The determination of vitamins in Food Supplements

The determination of vitamins in Food

Supplements

Food Supplements Directive

Food Supplements are defined by :

EC Directive 2002/46/EC (as amended) on the approximation of the laws of the Member States relating to Food Supplements

and

The Food Supplements Regulations 2003

Food Supplements Directive

• For the purposes of this Directive:• (a) "food supplements" means foodstuffs the purpose of which is to

supplement the normal diet and which are concentrated sources ofnutrients or other substances with a nutritional or physiological effect, alone or in combination, marketed in dose form, namely forms such as capsules, pastilles, tablets, pills and other similar forms, sachets of powder, ampoules of liquids, drop dispensing bottles, and other similar forms of liquids and powders designed to be taken in measured small unit quantities;

• (b) "nutrients" means the following substances:• (i) vitamins,• (ii) minerals.

Which vitamins are allowed?

• VITAMIN B1 - thiamin hydrochloride, thiamin mononitrate• VITAMIN B2 - riboflavin, riboflavin 5'-phosphate, sodium• NIACIN - nicotinic acid, nicotinamide• PANTOTHENIC ACID - D-pantothenate, calcium, D-pantothenate, sodium,

dexpanthenol (D+ Panthenol)• VITAMIN B6 - pyridoxine hydrochloride, pyridoxine 5'-

phosphate• FOLIC ACID - pteroylmonoglutamic acid

• NOTE: EC Directive (but NOT UK regulation) amended to FOLATE and adds calcium-L-methylfolate

• VITAMIN B12 - cyanocobalamin, hydroxocobalamin• BIOTIN - D-biotin

• VITAMIN C - L-ascorbic acid, sodium-L-ascorbate, calcium-L-ascorbate, potassium-L-ascorbate, L-ascorbyl 6-palmitate

Which vitamins are allowed?

• Shedule 1 of the Directive contains a positive list of vitamins which may be used & prescribes the units in which they should be expressed.

• Shedule 2 prescribes the forms of vitamins which may be used.

• VITAMIN A - Retinol, Retinyl acetate,Retinyl Palmitate, Beta-carotene µg RE

• VITAMIN D – Cholecalciferol, Ergocalciferol µg

• VITAMIN E - D-alpha-tocopherol, DL-alpha-tocopherol, D-alpha-tocopheryl mg ATEacetate, DL-alpha-tocopheryl acetate, D-alpha-tocopherylacid succinate

• VITAMIN K - Phylloquinone (phytomenadione) µg

Vitamin E

• Eight isomers exist: α, β, γ, δ Tocopherols and α, β, γ, δ Tocotrienols

• Eight stereoisomers exist for the tocopherols

• Synthetic forms: All-racemic α Tocopherol (dl α Tocopherol)• Natural form: RRR -α-Tocopherol (d-α Tocopherol)• Semi-synthetic: RRR-α-tocopherol formed by hydrolysis

& methylation of natural tocopherol esters

Biological activity

Biological activity of Tocopherol isomers

Relative Activity: α 100%, β 40-50%, γ 10%, δ 1-3%Tocotrienols : α 20-30%, β 4-5%, γ & δ <1%

Biological activity of alpha tocopherol:(IU)/mg

Natural RRR (d) - a -Tocopherol 1.49

RRR (d) - a -Tocopherol acetate 1.36

Synthetic All racemic (dl) -a-Tocopherol acetate 1.00

All racemic (dl) - a –Tocopherol 1.10

Activity is dependant on the assay/pharmacological attribute tested.

In human serum/plasma Natural tocopherol activity is 2 x synthetic tocopherol

Activity Nomenclature

International Units (IU)Activity of Vitamin E based on the rat fetal absorption test

1IU = 1mg of all-racemic alpha tocopherol acetate.

AlphaTocopherol equivalent α-TE or ATENational Research Council definition of Vitamin E in terms of naturally occuring alpha tocopherol.

1 ATE = 1mg of RRR alpha tocopherol

The Food Supplements directive requires ATE to be used.

Biological activity

1.1211.6710.60.891All rac α tocopherol

succinate

1.0011.4910.6711All rac α Tocopherol

acetate

0.9111.3510.741.11All rac α Tocopherol

0.8311.2310.811.211RRR α tocopherol

succinate

0.7411.1010.911.361RRR α Tocopherol

acetate

0.6711.00111.491RRR α Tocopherol

mgIUmgATEATE/mgIU/mgmgVitamer

Vitamin A activityQuantity of Source Material Vitamin A Activity (in

International Units) 1 µg all-trans-retinol (1 µg RAE) (synonym: vitamin A) 3.33 IU

1 µg of all-trans-retinyl acetate (synonym: vitamin A acetate) 2.91 IU

1 µg all-trans-retinyl palmitate (synonym: vitamin A palmitate) 1.82 IU

1 µg supplemental all-trans-beta-carotene (synonym: beta-carotene) i.e. as an isolate 1.67 IU

1 µg dietary all-trans-beta-carotene (synonym: beta-carotene) i.e. in plant (e.g. carrot powder), algal, or bacterial matrices

0.28 IU

1 µg supplemental all-trans-alpha-carotene or all-trans-beta-cryptaxanthin (synonyms: alpha-carotene and beta-cryptaxanthin) i.e. as isolates

0.83 IU

1 µg dietary all-trans-alpha-carotene or all-trans-beta-cryptaxanthin (synonyms: alpha-carotene and beta-cryptaxanthin) i.ein citrus fruit powder)

0.14 IU

Retinol Activity Equivalent (1 µg RAE): = 1 µg all-trans-retinol= 2 µg supplemental all-trans-beta-carotene = 12 µg dietary all-trans-beta-carotene = 24 µg dietary all-trans-alpha-carotene or all-trans-beta-cryptaxanthin

Types of food supplements

TabletsSwallowable

Coated

Uncoated

Chewable

Effervescent

CapsulesHard-gel

Soft-gel

Oil fillled

Paste filled

Gelatin

Non-gelatin

LiquidsOils

Syrups

Emulsions

Analytical problems

• Physical formulation – coatings, gels, oil, granulation• Chemical formulation – Effect of excipients and

additives, other vitamins, minerals.• Potential for absorption onto capsule shell.• Stability, lability• Calculation errors, expression errors• Overages• Different extraction methods• Different analytical methods • Tolerances

Source of methods

1. USP/ BP methods

2. CEN/ISO - Mainly food methods at present. Supplements added to remit of CEN Vitamins working group.

3. Published literature

4. Industry methods

Choice of method.

Before analysis:Single or multiple vitamin?

What are the supplement ingredients?

What is the target vitamin concentration?

What form of vitamin is present?

Is the form of vitamin important?

How is result to be reported? (w/w, per tablet, wt/vol)

Is it likely that overages have been used?

How old is the sample?

Should whole capsules or fill only be examined?

What sample weight is required?

Should tablets be ground?

Extraction

•Are all vitamins stable under the extraction conditions?

•Are minerals present?

•Is fill lipid or water soluble?

•Are the target analytes oil or water soluble

•Matrix interactions

Analysis

•Vitamin Forms

•Individual vitamers or total content

•Isomers

•Stability

•Detection method

•Recovery, Uncertainty etc

FSA study of Methodsfor the determination of food supplements

•To provide guidance in the selection of methods for the determination of food supplements

•To validate a method for the determination of b-group vitamins in Food Supplements

•To validate a method for the determination of oil-soluble vitamins in Food Supplements.

B1,B2, Niacin, B6 & Ca Pantothenate

• Extraction: • Whole capsules and whole/ground tablets• Citric acid containing EDTA and Tween 80. • 60°C for 10mins• Ultrasonicate for 20mins

Analytical Method

• HPLC• Lichrocart, Superspher 60RP (Merck)• Gradient elution

– A: Phosphate buffer ,containing Sodium heptane sulfonate , pH 3.– B: Acetonitrile/Phosphate buffer (60/40)

• UV-Diode Array Detection• B1, B2, B3, B12 - 260 nm• B6 - 290 nm 275 nm for all?• Ca Pantothenate - 205 nm

Water Soluble vitaminsChromatogram of B vitamins

Luna C18(2)Modified H2O/MeoH gradientDiode Array detection

Chromatogram of a multivitamin Tablet

Collaborative Trial

• Five test materials as blind duplicates.• Tablets and soft-gel capsules with & without minerals• Range of concentrations• Target Analytes : Thiamine, Riboflavin, Niacinamide, B6,

Ca Pantothenate.

• 16 laboratories – Mainly PA labs• HPLC conditions recommended but not mandatory

Bias – Comparison of study means with claim/input values

Thiamin

0.000.501.001.502.002.503.00

A B C D E

Sample

mg/

Tabl

et

Study mean Claim Input

Riboflavin

0.000.501.001.502.002.50

A B C D E

Sample

mg/

Tabl

et

Study mean Claim Input

Niacin

0.005.00

10.0015.0020.0025.00

A B C D E

Sample

mg/

Tabl

et

Study mean Claim Input

Vitamin B6

0.002.004.006.008.00

10.0012.00

A B C D E

Samplem

g/Ta

blet

Study mean Claim Input

Collaborative trial results

3.923.481.090.981.40E

2.700.881.310.860.53D

3.362.611.190.921.79C

3.512.221.181.371.38B

3.022.821.541.491.07A

HOR

Ca PantothenateRiboflavinVitamin B6NiacinThiaminSample

Reproducibility Horrat Values

1.132.120.740.970.49E

1.640.870.820.540.52D

2.541.170.911.400.81C

2.360.581.191.341.34B

1.571.091.401.631.10A

Hor

Ca PantothenateRiboflavinVitamin B6NiacinThiaminSample

Repeatability Horrat Values

Other B Vitamins

• Folic acid Determined by HPLC after neutral/alkaline extraction or Microbiological assay

• Biotin HPLC at 200nm for premixes after alkaline extraction or Microbiological assay

• Vitamin B12 HPLC in premixes. Too low in finished products for HPLC. Microbiological assay.

• Vitamin C Titrimetry (DCIP), Fluorimetry (OPD), HPLC after acid extraction – as AA (DTT) & UV 245 or as DHAA (OPDderivatisation)

A, D & E in supplements

• Samples saponified to convert esters to corresponding alcohols.

• Vitamins extracted using organic solvents

• A & E quantified directly

• D requires HPLC clean-up before analytical HPLC.

Analysis

ZORBAX ODS, 250 x 4.5 mm

Flowrate: 1ml/min

Mobile Phase: Acetonitrile / Dichloromethane / Methanol 70/20/10

Detector: Diode Array: Vit D-265nm, Vit A – 325nm, Vit E – 292nm

Prep-LC (D only)Partisil 5 PAC, 250 x 4mm

Mobile Phase: Hexane / Propan-2-ol 99/1

Detector: UV 265

Oil Soluble vitamins Chromatogram

Zorbax ODS

MeCN/DCM/MeOH

UV@280nm

Mixed Standard

Vitamin A -Retinol

Vitamin E – α -TocopherolVitamin D

Chromatograms for Vitamins A, D & E in Multivitamin & Mineral soft-gel capsule with CLO.

Collab trial

• Thirteen participants (Largely PA labs)• Six test samples of Tablets and capsules as blind

duplicates• Range of concentrations• Analytes: Vitamin A (ATR)

Vitamin E (Alpha tocopherol)Vitamin D

1025106510Claim

12.326.312.45.86.212.5Vitamin E

5.0D25.02.52.55.0Claim

4.6?5.94.82.76.1Vitamin D

800750750750300800Claim

663812744993361827Vitamin A

(Sample F)(Sample E)(Sample D)(Sample C)(Sample B)(Sample A)

Multivitamin Tablet

Multivitamin and Mineral Soft-Gel Capsule with Cod

Liver Oil

Multivitamin Soft-Gel Capsule with Cod Liver

Oil

Chewable Multivitamin and Mineral

Tablet

Chewable Multivitamin

Tablet with Iron and Calcium

Coated Multivitamin and Mineral

Tablet

Method Performance

Characteristic

Bias – Comparison with claimed values

Collaborative trial results

Method Performance

Characteristic

Coated Multivitamin and

Mineral Tablet

Chewable Multivitamin

Tablet with Iron and Calcium

Chewable Multivitamin and Mineral

Tablet

Multivitamin Soft-Gel

Capsule with Cod Liver Oil

Multivitamin and Mineral Soft-Gel Capsule with Cod

Liver Oil

Multivitamin Tablet

(Sample A) (Sample B) (Sample C) (Sample D) (Sample E) (Sample F)

n 13 12 13 13 12 13

outliers 2 0 0 2 1 0

n1 11 12 13 11 11 13

mean 605 362 1244 552 1167 1560

r 123 72 331 125 238 473

sr 44.1 25.8 118.3 44.8 85.1 169.0

RSDr 7.3 7.1 9.5 8.1 7.3 10.8

Hor 1.8 1.6 2.6 2.0 2.0 3.1

R 244 139 523 190 265 497

sR 87.1 49.7 186.8 67.8 94.7 177.6

RSDR 14.4 13.7 15.0 12.3 8.1 11.4

HoR 2.4 2.1 2.7 2.0 1.5 2.2

Vitamin A

Collaborative trial resultsVitamin D

Method Performance

Characteristic

Coated Multivitamin and Mineral Tablet

Chewable Multivitamin

Tablet with Iron and Calcium

Chewable Multivitamin and Mineral

Tablet

Multivitamin Soft-Gel

Capsule with Cod Liver Oil

Multivitamin Tablet

(Sample A) (Sample B) (Sample C) (Sample D) (Sample F)

n 11 9 10 11 11

outliers 1 1 2 2 1

n1 10 8 8 9 10

mean 4.48 2.70 5.97 4.41 10.8

r 0.96 0.63 1.30 1.32 4.7

sr 0.343 0.225 0.462 0.473 1.68

RSDr 7.7 8.3 7.8 10.7 15.6

Hor 0.9 0.9 1.0 1.3 2.1

R 1.80 1.06 2.08 2.89 5.7

sR 0.643 0.377 0.741 1.032 2.03

RSDR 14.4 14.0 12.4 23.4 18.8

HoR 1.1 1.0 1.0 1.8 1.7

Collaborative trial resultsVitamin E

Method Performance

Characteristic

Coated Multivitamin and

Mineral Tablet

Chewable Multivitamin

Tablet with Iron and Calcium

Chewable Multivitamin and Mineral

Tablet

Multivitamin Soft-Gel

Capsule with Cod Liver Oil

Multivitamin and Mineral Soft-Gel Capsule with Cod

Liver Oil

Multivitamin Tablet

(Sample A) (Sample B) (Sample C) (Sample D) (Sample E) (Sample F)

n 13 12 13 13 12 13

outliers 1 1 0 1 0 0

n1 12 11 13 12 12 13

mean 9.15 6.20 7.22 9.20 37.85 29.03

r 2.42 0.84 1.33 2.37 7.71 5.92

sr 0.860 0.301 0.476 0.845 2.754 2.113

RSDr 9.4 4.8 6.6 9.2 7.3 7.3

Hor 3.5 1.7 2.4 3.4 3.4 3.2

R 2.79 2.99 3.32 2.92 17.33 13.63

sR 0.995 1.069 1.18 1.044 6.189 4.869

RSDR 10.9 17.2 16.4 11.3 16.4 16.8

HoR 2.7 4.0 3.9 2.8 5.0 4.9

Conclusion

• Vitamin analysis can be easy but often isn’t.• Consider all available information• Consider output required• Staff should be trained and experienced• Examine results critically• Consultation before formal action?