range of supplement products2. Other methods for thiamin and riboflavin in supplements can be found in the scientific literature but may not be fully validated.

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Foods For foods, methods are generally designed to measure total thiamin and riboflavin content. Foodstuffs are extracted using acid hydrolysis to release bound forms followed by enzymatic conversion of the phosphates to free vitamins using takadiastase or acid phosphatase. The vitamins can then be determined using chemical methods, microbiological assay or HPLC. Feeds can be determined using similar procedures to those for food. There are a number of official or compendium methods for thiamin and riboflavin in food. AOAC3 methods rely on non-specific, chemical and microbiological procedures as shown below: Thiamin in human and pet foods Thiamin in grain products Thiamin in bread (& other foods) Thiamin in milk-based infant formula Riboflavin (Vitamin B2) in foods and vitamin preparations Riboflavin (Vitamin B2) in vitamin preparations Riboflavin (Vitamin B2) in foods and vitamin preparations Riboflavin in ready-to-feed, milk-based infant formula AOAC 942.23 AOAC 953.17 AOAC 957.17 AOAC 986.27 AOAC 981.15 AOAC 940.33 AOAC 970.65 AOAC 985.31 Fluorimetry Fluorimetry Fluorimetry Fluorimetry Automated Microbiological Fluorimetry Fluorimetry

Much of the nutritional data available for foods has been obtained using classical, microbiological assay. These assays are still in use at LGC 4and in other laboratories5. Used carefully, microbiological assays produce a reliable estimation of the vitamin content of foods and feeds but the methods are long and require experience to obtain good results. Even then, the analytical precision can be poor compared to more modern techniques. HPLC procedures have been developed and are published in the literature. These and other methods have been reviewed by a number of authors6,7. Notable, collaboratively studied methods have been published by Arella et al8 (food) and by the Analytical Methods Committee of the Royal Society of Chemistry9 (feed) , the former being the basis of two CEN procedures. CEN procedures are available as follows: EN 14122: 2003 Foodstuffs - Determination of vitamin B1 by HPLC. EN 14152: 2003 Foodstuffs - Determination of vitamin B2 by HPLC In these procedures, the vitamins are extracted by acid hydrolysis followed by enzymatic conversion of the phosphates to free vitamins which are then determined using HPLC with fluorescence detection. Riboflavin is naturally fluorescent whereas thiamin has to be converted to thiochrome before detection. This can either be done using pre-column or post- column derivatisation and both options are given. Aspects of these and related procedures are discussed later in part II of this report.

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Part II : Evaluation of methods for the determination of thiamin and riboflavin in foods by HPLC.
Published, HPLC methods for the determination of riboflavin and thiamin by HPLC generally rely on similar extraction principles. Samples are hydrolysed in acid to release the vitamins from bound forms. An enzymatic hydrolysis is then carried out to convert phosphate forms to free vitamins. Riboflavin is then injected directly onto the HPLC system using fluorescence detection. Thiamin has to be converted to thiochrome to facilitate the use of a fluorescence detector for improved sensitivity and specificity. Differences arise in the following areas: Acid hydrolysis conditions Enzymes used and hydrolysis conditions Conversion to thiochrome HPLC

The CEN standards for foodstuffs incorporate all of these steps and are presented as separate standards. In practice, it is convenient to combine the extraction steps and the HPLC can then be carried out on the same HPLC system although not in the same run. Formation of the fluorescent derivative, changes to the detector wavelengths, and perhaps to the mobile phase composition are required for the analysis of thiamine. The combined method was evaluated to assess its suitability for the determination of thiamin and riboflavin in foods. Certified reference foodstuffs were used as test samples. Sample extraction (Joint extraction for riboflavin and thiamin) 5g of sample (wholemeal flour, milk powder and mixed vegetables) and 2.5g for pigs liver were weighed into each of five, 100ml Duran bottles. Empty bottles were added as blanks. 50 ml of 0.1M HCl was added to each bottle, swirling to disperse the contents. The bottles were then autoclaved at 121C for 30 min and cooled in cold water at room temperature. The pH was then adjusted to 4.5 +/-0.1 with 1M NaOH. For each sample, 5ml of a mixed-enzyme preparation* (takadiastase and acid phosphatase) was added to two of the bottles and to a blank. Taka-diastase only, acid phosphatase only or no enzymes were added to the remaining three bottles (& blanks). *The mixed enzyme was prepared by dissolving 4g of taka-diastase {from Aspergillus Oryzae (Pfaltz and Bauer; T00040)} and 0.4g of acid phosphatase {from wheatgerm (Sigma P3627)} in 100ml distilled water using a magnetic stirrer. Single enzyme solutions were prepared in water at the same concentrations.

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The bottles were capped and incubated at 37C overnight. On the following day, the bottles were placed in the autoclave at 100C for 5 mins to deactivate the enzymes before cooling in water to room temperature. The extracts were quantitatively transferred to 100ml volumetric flasks, made to volume with distilled water, mixed and filtered through a Whatman 42 filter paper. For riboflavin the extracts were filtered through a 0.45m PTFE syringe filter and injected onto the HPLC. Thiamin was determined after derivatisation to thiochrome, as described later. Determination of Riboflavin Standards A stock solution of riboflavin was prepared by dissolving 20 mg (+/- 0.1mg) in 50ml of 0.1M HCl. This was stirred for 15 min on a magnetic stirrer, protected from light with aluminium foil. It was then transferred to a 500ml flask with ~400ml water, placed in an ultrasonic bath until dissolved and made up to volume with water. This produced a nominal concentration of 40g/ml. A stock standard of riboflavin 5-phosphate was also prepared by dissolving 20 mg (+/- 0.1mg) in 50ml of 0.1M HCl. A 5ml aliquot of the riboflavin stock was diluted to 20ml with water containing 1.75ml of 0.05M sodium acetate (pH 4). The concentration of riboflavin was determined by measuring its absorbance spectrophotometrically at 444nm and calculating the concentration using an extinction coefficient of 328. Riboflavin 5 phosphate is very soluble in water, however riboflavin is much less soluble and requires stirring and ultrasonication for dissolution. To overcome this, an alternative procedure was applied. Riboflavin is very soluble in dilute alkali but is not stable at this pH. Riboflavin (20mg) was dissolved in 5ml of 0.1M sodium hydroxide. 50ml of 0.1M HCL was then immediately added and the standard made up to 500ml with water. A concentration check was then carried out as above. The concentration of the two standards was equivalent, therefore the latter approach is recommended. The stock standard of riboflavin retained 96% of its original concentration (as determined by spectrophotometry) after one week at ~ 4C in a dark fridge. Intermediate and working standards for riboflavin were prepared by dilution in acidified water. Calibration standards covering the range 0.02 to 1.0g/ml were prepared. A single standard for riboflavin 5-phosphate with a concentration of 1.0 g/ml was also prepared.

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HPLC The standards were injected onto an HPLC system using the following conditions: Column Mobile phase Flow rate Injection volume Oven temperature Detection Genesis C18; 150 x 4.6mm, 3n 0.01M KH2PO4/MeCN/MeOH (60/10/30) 0.7 ml/min 50l 25C Fluorescence Ex: 450nm Em: 520nm

Under these conditions both riboflavin and riboflavin 5-phosphate were detected with retention times of 3 and 2.2 minutes respectively. Note that in published papers, several different reverse phase columns and mobile phases have been used. The chromatography should be optimised to obtain acceptable retention, good peak shape and resolution from interfering peaks, if present. Riboflavin phosphate contains several small impurity peaks, which should be taken into account if this is used as a standard. The material contains mainly riboflavin-5 phosphate with small amounts of free riboflavin, riboflavin diphosphates, lumiflavin and other volatile impurities. It is not normally necessary to use riboflavin phosphate standards as the extraction contains an enzymatic dephosphorylation step to convert phosphates to free riboflavin. It may be required, however if determining fortified drinks by direct injection as riboflavin phosphate is often added because of its superior solubility. The phosphate can also be used as a basic check of the dephosphorylation enzyme activity although it should not be assumed that the activity will be the same in a sample matrix. The calibration curve obtained for riboflavin was linear over the range studied as shown below:
y = 84187x - 673.84 2 R = 0.9999

Riboflavin
90000 80000 70000 60000 Area 50000 40000 30000 20000 10000 0 0.00 0.20 0.40 0.60

0.80

1.00

Concentration

Fig 1: Riboflavin calibration curve

The limit of detection based on 3 x SD of a blank solution was 0.004g/ml.

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Determination of thiamin Standards A stock solution of thiamin (chloride) hydrochloride was prepared by dissolving 25 mg (+/- 0.1) in 250ml of 0.1M HCl to give a concentration of 100g/ml. A thiamin phosphate standard was also prepared. A 1ml aliquot of the thiamin stock was diluted to 20ml with 0.1M HCl. This was used as an intermediate standard from which to prepare the calibration standards, and as a check solution to confirm the concentration by spectrophotometry. The absorbance was measured at 247nm against 0.1M HCl and the concentration calculated using an extinction coefficient of 421. Calibration standards were prepared with concentrations ranging from 0.02 to 1.0g/ml by diluting with water. Derivatisation Thiamin and its phosphates are not naturally fluorescent therefore a derivatisation step must be used to convert them to thiochrome. Thiamin is oxidised to thiochrome using potassium hexacyanoferrate(III) in alkaline conditions. This can either be carried out before (pre-column) or after (post-column) chromatographic separation. Pre-column derivatisation. For thiamin 2ml of sample extract was transferred to a 25ml flask, 5ml of K3Fe(CN)6 (0.04 % in 15 % NaOH ) was added and the extract was shaken for 1 min. 10ml of H3PO4 (10%} was added and the extract made to volume with water. All solutions were filtered through a 0.45m PTFE syringe filter for HPLC. HPLC The HPLC system described under Riboflavin was used except that the fluorescence detector was set to Ex 365nm & Em 435. The calibration was linear over the range studied as shown below. Note that there was no resolution between thiamin and thiamin phosphate however, the phosphate would be converted to free thiamin during sample extraction.

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Thiamin pre column

400000 350000 300000 250000 Area 200000 150000 100000 50000 0 0.000 0.020 0.040

y = 5E+06x - 4504.8 R 2 = 0.9989

0.060

0.080

0.100

Concentration

Fig 2: Calibration curve of thiamin after pre-column conversion to thiochrome

Post column derivatisation Post-column derivatisation was carried out by adding a T-piece and a 125cm reaction coil to the end of the HPLC column. The derivatisation reagent (as pre-column) was pumped into the Tpiece at 0.3ml/min and the reaction coil was maintained at 37C. The thiochrome retention time was ~ 3 minutes. The calibration curve was linear over the range studied.
THIAMIN POST COLUMN y = 3E+06x - 51324 R2 = 0.9995

1800000 1600000 1400000 1200000

Area

1000000 800000 600000 400000 200000 0 0.000 -200000 0.100 0.200 0.300 0.400 0.500 0.600

Concentration (ppm )

Fig 3: Calibration curve of thiamin after post-column conversion to thiochrome

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Pre-column vs. post-column derivatisation The advantages of post-column derivatisation are a reduction in sample handling, standardisation of the derivatisation, lack of delay between derivatisation and analysis and protection of the analytical column from aggressive reagents. Disadvantages are the need for a T-piece, reaction coil and supporting equipment, the potential for damage to the T-piece, coil and reagent pump by the alkaline, oxidizing reagent, sample dilution and peak broadening caused by the added reagents and increased dead-volume between the column and detector. The choice is influenced by the equipment and expertise available and the number of samples to be analysed. If large numbers of samples are routinely analysed, the post-column method is quicker, more reproducible and easier to perform once the set-up and optimisation has been done. Also, the analytical column is not degraded by the derivatisation reagents. For smaller sample numbers, especially where systems are not left intact, the pre-column derivatisation may be easier to use particularly if the same system is used for riboflavin. The precolumn derivatisation was used in this study. Method Performance The sample extracts and the standards (both derivatised in the case of thiamin) were analysed by HPLC. The results obtained are shown below:

Table 1: Riboflavin results (mg/Kg)

Reference material CRM 121 CRM 421 CRM 485 CRM 487

Description Wholemeal wheat flour Milk powder Mixed vegetables Pig liver

Certified value n/a 14.5 n/a 106.8

NO ENZYME

TAKA DIASTASE

ACID PHOSPHATASE

Replicate 1 0.3 14.5 1.8 63.0

Replicate 2 0.2 14.0 1.5 78.0

Mean 0.3 14.2 1.7 70.5

0.3 14.6 1.8 33.2

0.4 14.8 1.8 85

0.3 14.8 1.7 75

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Table 2: Thiamin (mg/Kg)

Reference material CRM 121 CRM 421 CRM 485 CRM 487

Description Wholemeal wheat flour Milk powder Mixed vegetables Pig liver

Certified value 4.63(0.39) 6.51(0.48) 3.07(0.34) 8.6 (1.1)

NO ENZYME

TAKA DIASTASE

ACID PHOSPHATASE

Replicate 1 5.0 7.7 3.7 6.8

Replicate 2 4.0 5.6 2.9 5.0

Mean 4.5 6.6 3.3 5.9

4.9 4.2 2.7 5.6

5.2 6.2 2.9 6.5

4.9 6.0 3.2 7.0

Some enzymes contained small amounts of thiamin or riboflavin, which were corrected using the results obtained for the blank assays. For riboflavin, the results for milk powder agreed with the certified value. The riboflavin is either free or is fully converted by acid hydrolysis as all of the results are similar. In pigs liver, the results are low, especially where no enzymatic hydrolysis was used. Even with enzymatic hydrolysis, the recovery is only ~ 70-80%. It has been suggested 10 that low results are obtained form pork products unless a protease is used additionally but this was not tested. The remaining samples were not certified for riboflavin and only very small results were obtained as expected. For thiamin, all of the results agreed with the certified values although there was some variability between duplicates. The pig liver was again slightly low. The method for riboflavin worked well with clean chromatograms such as that shown below:

Fog 4: Chromatogram of milk powder extract showing riboflavin peak

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Clean-up was not required but if necessary, a sample clean-up and concentration step could be carried out as follows:
Condition a 500mg C18 SPE cartridge with 5ml of MeOH. Load 10 ml of extract Wash with 10ml of 0.05M sodium acetate Elute with 2ml of 70% MeOH in water

(NB/ Riboflavin recovery should be confirmed with the matrix used)

The chromatography for thiamin was acceptable but there were several peaks due to the derivatising reagents and there were a number of late eluting peaks which extended the run time.

Fig 5: Chromatogram of milk powder extract showing thiochrome peak

Thiochrome formation If post-column derivatisation is used, the method described above works well but equipment setup is more difficult. For this reason, pre-column derivatisation is often used. In this case, the procedure used for the formation of thiochrome can have a direct effect on the results and analytical precision obtained. The conversion of thiamin to thiochrome reputedly occurs in a matter of seconds, however the samples may be kept for several minutes or even hours whilst awaiting injection during an HPLC run. Even if a cooled autosampler is used, the thiochrome can continue to react with excess reagents to produce sulphide compounds leading to significant losses. If the thiochrome extracts are injected directly, the high alkalinity will rapidly degrade the HPLC column therefore this is not recommended. Neutralisation of the extract with phosphoric acid as carried out above, prevents column damage due to pH and stops the thiamin degradation but artifacts due to excess reagents remain.
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The derivatisation conditions have been studied by a number of workers and possible clean-ups have been proposed. These include extraction of the thiochrome into iso-butanol prior to HPLC or SPE clean-up on a C18 cartridge. These clean-ups were evaluated as described below: The following procedures were used: Iso-butanol extraction 2ml of sample extract was vortexed with 5 ml of water-saturated isobutanol for ~10 seconds. 5ml of oxidant solution (0.04% K3FeCN6 in 15% NaOH) was added and the solution was mixed by vortexing for another ten seconds. The two layers were allowed to separate (centrifuged if required) and an aliquot of the upper (isobutanol ) phase was placed in an HPLC vial for analysis. Pre-mixing with iso-butanol ensures that the thiochrome is transferred to the isobutanol layer as quickly as possible to prevent any degradation. SPE Clean-up 2ml of sample extract and 5ml of the oxidant solution were vortexed together for 10 seconds. 10ml of 10 % phosphoric acid was added and the solution vortexed again. The extract was then loaded immediately onto a 500mg C18 SPE cartridge pre-conditioned with MeOH & water. The cartridge was washed with 10 ml of 0.05M sodium acetate solution and the thiochrome was eluted with 8 ml of 70:30 methanol/water solution. Both cleanup methods worked well and produced sharper thiochrome peaks with fewer interferences. The isobutanol clean-up is easier to perform and perhaps more reproducible, but either technique can be used. The preparation time in either case was less than ten minutes . Cleaned extracts were stable for several days when stored in a fridge.

Fig 6: Pork liver before clean-up

Fig 7: Pork liver after iso-butanol extraction

Results obtained for the CRM foodstuffs using the butanol clean-up agreed well with the certified values except for the pigs liver which was still low as shown below:
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Table 3: Thiamin results for CRM foodstuffs using optimised procedure.

CRM CRM 485 Mixed Vegetables CRM 121 Wholemeal Flour CRM 487 Pigs Liver CRM 421 Milk Powder

Thiamin Content ug/g (as is) 2.7 4.2 5.9 5.2

Certified Value ug/g (%DM) 3.1 4.6 8.6 5.9

Enzymes for enzymatic hydrolysis The choice of enzymes is not straightforward as these are available from many sources and the activity varies. The use of enzymes in the determination of thiamin and riboflavin has been investigated by various workers in recent years but because of the changing nature of enzyme availability and the variety of samples to which they must be applied, a definitive answer is still not available. Taka-diastase has been used widely and is recommended in the CEN standards. However, takadiastases consist of a number of enzymes (primarily amylases), and phosphatase may be present essentially as an impurity. The activity therefore varies with the source, the production methods and perhaps with different production batches. It is generally agreed at present, that takadiastase form Pfaltz and Bauer (T00040) is most widely applicable for thiamin in foods although this is expensive to use on a regular basis. Clara-diastase (Fluka 27540) has also been found to be widely applicable although this has now been discontinued. Fluka have a direct replacement (Clarase 300 Cat no.86959) although it is not known whether this is directly equivalent for the determination of thiamin and riboflavin. At LGC, a mixed enzyme containing taka-diastase (currently from Pfaltz & Bauer) and acid phosphatase (from wheat germ Sigma P3627) has been used successfully for many years. Several other sources of takadiastase and phosphatase exist with varying activities and the activity may also vary between sample types. The selection of enzymes is not straightforward. For samples containing free thiamin or riboflavin, enzymes are not required. In other cases, release of bound forms and dephosphorylation is enabled simply by acid hydrolysis whereas in many samples, enzymatic hydrolysis is usually required for full extraction of the vitamins. The sample matrix may also affect the activity as will the presence of other enzymes. Hasselmann et al10 showed that a mixture of papain (protease), - amylase and acid phosphatase could be used to obtain good results in a range of foods and suggested that protease was required for full release of thiamin and riboflavin from pork meat. This may explain the low results obtained from pigs liver in the current work.

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No standardised enzyme mixture is yet available and the analyst must confirm that the target analytes are being fully extracted under the analytical conditions used. This can be done by analysis of known samples, CRMs etc or by carrying out method optimisation experiments. If such information is not available, a generally applicable enzyme such as that supplied by Pflatz and Bauer should be used but the potential for incomplete release should be realised. In particular, it has been reported that products containing chocolate may give rise to low thiamin recovery.8

Summary
Thiamin and riboflavin in supplements can be determined by HPLC with UV detection after an acidic extraction. Methods can be found in the USP national formulary and a method has been recently developed at LGC for this purpose. In foods and feeds, samples can be analysed either by HPLC or by microbiological assay. A joint extraction procedure can be used for both thiamin and riboflavin. An enzymatic hydrolysis is required and care is needed to select appropriate enzymes. If HPLC is used, riboflavin is analysed directly by HPLC with fluorescence detection whereas thiamin must be converted to thiochrome before HPLC. A clean-up step for thiamin is recommended to improve the chromatography, preserve column life and improve repeatability. A protocol based on CEN, AMC, LGC and other published procedures is shown overleaf:

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Note: Laboratories making use of this procedure should insert their own Health & Safety, training and validation sections

Determination of Thiamin and Riboflavin in foods and feeds

1. Principle Thiamin and riboflavin are extracted using acid and enzymatic hydrolysis and are then determined using HPLC with fluorescence detection. Riboflavin is analysed directly whereas thiamin is derivatised to thiochrome before HPLC. 2. 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 Reagents Methanol Acetonitrile Hydrochloric acid, 0.1M Sodium hydroxide, 0.1M Sodium hydroxide solution (15% w.v) Dissolve 15g of NaOH in 100ml of water. Phosphoric acid, H3PO4, 10% w/v solution Dilute 10g of concentrated metaphosphoric acid to 100ml with water. Isobutanol (water saturated) Add water to the isobutanol and shake. Ensure that water remains in the flask. Potassium hexacyanoferrate III (1% solution) Dissolve 1g of K3FeCN6 in 100ml of water. Alkaline potassium hexacyanoferrate III solution (0.04% w/v K3FeCN6 in 15% w/v NaOH) Dilute 2ml of 1% K3FeCN6 to 50ml with 15% NaOH. Sodium acetate solution, 0.1M & 0.05M Dissolve 1.36 g or 0.68g of sodium acetate trihydrate in 100 ml of water. Potassium dihydrogen phosphate (KH2PO4), 0.01M Dissolve 1.36 g of KH2PO4 in 1 litre of water. (pH ~5-6) Methanol/water 70/30 Takadiastase (from Aspergillus Oryzae , e.g. Pfaltz & Bauer, T00040} Acid phosphatase (from wheatgerm, e.g. Sigma P3627) Mixed enzyme preparation Suspend 4g of taka-diastase and 0.4g of acid phosphatase in 100ml purified water and stir for 15mins using a stirrer. HPLC mobile phase (0.01M KH2PO4/MeCN/MeOH {60/10/30}) Mix 600ml of 0.01M KH2PO4 with 100ml of acetonitrile and 300ml of methanol. Degas before use.

2.16

3. 3.1 3.1.1 3.1.2

Standards Standard materials Thiamin hydrochloride (USP or similar) Riboflavin (USP or similar)

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3.2 3.2.1

Stock Standards Thiamin (100g/ml) Dissolve 25 mg of thiamin (+/- 0.1mg) in 250ml of 0.1M HCl. Riboflavin (100g/ml) Weigh 25 mg (+/- 0.1mg) of riboflavin into a 250ml volumetric flask. Add 5ml of 0.1M NaOH and swirl to dissolve. Immediately add 25ml of 0.1M HCL and make up to 250ml with water. Protect from light. Concentration check Dilute 10ml of thiamin stock standard to 100ml with 0.1M HCL. Measure the absorbance in a 1cm cell at 247nm against a blank solution of 0.1M HCl. Calculate the concentration using an extinction coefficient (E11%) of 421. Pipette 10ml of riboflavin stock solution into a 100ml volumetric flask. Add 1.75ml of 0.05M sodium acetate and dilute to volume with water. Prepare a blank solution using 1ml of 0.1M HCL and 1.75ml of sodium acetate in 100ml of water. Read the absorbance at 444nm in a 1cm cell against the blank solution and calculate the concentration using an extinction coefficient (E11%) of 328.

3.2.2

3.3 3.3.1

3.3.2

3.4

Calibration Standards Prepare calibration standards for each vitamin by diluting the stock standards with acidified water to give a suitable calibration range (0.02 1 or 1 - 10 g/ml as desired). Note that the thiamin standards must be derivatised before HPLC.

4. 4.1

Extraction Weigh 5g of sample into a 100ml Duran bottle. Add 50 ml of 0.1M HCl and swirl to disperse the contents. Cap, autoclave at 121C for 30 min and cool in cold water to room temperature. Adjust the pH to 4.5 +/-0.1 with 1M NaOH. A reagent blank should be included. Add 5ml of mixed-enzyme preparation to each sample extract, mix, cap and incubate at 37C for 18 hours. After incubation, heat the bottles to 100C for 5 mins to deactivate the enzymes before cooling in water to room temperature. Transfer the extract to a 100ml volumetric flask and dilute to 100ml with water. Filter through a Whatman 42 filter paper. Analysis can be carried out either by microbiological assay or by HPLC.

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4.2

For riboflavin, filter an aliquot of the extract through a 0.45m PTFE syringe filter for HPLC using riboflavin as an external standard. Thiamin is derivatised to thiochrome and cleaned before HPLC, two options are available:

4.3

4.3.1 Option 1: Isobutanol extraction Transfer 2ml of extract to a 15ml centrifuge tube. Add 5ml of water-saturated isobutanol and vortex for 10 seconds. Add 5ml of oxidant solution (0.04% K3FeCN6 in 15% NaOH) and vortex for another 30 seconds. Allow the layers to separate (centrifuge if required) and transfer an aliquot of the upper (isobutanol ) phase to a vial for HPLC. 4.3.2 Option 2: SPE Clean-up Add 5ml of the oxidant solution (as above) to 2ml of sample extract in a 20ml tube or suitable vessel and shake or vortex for 30 seconds. Immediately, add 10ml of 10 % phosphoric acid and mix by shaking. Load the entire extract onto a 500mg C18 SPE cartridge, pre-conditioned with 5ml MeOH and 5ml of water. Wash the cartridge with 10 ml of 0.05M sodium acetate solution and elute the thiochrome with 8 ml of 70:30 methanol/water. Dilute to 10ml with methanol/water. Filter the extract using a 0.45n filter for HPLC. 4.3.3 Inject the samples onto the HPLC system using external thiamin standards which have been derivatised in the same way as the samples. . (Alternatively, the sample extracts can be injected without derivatisation onto an HPLC system set-up for post column derivatisation using the same reagents)

HPLC Conditions The following HPLC conditions have been found to be suitable.

Column Mobile phase Flow rate Injection volume Oven temperature Detection

Genesis C18; 150 x 4.6mm, 3n 0.01M KH2PO4/MeCN/MeOH (60/10/30) 0.7 ml/min 50l 25C Fluorescence Riboflavin Ex: 450nm Em: 520nm Thiamin Ex 365nm Em: 435nm

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Other reverse phase HPLC systems are available. These are mainly C18 columns with methanol/water or ion-pair mobile phases. C18 amine columns using CHCl3 / MeOH have also been used for thiamine.

6.

Calculation Prepare calibration curves (3-5 points) for the range of interest for each vitamin and determine the vitamin concentration of sample test solutions by interpolation. Calculate the sample concentration talking into account the weights and dilutions used.

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References
1

United States Pharmacopoeia National Formulary, 2007 www.USP.org.

Publication pending ;[Collaborative study report published in the Food Standards Agency Information bulletin No 82.]

3 Official Methods of Analysis of AOAC International, 18th Ed. www.AOAC.org.

4

Bell, J., J. Lab. Practice, 1974, 23, 5, 235-242

FDA manual of methods for the microbiological analysis of selected nutrients; 1996, published by AOAC International, ISBN 0-935584-61-7. Eitenmiller, R. & Landen, W. ; in Vitamin Analysis for the Health and Food Sciences, 1999, CRC Press, ISBN 0-8493-2668-0 De Leenheer, A., Lambert, W., Bocxlaer, J., in Modern Chromatographic analysis of the Vitamins, 3rd Edition, 2000, Marcel Dekker Inc., ISBN 0-8247-0316-2. Liquid chromatographic determination of vitamins B1 & B2 in foods. A collaborative study; Arella,F., Lahely,S., Bourguignon,J., Hasselmann, C.; 1996, Food Chemistry, 56(1),81-86. Determination of thiamin and riboflavin in pet foods and animal feedingstuffs - Analytical Methods Committee, Analytical Division, The Royal Society of Chemistry, Analyst, 2000, 125, 353360. Extraction procedures for the liquid chromatographic determination of thiamin, riboflavin and vitamin B6 in foodstuffs, Ndaw, S., Bergaentzle, M., Aoude-Werner, D., Hasselmann,C ; Food Chemistry, (2000), 71, 129-138.
10 9
8 6

The information in this report has been published in good faith and reasonable attempts have been made to ensure its validity. However, neither the author nor LGC can assume responsibility for the consequences of the use of this information by third parties.

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