Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Table 1 Fat- and water-soluble vitamins: forms, function, and potential health benefits

Vitamin Vitamers and related forms Function Potentialhealth benefits

Fat-soluble vitamins

A Retinal, retinoic acid, retinol cis-isomers,

b-carotene, other carotenoids

Needed for normal vision, reproduction, and various tissues;

immune function

Protection against various cancers

D Cholecalciferol (D

), ergocalciferol (D

1-a-25-dihydroxycholecalciferol

Enhances calcium absorption; regulates calcium/phosphate

metabolism; promotes bone mineralization

Reduces risk of osteoporosis

E All-rac-a-tocopherol, b-, d-, g-tocopherols,

various tocotrienols

Prevents lipid oxidation of membranes and PUFAs from

autoxidation; normal immune function and healthy tissues

Reduces risk of cardiovascular disease,

precancerous lesions, and cancer

K Phylloquinone (K

), menaquinone (K

menadione (K

)

Essential for normal blood clotting, formation/maintenance of

healthy bones

Reduces risk of blood clotting and

osteoporosis

Water-soluble vitamins

Thiamin, thiamin monophosphate (TMP),

thiamin pyrophosphate (cocarboxylase, TTP)

complexed to proteins

Helps convert carbohydrates into energy metabolism;

required by brain, nervous system and heart

Important role in energy metabolism

Flavin mononucleotide (FMN), flavin adenine

dinucleotide (FAD)

Involved in carbohydrate, protein and fat metabolism; helps

release energy into cells

Important role in energy metabolism;

healthy skin

Niacin Nicotinic acid, niacinamide (nicotinamide),

nicotinamide adenine dinucleodite (NAD) and

nicotinamide adenine dinucleotide

diphosphate (NADP)

Involved in carbohydrate, protein and fat metabolism Reduces risk of arteriosclerosis

Pyridoxine, pyridoxal, pyridoxamine and

related phosphorylated forms

Essential for proper protein utilization; involved in

homocysteine metabolism

Reduces risk of cardiovascular disease

(with folate and B

) and osteoporosis

Cyanocobalamin, methyl- and

hydroxycobalamins, adenosylcobalamin

(coenzyme B

)

Needed for red blood cell formation, DNA and RNA syntheses,

involved in homocysteine metabolism

Prevents certain anemias and reduces risk

of cardiovascular disease

Folic acid (folacin) Pteroylmonoglutamic acid, 5-methyl- and

10-formyltetrahydrofolic acid, tetrahydrofolic

acid as both mono- and polyglutamates

Involved in amino acid interconversions and methylation

reactions, needed for red blood cell formation, DNA and

RNA syntheses, involved in homocysteine metabolism and

protects against neural tube defects

Prevents certain anemias and birth defects,

reduces risk of cardiovascular disease

(with B

and B

) and certain cancers

Pantothenic acid Coenzyme A, pantetheine and acyl carrier

protein (ACP)

Active in protein, carbohydrate and fat metabolism, formation

for certain nerve-regulators

General health and well-being

Biotin Biotin, desthiobiotin Active in protein, carbohydrate, and fat metabolism Helps develop healthy nails

C Ascorbic and dehydroascorbic acids,

isoascorbic acid

Helps form/maintain collagen; important for healthy tissues

and wound healing; enhances iron absorption; protects

against free-radical damage and infection

Protects against cancer, cardiovascular and

eye diseases; reduces symptoms of colds

and flu, and risks of osteoporosis

Adapted from Francis FJ (ed.) (2000) Vitamins: survey. In: Encyclopedia of Food Science and Technology, 2nd edn., vol. 4, pp. 2440–2449. New York: Wiley.

tbl0001

Table 2 Vitamins: commercial forms, food forms and rich dietary sources

Vitamin Major commerical forms Main food forms Rich dietary sources

Fat-soluble vitamins

A Retinol acetate & palmitate, b-carotene Retinly esters, carotenes (provitamin A compounds) Fatty fish, butter, margarines and liver

D Cholecalciferol, ergocalciferol Cholecalciferol Fatty fish, eggs, margarines

E(RRR)-a-Tocopherol, (RRR)-a-tocopherol acetate,

all-rac-a-tocopherol, all-rac-a -tocopherol

succinate

Tocopherols (a-, b-& g-forms) and tocotrienols (a-& b-) Vegetable oils

K 2-(R,S-)-Vitamin K

(cis/trans mixture), menadione

sodium bisulfite

Phylloquinone (K

), menaquinones (K

) Green leafy vegetables, beef, liver

Water-soluble vitamins

Thiamin chloride HCl, thiamin mononitrate, thiamin

diphosphate

Thiamin; thiamin mono- and pyrophosphates complexed to

proteins

Cereals, potatoes, liver, yeast extract

Riboflavin, riboflavin sodium phosphate Riboflavin; riboflavin mononucleotide, flavin adenine

dinucleotide complexed to proteins

Liver, milk, eggs, green vegetables

Niacin Niacin, niacinamide Nicotinic acid; nicotinamide adenine dinucleotide (NAD) and

its phosphate form (NADPH) complexed to proteins

Meat and meat products, cereals

Pyridoxine hydrochloride, pyridoxal-5

-phosphate Pyridoxine, pyridoxal and pyridoxamine and their

phosphorylated forms bound to proteins

Meat and meat products, eggs, cereals

Cyanocobalamin, hydroxycobalamin Methyl-, adenosyl-, hydroxy-, and sulfitocobalamins Kidneys, eggs, milk, and cheese

Folic acid (folacin) Folic acid Folic acid; 5-methyltetrahydrofolate, 10-

formyltetrahydrofolate, tetrahydrofolate as mono- and

polyglutamates

Leafy green vegetables, cereals, yeast, and

vegetable extracts

Pantothenic acid Calcium pantothenate,

D-panthenol Pantothenic acid, coenzyme A and acyl carrier protein (ACP) Liver, kidney, yeast, wheat germ, and egg

yolk

Biotin

D-Biotin Biotin and biotin bound to lysine and proteins Widely distributed especially liver, kidney,

yeast and eggs

C Ascorbic acid, ascorbyl palmitate, sodium

ascorbate

Ascorbic acid, dehydroascorbic acid and isoascorbic acid Citrus fruits and vegetables

Information from Francis FJ (ed.) (2000) Vitamins: survey. In: Encyclopedia of Food Science and Technology, 2nd edn. vol. 4, pp. 2440–2449. New York: John Wiley also Food Standards Agency (2002) McCance &

Widdowson’s The Composition of Foods, sixth summary edition. Cambridge: Royal Society of Chemistry.

tbl0002

Table 3 Recommended dietary allowances (RDA) and reference nutrient intakes (RNI) for vitamins in adult males and females

Category Age

(years)

A(mgofRE

perday)

D(mgofD

per day)

E(mgofa-TE

perday)

K(mg

per day)

(mgper day) B

(mg

per day)

Niacin (mg

perday)

(mg

per day)

(mg

per day)

Folate (mg

perday)

C(mg

per day)

RDA

Males 25–50 1000 5 10 80 1.5 1.7 19 2.0 2.0 200 (400) 60

Females 25–50 800 5 8 65 1.1 1.3 15 1.6 2.0 180 (400) 60

RNI

Males 19–50 700 1.0 1.3 17 1.4 1.5 200 40

Females 19–50 600 0.8 1.1 13 1.2 1.5 200 40

Taken from Food and Nutrition Board, National Research Council (1989) Recommended DietaryAllowances (10th edition), Washington, DC: National Academy Press; Report of the Panel on Dietary Reference Values of

the Committee on Medical Aspects of Food Policy (1991) Dietary Reference Values for Food Energy and Nutrients for the UK. London: HMSO.

RE, retinol equivalents.

TE, a-tocopherol equivalents.

Nicotinic acid equivalents.

Dietary folate equivalent (DFE) for adults in the USA, rising to 600 mg per day during pregnancy and 500 mg per day during lactation (See Folic Acid: Physiology). Taken from Food and Nutrition Board, Institute of

Medicine. National Academy of Sciences, Subcommittee on Folate, other B-Vitamins and Choline (1998) Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B- 6, Folate, Vitamin B-12, Pantothenic Acid, Biotin and

Choline. Washington, DC: National Academy Press.

tbl0003

from animal studies, and are generally revised every

5–10 years by panels of expert scientists. The recom-

mended intakes can vary from country to country

owing to a number of factors such as different inter-

pretations of investigations into the determination of

requirements, the level of the safety margin needed,

and the amounts needed to allow for individual vari-

ation in the general, healthy population. The recom-

mended dietary allowances (RDA) and recommended

nutrient intakes (RNI) used in the USA and the UK,

respectively, are given in Table 3 for adult males and

females. (See Coronary Heart Disease: Intervention

Studies; Dietary Reference Values; Dietary Require-

ments of Adults; Dietary Surveys: Surveys of National

Food Intake.)

0014 It is also becoming evident that some vitamins have

additional health benefits when given, via the diet or by

supplementation, at levels above those required for

eliminating classical deficiency states. Although, for

most vitamins, the optimal levels are not yet estab-

lished, for some vitamins such as folic acid, the health

benefits have been clearly demonstrated for taking

higher intakes to protect against spina bifida or other

neural tube defects. (See Cobalamins: Properties and

Determination; Physiology Folic Acid:Propertiesand

Determination; Physiology Food Fortification.)

Availability

0015 The way in which vitamins are absorbed and utilized

in the body can be affected by a number of factors

that can be broadly divided into dietary-related, and

physiological factors related to the host. The vitamin

may not be in a form that can be readily absorbed by

the body. For example, nicotinic acid occurs in cereals

in a form that is not absorbed from the gut. Other

components in the diet may also interact with the

vitamin, causing an enhancement, or depression, of

absorption. Thus, the nutritive value of a diet for

a given vitamin may be somewhat different from

the amount actually analyzed chemically. Thiamin

requirements are higher when the diet contains large

amounts of carbohydrate or alcohol. Similarly, when

the intake of polyunsaturated fat is high, vitamin E

requirements are also raised. (See Bioavailability of

Nutrients.)

0016 In general, for food tables, allowances are normally

made for reduced biological activities of different

forms of three vitamins: 13-cis-retinol and retinalde-

hyde (vitamin A), carotenes other than b-carotene

(provitamin A compounds), and tocopherols and

tocotrienols other than a-tocopherol (vitamin E).

Adverse Effects

0017 Along with increasing evidence of health benefits

from consumption of diets containing vitamins at

levels much higher than the RDAs, there is concern

over potential adverse effects and even toxicity.

Vitamins can be generally divided into two

groups: first, those vitamins with a safety level of

100 times or more the RDA for that vitamin for

which there is no clear indication of any adverse

effects, including most of the water-soluble vitamins

and the tocopherols; Second, those vitamins with

a safety level of about 10 times the RDA, above

which serious adverse reactions can occur. These vita-

mins include retinol, calciferols, phylloquinone, and

pyridoxine.

0018In particular, chronic intakes of vitamins A and D

are to be avoided. For example, an excessive intake of

vitamin D can lead to vitamin D intoxication, which is

characterized by an increase in serum 25-OH-D and

is associated with hypercalciuria and hypercaemia.

The safe upper limit for vitamin D is 25–50 mgper

day (1000–2000 IU per day). Pregnant women should

avoid large doses of vitamin A, either by avoiding

foods, such as liver, that contain high concentrations

of vitamin A, or by avoiding multivitamin supplements

that contain doses of vitamin A of > 5000 IU.

0019As for the water-soluble vitamins, there has been

some concern about excessive intakes for nicotinic

acid and pyridoxine. Vitamin B

intakes as low as

50 mg per day can be associated with possible neuro-

logical damage in patients. Similarly, the potential

benefits of lipid-lowering effects of high nicotinic

acid intakes (2–6 g per day) have to be considered

alongside the possible toxic effects, particularly for

the liver. One of the main concerns of folic acid

fortification is its potential to mask the diagnosis of

pernicious anemia, which is caused by vitamin B

malabsorption, at intakes of 1 mg per day, causing

progressive and irreversible nerve damage. (See

Bioavailability of Nutrients; Functional Foods.)

Refer to individual vitamins.

Effects of Processing on Vitamin Stability

0020Vitamin stability varies greatly and depends on a

number of factors such as temperature, oxygen, acid

or alkali strength, and so on. Table 4 provides general

information on stability (S) or instability (U) under

several conditions. Several processes have been de-

veloped to produce stabilized forms of the vitamin,

including spray drying in a suitable matrix (e.g.,

gelatin) and encapsulation.

0021Consumer interest over recent years in the nutri-

tional labeling of foods, and the increased awareness

in health benefits of vitamins, has focused attention

on the effects of food processing on vitamin stability.

It should be remembered also that one of the major

benefits of processing is that it has a marked effect on

preserving vitamin concentrations by inactivating

VITAMINS/Overview 6051

food enzymes despite any immediate loss. (See Food

Composition Tables.)

0022 The content of vitamins in processed foods is deter-

mined initially by the content of the raw materials

used. There is a wide variability in vitamin concen-

trations of commercially grown foods. For example,

carrots may vary in their carotene concentration 100-

fold, and vitamin C can vary by as much as 35-fold.

This can be due to a number of factors, including

variety differences, climate and growing conditions,

maturity at harvest, and postharvest storage condi-

tions. Thus, the vitamin content of raw materials for

processing can vary enormously, in many cases

exceeding that found in normal processed food.

0023 The different chemical and physical properties of

the vitamins mean that they vary widely in their

degree of stability. In the pure crystalline form,

they retain their activity for long periods of time.

However, when foods are processed, vitamins are

subjected to a range of conditions that can be detri-

mental to their stability such as light, moisture,

oxygen, heat and pH, these being typical environ-

mental factors to which the food will be subjected in

the food chain (see Table 4). The most labile vitamins

are retinol, ascorbate, folate, and riboflavin. Niacin

and biotin are the most stable, but information

on some of the vitamins, especially vitamin D, is

incomplete.

Domestic Cooking

0024Typical percentage losses of vitamins on cooking for

selected food groups are given in Table 5.

tbl0004 Table 4 Vitamin stability under different conditions

Vitamin Oxygen Light Heat Acid (pH<7)

Alkali (pH>7)

Metals

Reducingagents

AUUUUS

DUUUUU

EUUSSS

KSUSSU

USUS U UU

SUUS U UU

Niacin S S S S S U S

SUUS S SU

UUUS S

Folic acid U U U U S S S

Pantothenic acid S S U U U

Biotin S S U S S S

CUUUSUUS

Adapted from Chichester CO (1973). Nutrition in food processing. In: Rechcigi M (ed.) Food Nutrition and Health. World Review of Nutrition and Dietetics, vol.

16, pp. 319–326. Washington, DC: Karger-Basel.

In the absence of oxygen.

Information from van der Meer MA (1972) Voeding 33: 277. S, stable; U, unstable.

tbl0005 Table 5 Typical percentage losses of vitamins on cooking for selected food groups

Vitamin Cereals Milk Meats Fish Vegetables Fruit

Boiling Baking Boiling

Baked dishes Meat, grilled or fried Baking Boiling Stewing

A0000

D 0

E202000

40 25

10 25 20 30 35 25

40 15 10 15 20 20 20 25

Niacin 40 5 0 5 20 20 30 25

40 25 10 25 20 10 40 20

5 5 20 10

Folate 50 50 20 50 20 40 80

Pantothenate 40 25 10 25 20 20 25

Biotin 40 0 10 10 25

C5050 4525

Adapted from Food Standards Agency (2002) McCance & Widdowson’s The Composition of Foods, sixth summary edn. Cambridge: Royal Society of

Chemistry.

In milk-based drinks and custards.

15% in bread-making and toasting.

6052 VITAMINS/Overview

0025 Vitamin C is one of the most labile of all the vita-

mins and is readily leached into water on boiling,

particularly when the surface of the food has been

cut or damaged. Vegetables are an important source

of vitamins in the diet, and typical losses on boiling

vegetables are quite variable. The use of microwaving

can reduce vitamin losses for cooked vegetables (see

below). (See Cooking: Domestic Techniques.)

0026 Meat is also an important source of several vita-

mins. A considerable amount of B-group vitamins,

especially thiamin, can be lost when meat is grilled

or fried (up to 40%). Losses are less when pot

roasting and braising are used. Some vitamins that

leach into the meat juices during cooking will not be

lost if the sauce or the gravy is eaten as part of the

actual dish. This means that average losses in meat

dishes are no higher than for grilled or fried meat,

even though the cooking times are longer. (See Meat:

Dietary Importance.) Similarly, when bread is baked,

30% of the thiamin content can be destroyed, and

this can increase to as much as 80% if alkaline baking

powders are employed.

0027 Microwave cooking can result in retention of vita-

mins equal to, or greater than, conventionally cooked

vegetables. However, a number of studies have shown

that cooking time, water volume used, etc. affect

considerably vitamin retention with microwave

cooking, as with other cooking procedures. For meat

and meat products, the effect of microwave versus

conventional cooking on vitamin retention seems to

be less pronounced. A higher degree of riboflavin

retention and a lower degree of thiamin retention

have been found, but no difference in niacin concen-

trations. (See Cooking: Domestic Use of Microwave

Ovens.)

Other Processing Methods

0028 There has been only limited work reported on the

effects of extrusion cooking on vitamin retention. In

one of the few studies, the retention of B-group vita-

mins was investigated during crispbread production.

The destruction of the more labile vitamins (thiamin,

folate, B

, and B

) was directly proportional to the

energy input, although other factors, such as moisture

content, mass temperature, screw geometry, and rate

of throughput, are also thought to influence reten-

tion. It appears that retention can be improved by

increasing both the rate of throughout and moisture

content. However, carotenoids in corn starch are

fairly stable to extrusion, although some of the

forms can be oxidized further. Losses of vitamin C

during extrusion of 25–40% have been found. (See

Extrusion Cooking: Chemical and Nutritional

Changes.)

0029Radiation-induced changes in vitamin concentra-

tions in foods during irradiation have been reviewed

extensively in the literature. At low irradiation doses

(< 1 kGy) used for disinfectant treatment, losses are

negligible compared with the larger variations in the

vitamin content of the raw materials. The most

thermolabile vitamins are thiamin, tocopherols,

and ascorbate, but, with the medium-dose range

(1–10 kGy) used to control microbial contamination,

losses can be minimized. (See Irradiation of Foods:

Applications.)

0030It is well known that fermentation can increase the

B-vitamin content (e.g., folates) of some foods, such

as cheese, milks, yogurts, and other dairy foods, and

this is a potential area for production of foods with

enhanced health benefits. There does appear to be

some potential for producing significant amounts of

vitamins with various fermentation conditions and

organisms.

See also: Antioxidants: Natural Antioxidants; Synthetic

Antioxidants; Bioavailability of Nutrients; Cobalamins:

Properties and Determination; Physiology; Coenzymes;

Dietary Reference Values; Dietary Requirements of

Adults; Dietary Surveys: Surveys of National Food

Intake; Folic Acid: Properties and Determination;

Physiology; Food Fortification; Hypovitaminosis A;

Pregnancy: Maternal Diet, Vitamins, and Neural Tube

Defects; Vitamin B

: Properties and Determination

Further Reading

Ball GFM (1998) Bioavailability and Analysis of Vitamins

in Foods. London: Chapman & Hall.

Bates CJ and Heseker H (1994) Human bioavailability of

vitamins. Nutrition Research Reviews 7: 93–127.

Chichester CO (1973) Nutrition in food processing. In:

Rechcigi M (ed.) Food Nutrition and Health. World

Review of Nutrition and Dietetics, vol. 16, pp. 319–326.

Washington, DC: Karger-Basel.

Francis FJ (ed.) (2000) Vitamins: survey. In: Encyclopedia

of Food Science and Technology, 2nd edn. vol. 4,

pp. 2440–2449. New York: John Wiley.

McCance RA and Widdowson EM (2001) The Compos-

ition of Foods, 6th summary edn. London: HMSO.

Taylor TG and Jenkins NK (eds) (1986) Food processing.

In: Proceedings of the XII International Congress of

Nutrition, pp. 786–803. London: John Libbey.

Van den Berg H, Heseker H, Lamand M, Sandstru

m B and

Thurnham D (1993) Flair Concerted Action No 10 Vita-

min Status Papers. International Journal for Vitamin and

Nutrition Research 63: 247–316.

Walter P, Sta

helin H and Brubacher G (eds) (1989) Elevated

Dosages of Vitamins. Toronto: Hans Huber.

Ziegler EE and Filer LJ (eds) (1996) Present Knowledge in

Nutrition, 7th edn. Washington, DC: ILSI Press.

VITAMINS/Overview 6053

Determination

R Eitenmiller and L Ye, University of Georgia, Athens,

GA, USA

Vitamins and Food Composition

0001 From a metabolic and human health aspect, under-

standing of the roles that vitamins play is rapidly

expanding. Concurrently, new and more precise

information on food composition has been required

by the scientific community. One of the more chal-

lenging areas of food composition research is the

development and application of methods suitable to

accurately assay water- and fat-soluble vitamins in

foods. We have seen this area of bioanalytical chem-

istry expand rapidly over the past three decades to the

point that, for most of the vitamins, we can confi-

dently provide accurate measurements from most

matrices. In the following discussion, we present a

brief review of useful approaches for the analysis of

vitamins in food. Also included are tables giving per-

tinent details of methods useful for the analysis of the

specific vitamins. These approaches from the current

literature show the state-of-the-art methods de-

veloped by leading vitamin researchers. Because of

the significance of microbiological and chromato-

graphic techniques to the area, our discussion is

limited to these analytical approaches.

Analytical Methods

0002 Methods available for vitamin assay can be classified

as bioassays using animals or humans, microbio-

logical assays that are based upon specific vitamin

requirements of bacteria, yeast, molds, or proto-

zoans, and physicochemical methods that are based

on instrumental methods of analysis, including, but

not limited to, spectrophotometric, fluorometric

radiometric, immunological, and chromatographic

techniques. Most recent advances in vitamin analysis

have come through development and application of

high-performance liquid chromatographic (LC) tech-

niques specifically designed for food analysis. Factors

influencing the suitability of a method for analysis of

a vitamin(s) in food include the specificity of the

assay, sensitivity of the method and levels of the vita-

min in the food matrix to be assayed, the method’s

proven value as documented by validation studies

including precision and accuracy, the applicability of

the method to the food matrix, numbers of samples to

be assayed in relation to manpower and sample

throughput needs, availability of required instrumen-

tation, and cost requirements. While excellent,

sophisticated methods are available for most vita-

mins, these techniques are often not available for

limited analytical efforts because of initial set-up eco-

nomic considerations. In such instances, lower-cost,

older methods of analysis must be utilized. This

should not be considered as a limiting factor of the

quality of the data because, if properly conducted,

older methods based on wet chemistry or microbio-

logical assay can provide data of excellent quality.

0003Because of the instability of many vitamins to oxi-

dation, light inactivation, pH, heat, and various other

chemical and environmental factors, precautions must

be taken throughout the analysis to ensure stability of

the analyte. For this reason, it is important that the

analyst be thoroughly informed about the chemical

properties of the specific vitamin being assayed

(See Vitamins: Overview). Additionally, because the

stability of the vitamin must be considered throughout

the sample-handling period (collection through

storage through analysis), poor-quality data will be

obtained if degradative processes occur through im-

proper maintenance of conditions necessary to ensure

vitamin stability. This outcome negates the worth of

even the most advanced analysis technique.

Extraction Methods

0004Except for bioassays that require the feeding of intact

food samples, the vitamin(s) must be extracted from

the food matrix in a medium designed to stabilize the

vitamin prior to the determinative analytical step.

Because of the diverse stability characteristics of the

vitamins, specific extraction procedures have been

developed over the years that accomplish efficient

extraction of the various vitamins. Extraction media

must be compatible with the determinative method

and at the same time stabilize the vitamin against

degradation. Table 1 outlines some common ap-

proaches used to extract the various vitamins along

with the primary routes that might lead to degrad-

ation. Most extraction procedures use a combination

of physical and chemical effects to ensure analyte

stability. The overall extraction includes physical

treatment combined with use of the extraction solv-

ent to remove the vitamin with a high rate of recovery

from the sample matrix, provide stability to the

extracted vitamin, and provide the chemical environ-

ment required for compatibility with the determina-

tive procedure. Physical disruption to reduce the

particle size of the food sample is often accomplished

through grinding, sonication, or other means.

Following the initial sample treatment, solubilization

of the vitamin into the extraction solution is com-

pleted by heat, acid, alkali, or enzyme digestion,

often applied in combination. Extraction procedures

6054 VITAMINS/Determination

are available for single vitamins and for multianalyte

assays in which more than one vitamin is assayed

from the same extract.

Water-soluble Vitamins

Microbiological Analysis

0005 The microbiological assay of water-soluble vitamins

represents one of the earliest approaches for accurate

analysis of water-soluble vitamins in foods. Except

for vitamin C, microbiological assays are available

for each of the water-soluble vitamins. These methods

were originally developed in the 1940s concurrently

with, or shortly after, the development of bioassays

for the different vitamins. The assays have withstood

the test of time, often being applicable to the analysis

of low-concentration samples that are problematic

for other methods. Microbiological assays are highly

specific and sensitive, although less technologically

advanced, compared with many physicochemical

procedures.

0006All microbiological vitamin assays are based upon

the development of a dose–response curve resulting

tbl0001 Table 1 Extraction procedures applicable to vitamin analysis of food

Method Example Primaryroutes of degradation

Fat-soluble vitamins

Vitamin A, vitamin A esters,

provitamin A, carotenoids,

vitamin E, vitamin D

Direct solvent extraction Hexane or ether containing an

antioxidant. Maintain

darkness.

Oxidation

Light

Heat

Acid

Vitamin A, vitamin A esters,

provitamin A, carotenoids,

vitamin E, vitamin D

Saponification Reflux at 70



C in ethanolic KOH

containing antioxidant.

Extract digest with hexane or

combination of organic

solvents containing an

antioxidant.

Oxidation

Light

Heat

Acid

Vitamin K Hydrolysis and solvent

extraction

Lipase hydrolysis. Pentane

extraction. Maintain pH

control. Solid-phase clean-up.

Unstable at alkaline pH

Water-soluble vitamins

Ascorbic acid Metaphosphoric acid

extraction

Solvent usually contains

reducing agent and metal

chelating agent

Oxidation

Temperature

Light

Ascorbic acid

Oxidase

Thiamin Acid hydrolysis followed by

enzyme hydrolysis of

phosphate esters

0.1 N HCl, autoclave,

phosphatase digestion

Heat

Unstable at alkaline pH

Riboflavin Acid hydrolysis 0.1 N HCl, autoclave protect from

light

Light

Heat

Niacin Acid or alkaline hydrolysis HCl or H

digestion cereals –

alkaline digestion

Few stability problems

Vitamin B

Acid extraction or enzymatic

hydrolysis of phosphate

esters following extraction

in buffered solvent

Plant products – 0.44 N HCl,

autoclave. Animal products –

0.055 N HCl, autoclave.

pH, stable in acid solutions,

light

Folate Multienzyme digestion in the

presence of a reducing

agent

Trienzyme digestion with

Pronase

a-amylase and

conjugase

Oxidation

Light

Heat

Avoid pH below 5.0

Vitamin B

Phosphate buffer containing

reducing agent

Autoclave in phosphate buffer

containing citric acid and

sodium metabisulfate

Light

Optimum stability between pH

4.0 and 7.0

Oxidation

Biotin Acid hydrolysis Autoclave in 2 N H

(plant

products) or 6 N H

(animal products)

Oxidation

Stable between pH 4.0 and 9.0

Pantothenic acid Multienzyme digestion Digestion with alkaline

phosphatase and

pantotheinase

Stable between pH 5.0 and 7.0

VITAMINS/Determination 6055

from the growth of a microorganism in a medium

containing the vitamin that is essential for the specific

microorganism. The microorganism must have an

absolute nutritional requirement for the vitamin that

is not modified by other components in the food

extract. If this requirement is met by the microorgan-

ism, a basal medium can be formulated that provides

all of the growth requirements for the microorganism

except for the vitamin being assayed. When a stand-

ard or an extract of the material to be assayed is

added to the clear growth medium, the microorgan-

ism grows in proportion to the amount of added

vitamin. Over a defined vitamin concentration range,

the growth response will be directly proportional to

the amount of vitamin in the growth medium. Within

this range, sample and standard solutions can be

accurately compared. The dose–response curve is de-

veloped by adding known amounts of standard to the

growth medium. Unknown levels in the sample are

calculated from the dose–response curve using the

growth response resulting from the vitamin added to

the growth medium through a known amount of

sample extract. Growth is measured by turbidity,

by production of a metabolite such as lactic acid, by

respiration, or gravimetrically.

0007 Characteristics that microorganisms must possess

to be useful for water-soluble vitamin assay include:

(1) a specific requirement for vitamin forms that

are biologically active in higher animals; (2) inability

to synthesize the vitamin; (3) inability to utilize

metabolites that are not biologically active for the

human; (4) inability to utilize compounds that are

structurally related to the vitamin that are not bio-

logically active; (5) a rapid growth cycle; (6) a growth

response that is not easily modified by other sub-

stances present in the food extract (stimulators or

inhibitors); (7) genetic stability and (8) nonpathogeni-

city. A list of accepted microorganisms available from

the American Type Culture Collection (ATCC),

Rockville, MD is provided in Table 2. Table 2 also

lists the Association of Official Analytical Chemists

(AOAC) International methods for each water-

soluble vitamin assay.

0008 Microbiological vitamin analysis remains an im-

portant tool for laboratories involved in food-

composition studies. Advantages of the analytical

approach include: (1) high sensitivity; (2) capacity to

assay naturally occurring levels from complex food

samples without concentration of the extract; and

(3) low cost due to minimal laboratory requirements

for space, materials, and instrumentation. Disadvan-

tages include (1) labor intensiveness; (2) poor preci-

sion; (3) difficulty to automate, which leads to low

sample throughput; (4) varying growth responses of

the microorganism to different vitamin forms,

including bound forms and multichemical forms (e.g.,

folates); and (5) susceptibility of the assay micro-

organism to stimulators or inhibitors in the food

extract that produce drift. In relation to human nutri-

tion, the assays do not provide information on the

bioavailability of the vitamin from the food sample.

Chromatography for the Assay of Water-soluble

Vitamins

0009High-performance LC methods are available for

analysis of water-soluble vitamins in foods. These

methods offer speed, selectivity, accuracy, precision,

and, depending upon the chemical properties of the

specific vitamin, a high degree of sensitivity. Reversed-

phase chromatography systems using C-18 or other

bonded supports with varying adsorption properties

are widely applied to specific analytical methods for

the analysis of water-soluble vitamins. Specific appli-

cations depending on the chemical properties of the

analytes include ion exchange for vitamin C, folates,

and niacin, PLRP-S (polymeric styrene reversed-

phase macroporous supports) for riboflavin and vita-

min C, and C-18 for most water-soluble vitamins.

Useful mobile phases for water-soluble vitamin analy-

sis on reversed-phase systems are usually based upon

tbl0002Table 2 Microbiological assay of water-soluble vitamins

Analyte Assay organism AOAC International

method

Thiamin Lactobacillus

viridescens

ATCC 12706

Riboflavin Lactobacillus

rhamnosus (casei)

960.46 Chapter 45.2.06

ATCC 7469

Niacin Lactobacillus

plantarum

944.13 Chapter 45.2.04

ATCC 8014 985.34 Chapter 50.1.19

Vitamin B

Saccharomyces

uvarum

961.15 Chapter 45.2.08

ATCC 9080 985.32 Chapter 50.1.18

Folate Enterococcus

hirae

944.12 Chapter 45.2.03

ATCC 8043 992.05 Chapter 50.1.21

Lactobacillus

rhamnosus (casei)

ATCC 7469

Vitamin B

Lactobacillus

delbrueckii

952.20 Chapter 45.2.02

ATCC 7830 986.23 Chapter 50.1.20

Biotin Lactobacillus

plantarum

ATCC 8014

Pantothenic acid Lactobacillus

plantarum

945.74 Chapter 45.2.05

ATCC 8014 992.07 Chapter 50.1.22

From AOAC International (2000) Official Methods of Analysis of AOAC

International, 17th edn. Arlington, VA: Association of Official Analytical

Chemists.

6056 VITAMINS/Determination

methanol or acetonitrile, together with water, acetic

acid or buffers. Ion-pair chromatography, which

combines the principles of bonded-phase and ion-

exchange chromatography is often used to improve

analyte resolution, retention time, and peak shape.

0010 Often, the only limitation to application of LC to

water-soluble vitamins in foods is the low natural

levels that must be quantified. Detection systems

useful for LC analysis of the water-soluble vitamins

include UV, fluorescence, and electrochemical; how-

ever, the choice of the detection mode depends en-

tirely on the properties of the vitamin being assayed.

Low natural levels in foods are often below the detec-

tion capabilities of the LC system. In this case, sample

extract concentration using solid-phase extraction

(SPE), affinity chromatography, or other means such

as freeze-drying might be applied to bring the analyte

into the required concentration range. Often, with

low-concentration samples, microbiological assay

offers a workable solution to the analytical problem.

0011 Pre- and postcolumn derivatization can be used for

vitamins such as thiamin, folate, and others to induce

or increase the fluorescence of the analyte to increase

the sensitivity of the analysis. Fluorescence, because of

its sensitivity and selectivity, is a preferred detection

mode for LC procedures and should be incorporated

into an analysis protocol if chemically feasible. Multi-

analyte procedures useful for food analysis are mostly

limited to fortified foods or supplements in which

the analytes are present in high concentrations, as

opposed to low concentration in natural foods. Some

successful simultaneous assays are available for thia-

min and riboflavin, which possess excellent spectral

properties that make sensitivity less problematic.

0012 Because of the breadth and complexity of the ap-

plications of LC to water-soluble vitamin analysis,

examples of literature methods that have been proven

useful for food composition research are character-

ized in Table 3. The reader is urged to access the

Further Reading list for detailed information.

Fat-soluble Vitamins

0013 Chromatography presents versatile and highly useful

methods for the analysis of fat-soluble vitamins from

foods and other biological samples. The analytical

field has progressed from applications of paper

chromatography, thin-layer chromatography, and

gas chromatography to the almost universal use of

LC for fat-soluble vitamin analysis. Both normal-

phase and reversed-phase systems are applicable to

fat-soluble vitamin analysis.

0014 Efficient extraction of the fat-soluble vitamins

from the sample matrix is as important as the deter-

minative chromatography. With the fat-soluble

vitamins, many LC-based procedures require that

the vitamin be isolated from the lipid material prior

to injection of the vitamin extract on to the LC

column. Applicable extraction methods rely on alka-

line hydrolysis or saponification, enzymatic hydroly-

sis, solvent extraction, and supercritical fluid

extraction. Saponification is the most accepted

method for extraction of vitamin A, carotenoids,

vitamin D, and vitamin E from foods. Saponification

involves refluxing the sample with ethanolic KOH

solution in the presence of an antioxidant (pyrogallol

or vitamin C) at 70



C. The unsaponifiable fraction in

the aqueous digest containing the fat-soluble vita-

mins, sterols, carotenoids, and other material is ex-

tracted with hexane, ethyl ether, or a mixture of

hexane and ethyl acetate, leaving the fatty acid salts

and other water-soluble components in the aqueous

phase. Saponification has the ability to efficiently

destroy the sample matrix, which facilitates vitamin

extraction, frees cellular bound forms of the vitamins,

and destroys substances such as chlorophyls that

might interfere with the chromatography. Because of

the instability of vitamin K under alkaline conditions,

saponification cannot be used for extraction.

0015If the LC method is a normal-phase system, limited

amounts of lipid can be injected directly on to the

column. In this case, solvent extraction of the sample

can be used with direct injection of the extract con-

taining the fat-soluble vitamins on to the normal-

phase column. Direct solvent extraction has been

used to develop multianalyte procedures capable of

assaying several analytes including ester forms of

vitamin A (retinyl palmitate and acetate) and vitamin

E(a-tocopheryl acetate).

Vitamin A and Carotenoids

0016Most recently published methods for retinoids and

carotenoids use reversed-phase LC on C-18. The ad-

vantages of reversed-phase compared with normal-

phase LC include: (1) less sensitivity to changes in

retention time due to the presence of water; (2) more

easily cleared of contaminants; (3) more stable to

small changes in mobile phase composition; (4) more

quickly equilibrated to mobile phase composition

changes, permitting use of gradients; and (5) capable

of resolving compounds with a wide range of polar-

ities. Both isocratic and gradient mobile phases are

useful (see Table 4). For vitamin A (retinol) assay,

reversed-phase chromatography with simple, isocratic

mobile phases consisting of methanol:water, acetoni-

trile:water, or gradients based on these solvents can be

used. Resolution of retinol cis- and trans-isomers is

best accomplished with normal-phase LC.

0017The conjugated double bond system of vitamin A

and its closely related retinoids gives specific and

VITAMINS/Determination 6057