1. The Bridge to a New Generation of B-Vitamin Testing in foods
Steven McCrary and Jeffrey Shippar
Covance Laboratories, Inc.
International Vitamin Conference
12 May 2014, Washington, DC

2. About Covance Nutritional Chemistry and Food Safety Laboratories
Part of Covance, Inc., a leader in drug development since 1997
Vitamin Analysis since 1930 as WARF Institute analyzing vitamin D
About 400 employees at five sites performing analysis of foods, dietary supplements and ag biotech
| B-Vitamin Testing In Foods May 12, 2014

3. | B-Vitamin Testing In Foods May 12, 2014

4. B-Vitamins of Nutritional Relevance
B-Vitamin Overview
Water soluble vitamins
Old analytical methods (1920s), many microbiologically based
AOAC methodology
B-Vitamins of Nutritional Relevance
B-vitamin
Common Name B1
Thiamin B2
Riboflavin B3
Niacin B5
Pantothenic Acid B6
Pyridoxine B7
Biotin B8
Inositol B9
Folic Acid
B10
PABA
B12
Cyanocobalamin
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5. Current Methods – Microbiological
Advantages
All bio-active forms
Optimized extractions for each analyte
Highly sensitive, low LOQs
Low reagent and equipment costs
Disadvantages
Narrow growth curve
Overnight microbial growth step
Microbial response is variable
Separate assay for each analyte
Good technique needed
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6. Current Methods – HPLC-UV/Fluorescence
Advantages
Multiple analytes at once
Same day results
Less variability
Wider standard curve
Disadvantages
B-vitamins from one injection
varying levels analytes
differing wavelength
Different forms of the vitamins
Matrix interferences
Generally higher LODs compared to micro or LC/MS/MS methods
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7. Other Industry Methods
ELISA
Ligand-binding
Titrimetry
Fluorometry
Electrophoresis
GC/GC-MS
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8. New Methodology
B-vitamin profile by ultra high pressure liquid chromatography with tandem mass spectroscopy (LC/MS/MS) detection
Agilent 1290 UHPLC / 6490 triple quadrupole detector
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9. MS/MS Analysis Overview (Thiamine)
Product m/z=122
Precursor m/z [M+H]+=377
Product m/z=144
TY - CHAPJO - Topics on Drug MetabolismAU - Jinsong NiAU - Josh RoweTI - Microdosing Assessment to Evaluate Pharmacokinetics and Drug Metabolism Using Liquid Chromatography-Tandem Mass Spectrometry TechnologyY N UR ER -
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10. LC/MS/MS Overview – Electrospray Ionization
Agilent Technologies, Inc EN (Accessed 02MAY14)
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11. Newly Developed LC/MS/MS Method
Advantages
Specificity
Detection
Repeatability
Curve range
Turnaround time
Flexibility
Simultaneous determination of 7 B-vitamins B1
Thiamin B2
Riboflavin B3
Niacin B5
Pantothenic Acid B6
Pyridoxine B7
Biotin B9
Folic Acid 11
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12. Centrifuge extract and filter
Method Overview
Weigh
Sample
(1)
Weigh
Sample
(2)
Add
labeled ISTD
(3)
Add 1% ascorbic acid
(4)
Shake samples
(5)
Add
NH4OH and shake
(6)
Centrifuge extract and filter
Time (min) %B
0.5 1
0.6 10 5 85
5.1
7.5
Instrument:
Column: Zorbax SB-Aq 100 x 3.00 mm, 1.8 µm
Flow rate: 0.5 mL/min
Mobile Phase A: 20mM ammonium formate w/0.1% formic acid
Mobile Phase B: 20mM ammonium formate w/0.1% formic acid in Methanol
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13. LC/MS/MS Overview – Dynamic MRM
30 total MRMs
Maximum 19 concurrent
Minimum dwell time 27ms
Compound Name
Polarity
Precursor Ion
Product Ion
Res
Ret Time (min)
Collision Energy
Thiamine
Positive
265.11
121.9
Unit/Unit
2.18 8
80.9 28
Thiamine 13C4
269.11
122
Pyridoxine
170.08
152.1
2.31 12
134 24 77 40
Pyridoxine HCl 13C4
174.08
138.1
2.37
Nicotinic Acid D4
128.04
96.1
2.38
Nicotinic Acid
124.04
80.1
2.39 20
78.1 53 32
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14. LC/MS/MS Overview – Dynamic MRM (cont.)
Compound Name
Polarity
Precursor Ion
Product Ion
Res
Ret Time (min)
Collision Energy
Pantothenic Acid
Positive
220.12
202.1
Unit/Unit
2.95 4 90 8
71.9 16
Pantothenic Acid 13C3 15N
224.12
94.2
Niacinamide D4
127.06
84.1
3.22 24
Niacinamide
123.06 80
3.24 20 53 36
Biotin 13C5
250.1
126
4.06 28
Biotin
245.1
123
4.07
97.1 32
227.1
Folic Acid
442.15
295
4.12
176 44
120
Folic Acid 13C5
447.15
295.1
Riboflavin
377.15
243
4.28
198.1
172
Riboflavin 13C4 15N2
383.15
249
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15. Chromatography Niacinamide Thiamine Pyridoxine, nicotinic acid
Pantohenic acid

16. Chromatography Riboflavin Biotin Folic Acid
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17. Validation Plan and Data
Infant Formula
Powder
Ready to Feed Liquids
Dietary Supplements
Tablets (NIST 3280)
Capsules
Gummies (Pectin & Gelatin)
Complex Adult Meal Replacement
Drinks
Fortified Foods
Breakfast Cereals
Pet Food
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18. LC/MS/MS Data - NIST SRM 1849a Infant Formula
  Analyte
Range (mg/kg)
(mg/kg)
%RSD
Niacinamide
111
3.0
Pyridoxine
13.46
2.7
Pantothenic acid
68.6
3.4
Thiamine
13.1
3.2
Biotin
1.98
Riboflavin
20.4
2.9
Folic acid
2.31
2.8
National Institute for Standards and technology, Certificate of Analysis: Standard Reference Material 3280, Table 1, page 6 of (accessed May 2, 2014)
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19. Infant Formula Comparison: LC/MS/MS and Microbiological
Analyte
LC/MS/MS
(%RSD)
(mg/100g)
Micro result
% difference
Thiamine
4.1%
1.25 NA
Pyridoxine
3.1%
0.496
0.745
-33%
Pantothenic Acid
2.4%
5.45
5.35
+1.9%
Niacinamide
2.1%
7.65
8.21
-6.8%
Biotin
3.4%
0.0550
0.0581
-5.7%
Folic Acid
6.2%
0.157
-0.10%
Riboflavin
2.6%
1.23
1.56
-21.3%
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20. LC/MS/MS Data - NIST SRM 3280 Tablet
Analyte
Mass Fraction
(NIST)
Value (n=18)
%RSD
Thiamine HCl
mg/g
115 mg/g
2.9
Pyridoxine HCl
mg/g
1.88 mg/g
4.6
(Ca)Pantothenic acid
mg/g
7.21 mg/g
Niacinamide
mg/g
15.2 mg/g
3.8
Biotin
µg/g
2.32 µg/g
6.2
Folic acid
µg/g
38.1 µg/g
Riboflavin
mg/g
1.34 mg/g
3.6
National Institute for Standards and technology, Certificate of Analysis: Standard Reference Material 3280, page 8 of May 2, 2014)
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21. LC/MS/MS Data - NIST SRM 3280 Tablet Niacinamide spike recovery
Spike level (n=3)
% recovery
50%
101%
100%
99%
150% 21
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22. LC/MS/MS Data-Pectin Gummy Recovery Data
Analyte
Fortified/ blank
Innate %RSD
Recovery %
(LMH or 50,100,150)
Thiamine HCl
Blank NA
95%, 96%, 99%
Pyridoxine HCl
Fortified
2.8%
109%, 97%, 96%
Nicotinic Acid
105%, 108%, 101%
(Ca) Pantothenic Acid
100%, 102%, 102%
Niacinamide
Heavily fortified
3.0
100%, 100%, 96%
Biotin
2.2%
105%, 101%, 99%
Folic Acid
5.8%
94%, 110%, 98%
Riboflavin
102%, 101%, 100%
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23. LC/MS/MS Data - Gelatin Gummy Recovery Data
Analyte
Fortified/ blank
Innate %RSD
Recovery %
(LMH or 50,100,150)
Thiamine HCl
Blank NA
97%, 97%, 97%
Pyridoxine HCl
Fortified
1.5%
99%, 95%, 98%
(Ca) Pantothenic Acid
102%, 100%, 101%
Niacinamide
99%, 99%, 99%
Biotin
2.5%
95%, 101%, 98%
Folic Acid
3.8%
95%, 105%, 102%
Riboflavin
105%, 104%, 103%
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24. Newly Developed LC/MS/MS Method
Challenges
Extraction challenges
Quantitation limited by form
Cost
Instrumentation
Aglilent 1290 UHPLC ~$100k
Agilent 6490 Triple Quadrupole >$400k
Total >$500k
Isotopically labelled internal standards
[13C5] - Biotin (30mg) $9500
[13C5] - Folic acid $2800
[13C4] - Thiamine HCl (10mg) $1100
[13C3,15N] - Calcium pantothenate $ 550
[13C4] - Pyridoxine HCl $ 450
[13C4,15N2] – Riboflavin $1500
[D4]-Nicotinic acid $ 425
[D4]-Nicotinamide $ 525
Total >$25k
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25. Conclusions
LCMSMS analysis of B vitamins results in a greater level of specificity and efficiency
LCMSMS data may not compare to historical, bio-active based, B-vitamin data
A variety of analytical approaches will continue to be employed for B-vitamins
There is much more to learn
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26. Acknowledgements Chad Scheuerell Joe Polywacz Brent Rozema
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27. The Bridge to a New Generation of B-Vitamin Testing in Foods
Questions?
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28. Appendix – vitamin structures
Pyridoxamine
Pyridoxal
Pyridoxic acid
Nicotinic acid (B3)
Nicotinamide (B3)
Riboflavin (B2)
Thiamine (B1)
Pantothenic acid (B5)
PABA (4-Aminobenzoic acid, B10 )
Pyridoxine (B6)
Fig. 1 Chemical structures of B vitamins