New Approaches to Accurately
Measure B Vitamins
in Pet Foods
John Szpylka, Ph.D.; Sneh Bhandari, Ph.D.; Michael P.
Napolitano, Ph.D.; Tiffany Gallegos; Seaver (Blue-Fur
Shar Pei)
Mérieux NutrSciences
Silliker Laboratories
Copyright © 2015 Silliker Inc
Outline
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B vitamins in the pet food world
Current testing and known hurdles
Recent advances that overcome hurdles
Development work is underway
Survey of methods testing some pet foods
Conclusions
2
All Right! Give It To Me!!
B Vitamins
• The B vitamins
– help convert carbohydrates into glucose, the body’s fuel.
– assist in fat & protein metabolism.
– are necessary for healthy skin, hair, nerves, bone marrow,
proper weight control, and other health needs.
• B vitamins are not stored in the body.
– Must be ingested
The B Vitamins
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Vitamin B1 – Thiamine
Vitamin B2 – Riboflavin
Vitamin B3 – Niacin (nicotinic acid and niacinamide)
Vitamin B5 – Pantothenic acid
Vitamin B6 – pyridoxine, pyridoxal, pyridoxamine
Vitamin B7 – Biotin
Vitamin B9 – Folic Acid + 6 other forms
Vitamin B12 – various cobalamins, notably cyanocobalamin
Vitamin B4(ish) – Choline / Carnitine
AAFCO B Vitamins Guaranteed Analysis
B Vitamin
Cat Food (dwb) mg/kg
Dog Food (dwb) mg/kg
Thiamine (B1)
5
1
Riboflavin (B2)
4
2.2
Pantothenic Acid (B5)
5
10
Niacin (B3)
60
11.4
Pyridoxine (B6)
4
1
Folic Acid (B9)
0.8
0.18
Biotin (B7)
0.07
----
Cyanocobalimin (B12)
0.02
0.022
Choline (B4ish)
2400
1200
Current Analytical Methods
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Vitamins in fortifying ingredients
– Vitamins typically present in one form at high concentrations
– A wide range of scientific methods are used (HPLC, wet chemistry, others).
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Vitamins in pet foods/animal feeds
– Vitamins can be present in multiple forms (added and naturally-occurring)
– Each vitamin will be at a different concentration
– Knowing the total amount of a vitamin may be desired
• Typically, the fortifying form is dominant and can quickly be measured
• Measuring total amount is sometimes desirable for compliance and for setting production targets
Big Hurdle: Vitamins Exist in Multiple Forms
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Folic Acid & Folates
– Folic Acid and 6+ naturally occurring forms
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Glutamate chains of various lengths
Vitamin D
– 2 common forms (D2 & D3); each with pre-vitamin forms that can form naturally and during
extraction for analysis. Two types of 25-hydroxy forms will soon be added by FDA.
Vitamin B12
– Fortification by cyanocobalamin (a provitamin). Several other active forms.
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Niacin (3 common forms), B6 (3 predominant forms and as phosphate esters),
biotin (8 forms but only 1 natural), pantothenic acid (active and provitamin forms)
Historical Solutions
• Some methods only look at the forms used in fortification
– Acceptable if this level is above AAFCO minimum amount
• Some methods convert all forms into one measurable form
– Vit A: first analytical step converts all forms (except b-carotene) to one form
– B vitamins not so lucky
• Some methods measure each form and sum them somehow (folic acid B9)
– Vitamin E screens measure all forms (Be Careful: declaration is country specific)
• Some methods use bacteria growth test methods to measure how much of a
vitamin is present.
– Currently the Gold Standard for many B vitamins
Current Status & Hurdles
• Micro-based methods developed in response and are still being used
– Measures the sum of all bio-active forms
• Assumes bacteria behave similar to animals / humans
– These methods use long procedures which require chemistry and
microbiology expertise
• Liquid Chromatography methods being used and more being developed
– Historically targets only certain forms of the vitamin
• Commonly the form used to fortify products
– Recent advances looking at total amount of vitamins (includes all forms)
Folic Acid is for Fortification
The Many Other Forms of Folates
Microbiological Assays
Media lacking vitamin B9
Lactobacillus casei
22 hrs
Food Extract
Protease
&
Amylase
(>5 hrs)
Chicken Pancreas
Conjugase
(16 hrs)
Microbiological Assays
Calibration Plot
Turbidity
0.9000
(ABS)
0.7000
y = 0.2383x + 0.841
R² = 0.9966
0.5000
0.3000
0.1000
-0.1000
-3.5
-2.5
-1.5
-0.5
LN[Conc(ng/ml)]
0.5
Chromatographic Methods
BB915362
B96BB
B315
B2
a
a
a
a
a
a
Chromatographic Assays
LC/MS/MS Procedure
Homogenize
Protease
Amylase
2 internal stds
Buffer
& Heat
AOAC 2011.06
Extraction
via 96-well weak-anion
exchange SPE
Analysis by UPLC/MS/MS
M-R-M Transitions for each
monoglutamate vitamer
Vitamin B9 –Folic Acid
• Current FDA definition = sum of all forms
• Proposed definition for human food
– Factor in bioavailability
– Dietary Folate Equivalents (DFE)
Total DFE = (μg folates) + (1.7 x μg folic acid)
Vitamin B9 –Dietary Folate Equiv.
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New method needed
In the meanwhile, use production records
Proposed for human food.
Will guaranteed analysis be allowed to use DFE?
Recent Examination of AOAC 2011.06
Vitamin B9 Average
Vit B9 Micro Results
mcg/100g
mcg/100g
Protein supplement
244
209
TV Dinner
Frozen finished food
24
25
Choc Chunk Cookies
Cookies
54
52
Peanut butter
Peanut butter
71
80
Chocolate Milk Supplement
Dairy based supplement
57
53
AACC VMP2
Cake mix
797
784
AAFCO
Dry feed sample
350
348
Flour
Wheat Flour
241
230
Casein R1
Casein
16
Sample Name
Matrix
Protein Powder
Pet Foods
Folic Acid (ug/100g) – all forms
LC-MS
Micro Results
Dry Dog Food
224
213
Dry Cat Food
338
310
Wet Dog Food (chicken)
85
72
Wet Cat Food (turkey/cheese)
57
61
Wet Cat Food (liver/chicken)
85
82
Cat Food (liver/bacon)
74
119
Beef dog food
73
62
Dog treat
85
78
Switch to LC-MS/MS
• Most methods target only 1 or 2 forms
– Folic Acid
– 5-Methyl Folate
– There are 4 others
Methods for Folic Acid (Folate) Analysis
• AOAC Official Method 2011.06: Total Folates
in Infant Formula and Adult Nutritionals Trienzyme Extraction and UPLC-MS/MS
Quantitation
Vitamin B12
• Micro method used for years
• LC-UV methods for testing of concentrates
• Recently AOAC First Action Methods (LC-UV) allowable to test
Foods
– 2011.01
– 2011.09
– 2011.16
-2011.08
-2011.10
• Extraction step converts all forms of B12 into one form
– Therefore quantifies total vitamin B12 content
Some Future Hurdles
The Remaining Vitamins
• Some vitamin forms are in low concentrations
– Are these forms important if only a small portion of the
overall vitamin amount?
• Some vitamin forms not easily tested
• An ongoing battle: Encapsulation
– Always tell the lab of encapsulated vitamins
New Method: B Vitamin Screen
• A method is being developed to measure most of the B
vitamins in one analysis
– The Target: measure all in one analysis
LC/MS/MS Procedure
Homogenize
Taka-diastase
Protease
α-Amylase
Acid Buffers
& Heat
LC/MS/MS Procedure
LC/MS/MS Procedure
B1
B6-N
B3-N
B6-AL
B3-acid
Multi-B Standard Mixture
B6-OL
B9
B7
B5
B2
Sample 418: dry dog food, beef & chicken
30
B Vitamin Screen
• The superior selectivity of LC/MS/MS affords the
method to have a sample-level detection limit of
approximately 1 ppb for all the B vitamins
• The typical microbiological detection limit greatly
varies from ≈500 ppb (B3, B6, B5) to ≈2 ppb (B7, B9)
• 1 ppb is equivalent to 1 yard in the length of ten million
football fields!
B Vitamin Screen
• Samples currently used for development:
– dry dog food: lamb, beef, & chicken
– wet cat food, fish
– NIST infant formula (accepted control for vitamins)
• Samples to be included:
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dry dog food: chicken; turkey; chicken & vegetable
dry cat food: seafood; chicken
wet dog food: beef & rice; boar
wet cat food: chicken
Dry Dog Food: Lamb-Based
Vitamin
Microbiological
LC-MS/MS
AAFFCO Guaranteed
(dwb)
B1 (Thiamine)
16
----
1.0
B2 (Riboflavin)
15.2
11.2
2.2
B3 (Niacin)
63.0
21.8
11.4
B5 (Pantothenic)
47.8
38.8
10
B6 (Pyridoxine)
10.9
11.8
1.0
B7 (Biotin)
0.625
0.43
n/a
B9 (Folic Acid)
0.166
1.23
0.18
Dry Dog Food: Beef & Chicken
Vitamin
Microbiological
LC-MS/MS
AAFFCO
Guaranteed
B1 (Thiamine)
5.2
----
1.0
B2 (Riboflavin)
7.83
2.71
2.2
B3 (Niacin)
60.9
15.7
11.4
B5 (Pantothenic)
27.2
26.5
10
B6 (Pyridoxine)
1.52
3.04
1.0
B7 (Biotin)
0.20
0.16
n/a
B9 (Folic Acid)
0.125
0.272
0.18
Wet Cat Dog Food: Fish
Vitamin
Microbiological
LC-MS/MS
AAFFCO Guaranteed
(dwb)
B1 (Thiamine)
14.2
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5.0
B2 (Riboflavin)
20.5
12.7
4.0
B3 (Niacin)
132
82
60
B5 (Pantothenic)
64
52
5.0
B6 (Pyridoxine)
10
8.2
4.0
B7 (Biotin)
0.46
0.64
0.07
B9 (Folic Acid)
0.67
----
0.8
Conclusions
• B vitamins are important for you and your pet’s health.
• Testing has been a challenge for many years.
• The test methods are getting better and are giving more
information about what’s in your food.
• Methods are being combined to improve speed
Any Discussions?
Copyright © 2015 Silliker Inc