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

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Table 3 Selected HPLC methods for the analysis of water-soluble vitamins in various foods

Analyte Sample type Sample preparation HPLC parameters References

Column Mobile phase Detection Quality-assurance

parameters

Ascorbic acid

AA Food Dilute samples to an estimated AA

concentration of 100–800 ng ml

1

with

mobile phase. Filter.

Inersil ODS-3

5 mm

150  4.6 mm

100 mM KH

(pH 3)

containing 1 mM EDTA-

2Na:2H

O. 0.6 ml min

1

400 mV

Ag/AgCl

DL (ng)

On-column

0.5

% Recovery

>90

Journal of

Chromatography A

806 (1998)

361–364

AA, DHAA,

IAA, DHIAA

Various foods Extract sample with 1% MPA, 0.5%

oxalic acid (pH 2). For samples high in

starch, extract with 2% MPA, 1%

oxalic acid–EtOH (50:50). Centrifuge

Filter. Inject.

Jupiter C

5 mm

250  4.6 mm

2.3 mM DTMAC:2.5 mM

EDTA in a 66 mM

phosphate–20 mM acetate

butter pH 4.5, 1.2 ml min

1

Postcolumn

derivatization of

dehydroforms

with OPD

fluorescence

350 mm

430 nm

RSDr (%)

1.1–1.6

RSD

(%)

1.3–3.5

Journal of

Chromatography B

730 (1999)

101–111

AA Various foods Dilute samples to an estimated AA

concentration of 1–10 mgml

1

with

mobile phase. Filter.

Inersil ODS-3

5 mm

150  4.6 mm

20 mM MSG (pH 2.1) or GMP

(pH 2.1) 0.8 ml min

1

400 mV

Ag/AgCl

% Recovery >90 Journal of

Chromatography A

881 (2000) 317–

326, 327–330

Thiamin

Thiamin,

TMP, TPP,

TTP

Various foods Add amprolium (IS), homogenize with

5% sulfosalicyclic acid. Centrifuge.

Filter water layer.

Perkin Elmer

3 mm

300  3mm

Gradient 0.1 M Na

, pH 5.5,

6 min 0.1 M Na

, pH 2.6,

19 min

Thiochrome

postcolumn

Fluorescence

339 nm

432 nm

Journal of

Micronutrient

Analysis 2 (1986)

189–199

Thiamin Various foods Add HCl, digest, 100



C, 30 min. Adjust pH

to 4–4.5. Digest with takadiastase,



C, 3 h.

Lichrosphere

100 RP 18

5 mm

125  4mm

Isocratic H

/KH

,10

2

M, pH 2.8 : MeOH (85:15)

with 5 mM Hex sulfonic acid

and 0.1% triethylamine

254 nm Journal of Liquid

Chromatography

and Related

Te c hn o l o g y 19

(1996) 2155–2164

Thiamin Cooked

sausages

Homogenize 10 g ground sample with

60 ml 0.1 N HCl. Autoclave, 20 min.

Cool. Adjust pH to with 2.5 M NaOAC.

Add 5 ml 6% claradiastase, incubate,

3 h. Add 2 ml 50% TCA, 90



C, 15 min.

Filter.

Spherisorb

5 mm

250  4mm

Phosphate buffer (5 mM, pH

7.0):MeCN (70:30) 35



0.6 ml min

1

Thiochrome

precolumn

derivatization

Fluorescence

360 nm

430 nm

% Recovery > 90 Journal of Agricultural

and Food

Chemistry 47

(1999) 170–173

Riboflavin

FMN, FAD

Wine,

beverages

Filter sample through Millex-GV

(0.22 mm). Dilute the sample up to four

times with water. Inject.

Hypersil

ODS C

5 mm

200  2.1 mm

Gradient (A) 0.05 M NaH

(pH 3.0) (B) MeCN

Fluorescence

265 nm

525 nm

QL (mgl

1

)

Riboflavin–1.7

FAD – 6.6

FMN – 2.8

Journal of

Chromatography A

823 (1998)

355–363

tbl0003

Riboflavin Cooked

sausages

Homogenize 10 g ground sample with

60 ml 0.1 N HCl. Autoclave, 20 min

Cool. Adjust pH 4–4.5 with 2.5 M

NaOAC. Add 6% claradiastase,

incubate, 50



C 3 h. Add 2 ml 50% TCA,



C, 15 min. Filter

Spherisorb

ODS-2

5 mm

250  4mm

5 mM HEP sulfonic acid (pH

2.7):MeCN (75:25) 35



0.6 ml min

1

Fluorescence

277 nm

520 nm

DL (mg/100g) 0.015

% Recovery >95

Journal of Agricultural

and Food

Chemistry 47

(1999) 1067–1070

Riboflavin

FMN, FAD

Various food Add 19 ml MeOH–CH

(9:10) to 12 g

sample and shake, 1 min. Add 9 ml 0.1

MNH

OAC (pH 6.0) and shake, 1 min.

Centrifuge at 4



C, 20 min. Filter.

Alphabond

10 mm

300  4.6 mm

MeOH: 0.05 M NH

OAC (pH

6.0) (30:70) gradient 1 ml

min

1

Fluorescence

450 nm

530 nm

DL (ng ml

1

)

Riboflavin, FMN –

1 FAD – 6 %

Recovery >95

Journal of

Chromatography A

881 (2000)

285–297

Niacin

Niacin Infant formula

wheat flour

Weigh sample containing 100–200 mg

niacin. Add 8 mL DMW, 2 mL (1þ1)

, mix. Autoclave, 45 min. Cool.

Adjust pH to 6.5 with 7.5 N NaOH.

Adjust pH 1 with H

. Filter.

PRP-X100

Anion-

exchange

250 

4.1 mm

0.1 M NaOAC–HAC (pH 4–4.2)

1.5 ml min

1

260 nm DL (mgml

1

)0.2%

Recovery 95–107

Journal of AOAC

International 82

(1999) 128–133

Niacin Various foods Add 30 ml 0.1 M HCl to 1–5 g ground

sample, heat at 100



C, 1 h. Cool.

Dilute. Filter. Autoclave, 1 h. Cool.

Adjust pH to 4.5, dilute. Filter.

Lichrospher

100 RP-18

5 mm

250  5mm

0.07 M KH

, 0.075 M

hydrogen peroxide, 5.10 

6

M copper (II) sulfate

solution 1 ml min

1

Postcolumn

derivatization

Fluorescence

322 nm

380 nm

% Recovery 90–107

%CV<4

Food Chemistry 65

(1999) 129–133

Nicotinic

acid,

nicotinamide

Cooked

sausages

Add 30 ml water, mix and homogenize,

2 min. Dilute with water and centrifuge.

Filter upper solution. Add 1 ml

saturated zinc sulfate solution and 1 ml

1N NaOH. Dilute with water, let stand

30 min. Filter.

Spherisorb

ODS-2

5 mm

250  4mm

HEP sulfonic acid (5 mM,

pH 3.3):MeCN (75:25) 35



0.65 ml min

1

261 nm % CV 2.3–4.5 %

Recovery 93–95

Journal of Agricultural

and Food

Chemistry 48

(2000) 3392–3395

Vitamin B

PL, PM, PN,

PLP, PMP,

PNP, 4-PA

Egg, milk,

baker’s

yeast

extract,

yeast cell-

free culture

media

Baker’s yeast extract – 50 mg sample,

add 50 m l1mMiso-PL, 3 ml of 1 M

ClHO

. Egg yolk – 2 g sample, add

100 ml1mMiso-PL, 6 ml 1 M ClHO

;

Milk – 2 g sample, add 10 ml 1 mM iso-

PL, 1 ml 1 M ClHO

; mix. Centrifuge.

Decant supernatant, adjust pH to 3–4

with 10 M KOH. Refrigerate.

Centrifuge. Filter

Phenospher

ODS-2

5 mm

250  4.6 mm

0.15 M NaH

Adjust pH with

70% ClHO

to 2.5 1 ml min

1

Postcolumn

derivatization

sodium bisulfite

Fluorescence

290 nm

389 nm

% Recovery 89–102 Journal of

Chromatography A

790 (1997) 83–91

PMP, PM,

PL, PLP PN

Cooked

sausages

Extract with 5% MPA. Centrifuge Filter. Hypersil

BDS C

5 mm

Isocratic 0.05 M KH

(pH 3.2): MeCN (99:1) 0.8 ml

min

1



Fluorescence

290 nm

395 nm

% Recovery 92–100 Journal of Agricultural

and Food

Chemistry 49

(2001) 38–41

Continued

Folate

Folic acid,

5-CH

-H

folate,

5-CHO-H

folate,

10-CHO-H

folate,

10-CHO-

folate

Cereal foods Add 10 volumes Hepes/Ches buffer, pH

7.85 (50 mM Hepes, 50 mM Ches)

containing 2% AA and 10 mM 2-MCE,

vortex, boiling water bath, 10 min.

Cool. Homogenize, Digest rat plasma

conjugase and a-amylase (37



C, 4 h)

followed by protease (37



C, 1 h),

boiling water bath, 5 min. Cool.

Centrifuge. Resuspend residue in

extraction buffer. Centrifuge. Filter

(Whatman #1 flush N

Ultramex C

5 mm

250  4.6 mm

Gradient (A) MeCN (B) 0.033 M

, pH 2.3 0–8 min

Isocratic 5%A 8–33 min

Linear gradient to 17.5% A

1 ml min

1

PDA

Monitor at 280 nm

% Recovery 87–107 Journal of Agricultural

and Food

Chemistry 45

(1997) 407–413

Biotin

d-Biotin,

d-biocytin

Various foods Ground sample (5–10 g) for high-starch

sample, add 100 mg takadiastase

(60.5 U mg

1

), add reduced glutathione

(300 ml), EDTA (300 ml), citrate buffer

(30 mL) and papain (3 ml). Shake and

incubate, 37



C, overnight. Cool. Filter.

Lichrospher

100

RP 18

5 mm

250  5mm

0.1 M phosphate buffer (pH

6):MeOH (81:19) 0.4 ml min

1

Post column

derivatization

with avidin-

fluorescein

isothiocyanate

Fluorescence

490 nm

520 nm

% Recovery 90–104

for d-biocytin

92–106 for d-biotin

Food Chemistry 65

(1999) 253–258

Pantothenic acid

Pantothenic

acid

Milk, infant

formulas

Add 5 ml warm water (<40



C) to 0.5 g

sample, vortex. Stand, 20 min. Add

0.5 ml HAC (3%), vortex. Stand, 20 min.

Centrifuge. Filter.

Nomura

Develosil

ODS-MG-5

5 mm

250  4.6 mm

0.1 M KH

(pH 2.25): MeCN

(95:5) 1.0 ml min

1

200 nm

205 nm

240 nm

DL (mg per 100 g)

0.3

% Recovery 98–

100

Food Chemistry 69

(2000) 201–208

Table 3 Continued

Analyte Sample type Sample preparation HPLC parameters References

Column Mobile phase Detection Quality-assurance

parameters

tbl0004 Table 4 Selected HPLC methods for fat-soluble vitamins in foods

Analyte Sampletype Sample preparationandclean-up HPLC parameters References

Column Mobile phase Detection Quality-assurance

parameters

Vitamin A

Retinol Milk powder,

flour

Mix 1.25 g sample with 5 ml

aqueous NaOH (50%), heat



C, 3 min. Add 25 ml EtOH

and 0.5 ml hydroquinone (20%

in EtOH), heat, 80



C, 30 min.

Cool. Add water and extract

with Et

O-PE (1:1). Reextract.

Filter. Evaporate and redissolve

in MeOH.

LiChrosorb

RP-18

5 mm

125  4.5 nm



Isocratic MeCN:water

(80:20) 0.8 ml min

1

325 nm %Recoveries Flour:

89–108 Milk

powder: 91–101

QL <32 mg per

100 g

Chromatographia 47

(1998) 716–720

Retinol Human milk Add 1 ml EtOH (containing 2%

BHT) and 500 ml KOH (12.5 M) to

500 ml sample. Saponify, 80



25 min. Extract with 1 ml Hex:t

oluene (1:1). Centrifuge. Inject

200 ml of the organic phase

Grom-Sil-CN-

2PR

5 mm

250  4.6 mm

Hex:IPA (98:2) 2 ml min

1

Fluorescence

325 nm

480 nm

RSD (%) Intra – 1.9

Inter – 2.3

% Recovery 102

Journal of

Chromatography A

898 (2000)

179–183

Carotenoids

a-, b-carotene;

13-cis-b-carotene;

9-cis-b-carotene

Green

vegetables

Blend with water containing 0.5%

AA. Extract with A:PE (3:2)

containing 0.5% BHT. saponify

extract, ambient, 15 min. Wash 3

 with 10% NaCl solution. Dry

over Na

. Evaporate.

Dissolve in MeOH:CH

(9:1).

Vydac TP-201

5 mm

250  4.6 mm

Isocratic prepared

isomers – MeOH:CH

water (79:15:6) sample–

MeOH: CH

: water

(80:12.5:4.8) 1 ml min

1

450 nm % Recovery

96.4–103.5

Food Chemistry 55

(1996) 63–72

All-trans-lutein,

zeaxanthin,

cryptoxanthin,

lycopene, a-,

b-carotene

Human milk Add 1 ml EtOH and 1 ml Hex to 1 ml

sample, vortex, centrifuge.

Remove upper organic layer.

Repeat one more time with 1 ml

Hex. Extract Hex layer twice

with 1 ml EiOH:water (9:1). Dry

the remaining Hex. Combine

the residue with the aqueous

phase from first extraction step.

Saponify, 37



C, 2 h. Extract with

1 ml Hex 4. Combine the

ethanol and Hex extract, dry,

redissolve in 0.2 ml IPA. Inject.

column

5 mm

250  4.6 mm

Gradient Solvent A

MeOH:water (90:10) with

0.04%NH

OAC Solvent B

MeOH:MTBE:water

(8:90:2) with 0.01%

OAC 1 ml min

1

450 nm % Recovery >90 International Journal

for Vitamin and

Nutrition Research

70 (2000) 79–83

Continued

15-cis-b-carotene;

13-cis-b-carotene;

all-trans-a-,

b-carotene;

9-cis-b-carotene

Carrot juices and

vitamin

supplemented

drinks

Extract sample with A:Hex (1:1).

Add NaCl solution (10%) to

remove the emulsion. Wash

Hex layer with water. Add BHT

as an antioxidant (0.1%). Dry

with Na

. Evaporate.

Redissolve in IPA. Inject

RP column

5 mm

250  4.6 mm

Linear gradient from 100% A

to 56% B within 50 min at

flow rate 1 ml min

1

Eluent

A MeOH:MTBE:water

(81:15:4) Eluent B

MTBE:MeOH: water

(90:6:4)

9-cis-b-carotene,

a-carotene –

445 nm

b-carotene –

452 nm 13-cis-

b-carotene –

471 nm b-apo-8

carotenal (IS) –

462 nm

% Recovery >90% Food Chemistry 70

(2000) 403–408

Vitamin D

Baby food Add D

or D

(IS). Saponification,



C, 30 min. Extract with

O:PE (1:9). Evaporate.

Redissolve in iOCT Clean-up.

Semi-Prep LC Polygosil 60–

5 mm,

250  8 mm.

Two Shandon

ODS-5

columns in

series

5 mm

250  4.6 mm

Isocratic MeOH (100%)

1 ml min

1

265 nm QL

350–420 IU kg

1

Recovery 84

DL (ng)

On-column ¼2

NetherlandsMilkand

Diary Journal 48

(1995) 31–39

Infant formula,

enteral

nutritionals

Add D

(IS). Saponification, 60



30 min. Extract with Hex.

Evaporate. Clean-up SPE B & J

Silica 9054. Evaporate.

Redissolve in MeCN.

Vydac 201

TP54 C

5 mm

250  4.6 mm

(not

endcapped)

Gradient

MeCN:MeOH:EtOAC 0.7–

2.5 ml min

1

265 nm QL

8 IU/quart

% Recovery

>99

RSD

¼ 13.48%

Journal of AOAC

International 75

(1992) 566–571; 79

(1996) 73–80

Vitamin E

a-, g-, d-T Margarine,

reduced fat

products

Dissolve sample (5 g) with Hex

containing 0.1% BHT, add

MgSO

to remove water.

Stand in dark 2 h. Filter.

LiChrosorb

Si60

5 mm

250  4.6 mm

Isocratic Hex:IPA (99.1:0.9)

1.0 ml min

1

Fluorescence

290 nm

330 nm

QL (m g per 100 g)

a-T–39.8

g-T – 5.0

d-T – 3.0

% Recovery >97

Journal of Liquid

Chromatography

and Related

Te c hn o l o g y 21

(1998) 1227–1238

Table 4 Continued

Analyte Sample type Samplepreparation and clean-up HPLC parameters References

Column Mobile phase Detection Quality-assurance

parameters

a-T acetate retinyl

palmitate

Soy-, milk-based

infant formula,

medical foods

Matrix solid-phase extraction, C

LiChrosorb

Si60

5 mm

250  4.6 mm

Isocratic IPA:Hex (0.5:99.5)

for a-T acetate,

(0.125:99.895) for retinyl

palmitate 1.0 ml min

1

Fluorescence

a-T acetate

285 nm

310 nm

Retinyl palmitate

325 nm

470 nm

Recoveries >91% Journal of AOAC

International 81

(1998) 582–586; 82

(1999) 107–111;

Journal of Liquid

Chromatography

and Related

Te c hn o l o g y 21

(1998) 2853–2861

Vitamin K

Vegetables To 1–5 g sample, sonicate with

10 ml MeOH, 15 min. Centrifuge.

2 ml of the mix solution extract

with 4 ml volume carbonate.

Heat at 80



C, 1 h. Partition with

4 ml Hex (3). Evaporate Hex

layer. Redissolve in MeOH.

LiChrosorb

RP-8

10 mm

250  4.6 mm

Isocratic

100% MeOH

0.6 ml min

1

247 nm and particle

beam mass

spectrometry

DL (ng)

On-column

UV – 0.1

% Recovery 82–90

Fresenius Journal

of Analytical

Chemistry 355

(1996) 48–56

, MK-4, K

) Margarine,

butter,

vegetable oils

Add 20 ml of Hex to samples

(0.2 – 0.5 g or 5.0 g for fat-free

products). Homogenize. Filter.

Evaporate. Redissolve in

THF:MeOH (1:1).

YMC C

3 mm

250  4.6 mm

followed by a

zinc column

(20  4 mm)

Isocratic 100 ml of

þ900 ml of

MeOH þ5 ml of methanolic

solution containing

1.37 mg ZnCl

, 0.41 mg

NaOAC and 0.3 mg glacial

HAC 0.8 ml min

1

Fluorescence

243 nm

430 nm

% Recovery 93–99 Food Chemistry 69

(1999) 79–88

Milk- and soy-

based infant

formula

Matrix solid-phase extraction, C

Alltech C

3 mm

150  4.6 mm

followed by

a zinc

reduction

column (25 

3.2 mm)

MeOH:reductive ionic

solution (RIS) (950:5)

containing 10% Hex 1 ml

min

1

RIS – 2 M ZnCl

þ1

M NaOAC þ1 M HAC per

liter of methanol

Fluorescence

248 nm

418 nm

DL (pg) K

–12 QL

(pg) K

– 38%

Recovery 84–102

Journal of AOAC

International 82

(1999) 1140–1145;

Journal of Liquid

Chromatography

and Related

Te c hn o l o g y 23

(2000) 423–432

Multianalyte methods

All-trans retinol, a-,

b-, g-, d-T,

b-carotene,

13-cis-retinol

Italian cheese Saponification, 70



C, 30 min.

Extract with Hex:EtOAC (90:10).

Evaporate. Redissolve in

mobile phase.

Ultrasphere Si,

5 mm

250  4.6 mm

Isocratic – Hex: IPA (99:1)

1.5 ml min

1

gradient –

multilinear pump A Hex:

IPA (99:1), pump B Hex

(100%), 1.5 ml min

1

Tocopherols

fluorescence

280 nm

325 nm

Retinol

fluorescence

325 nm

475 nm

b-carotene

450 nm

DL (ng)

On-column

a-T – 0.9,

b-T – 0.73

g-T – 0.55 d-T – 0.56

13-cis-retinol – 0.09

b-carotene – 0.16

all-trans

retinol – 0.32

Analyst 119 (1994)

1161–1165

Continued

, a-T,

all-trans-retinol

Milk, milk

powder

(a) a-T, retinol saponification,

overnight, ambient. Extract with

Hex. Evaporate. Redissolve in

MeOH (b) K

lipase hydrolysis,

add alcoholic sodium hydroxide

and immediately extract with

Hex. Clean-up Sep-Pak silica.

Brownlee OD-

224 RP 18

5 mm

220  4.6 mm

Isocratic MeOH:water (99:1)

containing 2.5 mM

HAC:NaOAC, 1.25 ml

min

1

dual-EC glassy

carbon -1100 mV

þ700 mV vs

Ag/AgCl

DL (ng)

On-column

retinol – 0.06

–3.1

a-T – 0.19

Journal of

Chromatography

623 (1992) 69–74;

Analyst 120 (1995)

2489–2492

a-T, retinol Infant formula Saponification, overnight,

ambient. Extract (5) with Hex.

Wash extract with water and dry

over Na

. Filter. Evaporate,



C. Redissolve in MeOH.

Filter.

Spherisorb

ODS-2 C

5 mm

250  4.6 mm

Isocratic water:MeCN:MeOH

(4:1:95)

a-T – 292 nm

retinol – 323 nm

% Recovery

a-T – 86

retinol – 98

Journal of

Chromatography A

778 (1997)

243–246

All-trans retinol, 13-cis

retinol, a-, b-T,

b-carotene, retinyl

palmitate

Milk Saponification, 70



C, 7 min.

Extract with Hex:DIPE (75:25)

containing 0.04% BHT. Add

30 ml Milli-Q water, gently

inverted the tube, centrifuge.

Evaporate supernatant.

Redissolve in 2 mL Hex

containing 0.4% BHT b-T (IS).

Alltech Econo-

sphere silica

3 mm

150  4.6 mm

Isocratic 0.01% acetic acid,

0.5% IPA, 0.02 mg l

1

a-T in Hex 1.5 ml min

1

Vitamin A

fluorescence

330 nm

470 nm

Vitamin E

fluorescence

295 nm

330 nm

b-carotene 450 nm

% Recovery 98–100 Milchwissenschaft 53

(1998) 11–15

a-, g-T, a-, b-carotene,

lutein, zeaxanthin,

b-cryptoxantnin

Corn Add 6 ml EtOH with 0.1% BHT to

600 mg freeze-dried corn

powder. Heat at 85



C in water

bath, 5 min. Add 120 ml 80%

KOH, vortex and saponify at



C, 10 min. Add 3 ml cold

deionized water, cool. Extract

with 3 ml Hex (3). Wash Hex

layer with 3 ml deionized water.

Evaporate Hex layer.

Redissolve in 200 mL

CN:MeOH:CH

(45:20:35).

Vydac 201TP54

5 mm

150  4.6 mm

connected

with Nova-

Pak C

4 mm

150  3.9 mm

Isocratic

CN:MeOH:CH

(75:20:5) containing 0.05%

TEA and 0.1% BHT 1.8 ml

min

1

Carotenoids –

450 nm

Tocopherols –

290 nm

% Recovery 85–122 Journal of Liquid

Chromatography

and Related

Te c hn o l o g y 22

(1999) 2925–2934

Table 4 Continued

Analyte Sample type Sample preparationandclean-up HPLC parameters References

Column Mobilephase Detection Quality-assurance

parameters

a-, g-T, a-, g-, d-T3,

carotenoids

Red palm oil Add 5 ml MeOH, 0.3 ml 60% KOH,

1 ml Methanolic pyrogallol

(0.02%) to 400 mg sample. Heat

at 100



C in water bath, 30 min.

Cool. Add 10 ml water. Extract

with 10 ml ET

O(3). Wash

ether layer with 10 Ml water

(4). Add anhydrous Na

and filter. Evaporate.

Redissolve in HPLC mobile

phase.

YMC C

5 mm

250  4.6 mm

(A) MeOH:MTBE:water

(81:15:4)

(B) MTBE:MeOH (91:9)

0–45 min – 100% A to 50% A

45–56 min – 100% B

56–60 min – 100% A

1 ml min

1

Vitamin E – 295 nm

Carotenoids –

450 nm

% CV 2–25 for

carotenoids

0.6–20 for

tocopherols

Journal of Liquid

Chromatography

and Related

Te c hn o l o g y 23

(2000) 1873–1885

Vitamin A, E, D

, retinyl acetate,

retinyl palmitate,

a-T acetate,

ergosterol, 7-

dehydrocholesterol

Milk Add 5 ml EtOH with 0.025% BHT to

5 ml sample, sonicate. Extract

with 15 ml Hex (2). Wash Hex

layer with MeOH-water (9:1).

Filter and evaporate Hex layer.

Redissolve in 100 ml MeOH.

Inject 60 nl.

Hypersil C

BDS

3 mm

150  0.3 mm

(A) MeOH:water (99:1)

(B) MeOH: THF (70:30)

0–4 min – 0% B

4–10 min – 100% B

10–15 min – 100% B

15–17 min – 0% B

6 ml min

1

Wavelength (nm)

325–vitamin A

264–vitamin D

280–vitamin K

,E,

provitamin D

%Recovery 89–107

%CV 2–8

Journal of

Chromatography A

891 (2000)

109–114

a-, b-, g-, d-T a-T

acetate,

b-carotene, retinyl

palmitate

Fortified foods To 3 g sample, add 2 ml 80



water, sonicate. Add IPA,

MgSO

and Hex containing

0.003% BHT. Homogenize,

1 min. Filter. Evaporate.

Redissolve in Hex. Inject.

LiChrosorb

Si60

5 mm

250  4.6 mm

Isocratic 0.27% IPA in Hex

Gradient flow 0.9–1.5 ml

min

1

over 5.3 min

Fluorescence

(programmable)

a-T acetate

285 nm

315 nm

Tocopherol

290 nm

330 nm

Retinyl palmitate

325 nm

470 nm

b-carotene

PDA 450 nm

%Recovery 99–101 Journal of Agricultural

and Food

Chemistry 48

(2000) 4003–4008

Vitamin A, E, D

, retinyl acetate,

retinyl palmitate,

a-T acetate,

ergosterol, 7-

dehydrocholesterol

Milk, butter Milk – add 500 ml EtOH containing

0.025% BHT to 1 ml fresh or

2.5 ml fortified milk, sonicate,

2 min. Dilute with water. Clean-

up. Mega Bond Elut C

. Butter –

add 5 ml EtOH containing

0.025% BHT to 1 g sample.

Extract with 15 ml Hex (2).

Wash Hex layer with MeOH–

water (9:1). Evaporate Hex

layer. Redissolve in 1 ml MeOH.

Inject.

Extrasil ODS2

3 mm

150  2.1 mm

(A) MeOH:water (99:1)

(B) MeOH:THF (70:30)

0–20 min –0% B

20–22 min – 85% B

22–29 min – 85% B

29–33 min – 0% B

Flow rate gradient

0–5.5 min – 0.15 ml min

1

6.3 min – 0.2 ml min

1

Wavelength (nm)

325 – vitamin A

264 – vitamin D

280 – vitamin K

E, provitamin D

0.7 min – 325 nm

7–9.5 min –

264 nm 9.5–24 min

– 280 nm 24 min –

325 nm

DL (ng)

0.14–6.9

% CV 1.6–4.5

% Recovery 84–108

Analyst 125 (2000)

427–431

A, acetone; AA, ascorbic acid; BHT, butylated hydroxytoluene; CH

, methylene chloride; Ches, 2-[N-cyclohexylamino]-ethanesulfonic acid; ClHO

, perchloric acid; (Vitamin) D

, cholecalciferol; (Vitamin) D

ergocalciferol; DHAA, dehydroascorbic acid; DIPE, diisopropyl ether; DMW, demineralized water; EC, electrochemical; EtOAC, ethyl acetate; EtOH, ethanol; Et

O, diethyl ether; FAD, flavin adenine dinucleotide; FMN,

flavin mononucleotide; GMP, disodium guanosine-5

-monophosphate; HAC, acetic acid; HCl, hydrochloric acid; HEP, heptane; Hepes, N-[2-hydroxyethyl]pipera zine-N

-ethanesulfonic acid; Hex, hexane; HPLC, high-

strong ultraviolet (UV) absorbance properties. Most

LC procedures use UV aborbance between 320 and

380 nm for detection. Fluorescence (excitation:

320 nm, emission 470 nm) is an excellent detection

mode for retinol and retinyl esters. Carotenoids

absorb strongly in the visible region due to the long

conjugated double-bond system. For b-carotene,

450 nm is universally used for detection. Variable

wavelength, programmable UV/visible and photo-

diode array detectors have greatly aided the chroma-

tographic study of complex, natural carotenoid

mixtures characteristic of fruit and vegetable samples.

Vitamin D

0018 Vitamin D analysis of nonfortified foods is difficult

due to the low concentration and the relatively poor

spectral properties of the vitamin. Both normal- and

reversed-phase LC efficiently resolve vitamin D

previtamins, and hydroxylated metabolites if prop-

erly prepared extracts are available. Excellent

methods that use competitive protein binding assays

(CPBA) or radioreceptor assays (RRA) using (

metabolites are available for analysis of biological

matrices. These methods use extensive prepurifica-

tion of extracts by LC or solid-phase chromatog-

raphy. CPBA and RRA are specific and sensitive

enough to measure circulating levels of hydroxylated

vitamin D metabolites; however, the methods have

not been applied to food analysis. Radioimmune

assay with

125

I-labeled metabolites is used extensively

in clinical chemistry.

0019 Provided the extraction protocol provides an

extract that meets the detection limits of the assay,

reversed-phase LC with UV detection at 265 nm is

useful as the determinative assay. More recent

methods applicable to food analysis incorporate pre-

parative LC for sample clean-up and concentration

prior to LC resolution on C-18.

Vitamin E

0020 Vitamin E exists in nature as eight closely related

compounds (a-, b-, g-, d-tocopherols and correspond-

ing tocotrienols). Normal-phase LC on silica resolves

the eight vitamin forms. Reversed-phase LC cannot

resolve the b- and g-positional isomers; therefore,

most methods for food analysis use normal-phase

LC. Detection does not pose a problem because of

the strong fluorescence exhibited by the chromanol

family. a-Tocopheryl acetate is normally used for

food fortification. This compound is much more

stable than a-tocopherol and possesses much less

native fluorescence. However, highly sensitive fluor-

escence detectors that are now readily available can

quantify a-tocopheryl acetate at the levels that are

commonly used for food fortification.

Vitamin K

0021Methods for vitamin K analysis of foods use a com-

bination of adsorption chromatography for sample

clean-up, and reversed-phase LC on C-18 supports

for the quantification. Mobile phases are simple, iso-

cratic systems of methanol modified with dichloro-

methane. The mobile-phase composition depends on

the detection mode. If electrochemical detection is

used, an electrolyte must be added to the mobile

phase to provide for conductivity. More commonly,

vitamin K compounds are converted from the quin-

one form to the highly fluorescent hydroquinone

form by postcolumn zinc metal reduction. This con-

version allows the use of fluorescence for detection.

The instability of vitamin K at alkaline pH dictates

that saponification cannot be used for extraction.

Because lipids must be removed from vitamin K ex-

tracts before reversed-phase LC, novel lipid removal

procedures have been developed for vitamin K extrac-

tion. Recent methods (Table 4) use lipase hydrolysis

followed by solid-phase extraction to provide a suit-

able concentrated extract for LC resolution.

Multianalyte Procedures for Fat-soluble

Vitamin Analysis

0022Historically, fat-soluble vitamin analysis of foods

has been difficult due to methodology deficiencies

of poor detector sensitivity, poor precision due to

interferences, and labor intensiveness of the methods.

Technological advances in detectors and LC instru-

mentation together with research on extraction

methods for the fat-soluble vitamins from foods

have led to the availability of methods with improved

precision, specificity, and sensitivity. These improve-

ments have led to the development of multianalyte

approaches for fat-soluble vitamin analysis that are

especially useful for the analysis of foods fortified

with combinations of the fat-soluble vitamins. Such

methods, when incorporated into the laboratory an-

alysis program, can save a great deal of time and

present significant cost savings.

0023A recent multianalyte approach developed in our

laboratory at the University of Georgia is presented

to show the analytical power of multianalyte analysis.

The procedure is capable of simultaneously assaying,

retinol, retinyl palmitate, retinyl acetate, b-carotene,

natural vitamin E, and a-tocopheryl acetate. In this

method, the vitamins are extracted in 2-propanol

and hexane without saponification. Quantification is

by normal-phase LC with fluorescence detection of

retinol, the retinyl esters, a-tocopheryl acetate and

the vitamin E alcohols. b-Carotene is quantified at

450 nm. Fluorescence and photodiode array detectors

6066 VITAMINS/Determination

in series allow quantification of all analytes from a

single injection. Figure 1 shows the elution profile

obtained from a margarine sample fortified with reti-

nyl palmitate, b-carotene, and a-tocopheryl acetate.

See also: Ascorbic Acid: Properties and Determination;

Carotenoids: Occurrence, Properties, and

Determination; Cholecalciferol: Properties and

Determination; Cobalamins: Properties and

Determination; Retinol: Properties and Determination;

Riboflavin: Properties and Determination; Physiology;

Thiamin: Properties and Determination; Physiology;

Tocopherols: Properties and Determination; Physiology;

Vitamin K: Properties and Determination; Vitamins:

Overview; Vitamin B

: Properties and Determination;

Physiology