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?