Analysis of B-Vitamins by LC-MS/MS in Infant Formula

Analysis of B-Vitamins by LC-MS/MS in Infant Formula and Dietary Supplements
Chad Scheuerell, Jeff Shippar, John Austad and Brent Rozema
Covance Laboratories Inc., Madison, Wisconsin
Introduction
Results
Analytical methods for analysis of B vitamins have changed little over
the years. While some chemical analysis methods exist, microbial
analysis methods for individual B vitamins have been the standard
since the early-to-mid 1900s. These methods determine bio-available
levels of B vitamins based upon actual biological uptake. However,
they require extensive lab preparation, lengthy incubation times and
are not very rugged. In today’s food testing environment where
accuracy and speed are more critical than ever, change is required for
B vitamin analysis. Using ultra high pressure liquid chromatography
(UHPLC) and tandem mass spectrometry (MS/MS) detection (on an
Agilent 1290 UHPLC and 6490 triple quadrupole platform), combined
with quick and simple extraction, the next generation of B vitamin
analysis is here. Detection and quantitation of 8 different B vitamins
(thiamine, riboflavin, nicotinic acid, niacinamide, pantothenic acid,
pyridoxine, biotin and folic acid) in infant formula and dietary
supplements all in just a few hours of analytical time is now a reality.
This method has been validated on ready-to-feed infant formula (RTF), powder infant formula
(milk-based, soy-based and hypoallergenic protein sensitivity formulation), adult RTF nutritionals,
gummy supplements (gelatin and pectin based) and energy drinks. Included in this validation were two
certified reference materials (CRM), NIST 1849a Infant/Adult Nutritional Formula and NIST 3280
Multivitamin/Multielement Tablets. Spike recoveries were performed on any B-vitamin that was not present
in the matrix being tested. Curve ranges were set to accommodate the majority of samples tested without
the need for multiple dilutions or injections. Generally 1-10 ng/mL was used for the lowest standard
concentrations of each B vitamin.
Experimental
Samples are extracted by agitation using acidic aqueous-solvent
mixture, followed by the addition of a small amount of alkaline solution
to precipitate proteins and aid in chromatography. Extracts are
centrifuged and filtered. The LC-MS/MS analysis is performed with Jet
Stream Electrospray ionization. All compounds were analyzed in ESI+,
giving [M+H]+ precursor ions. Two or three transitions are collected
using dynamic MRM (dMRM) mode for each analyte for quantification
and identification purposes. The extracted samples are compared to
standards of known concentrations. Stable isotope internal standards
of each B vitamin are used in the quantitation.
(1)
(2)
(3)
(4)
(5)
(6)
Weigh
Sample
Add
labeled
ISTD
Add 1%
ascorbic
acid
Shake
samples
Add
NH3(aq)
and shake
Centrifuge
extract and
filter
Instrument: Agilent 1290 UHPLC coupled with an Agilent 6490 triple
quadrupole mass spectrometer.
Column: Zorbax Eclipse Plus C18 RRHT 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
Time (min)
% Mobile
Phase B
0.5
1
Figure 1. Extracted ion chromatograph (XIC)
of a mixed B vitamin standard.
Figure 2. Extracted ion chromatograph
(XIC) of a mixed B vitamin standard.
Note: These two XICs are of the same standard injection. It was split into two
chromatograms for display purposes only.
Table 1. MS/MS Conditions for the Tested B Vitamins
and Their Isotopically Labeled Internal Standards
Analyte
Compound
Name
Polarity
Precursor
Ion
Product
Ion
Res
Ret Time
(min)
Thiamine
Positive
265.11
121.9
Unit/Unit
2.18
8
Thiamine
Positive
265.11
80.9
Unit/Unit
2.18
28
Positive
269.11
122
Unit/Unit
2.18
8
Pyridoxine
Positive
170.08
152.1
Unit/Unit
2.31
12
Pyridoxine
Positive
170.08
134
Unit/Unit
2.31
24
Pyridoxine
Positive
170.08
77
Unit/Unit
2.31
40
Pyridoxine
HCl 13C4
Positive
174.08
138.1
Unit/Unit
2.37
24
Thiamine
13C
4
Collision
Energy
Nicotinic Acid D4
Positive
128.04
96.1
Unit/Unit
2.38
12
Nicotinic Acid
Positive
124.04
80.1
Unit/Unit
2.39
20
Nicotinic Acid
Positive
124.04
78.1
Unit/Unit
2.39
24
Nicotinic Acid
Positive
124.04
53
Unit/Unit
2.39
32
Pantothenic Acid
Positive
220.12
202.1
Unit/Unit
2.95
4
Pantothenic Acid
Positive
220.12
90
Unit/Unit
2.95
8
Pantothenic Acid
Positive
220.12
71.9
Unit/Unit
2.95
16
Pantothenic Acid
13C 15N
3
Positive
224.12
94.2
Unit/Unit
2.95
8
Niacinamide D4
Positive
127.06
84.1
Unit/Unit
3.22
24
Niacinamide
Positive
123.06
80
Unit/Unit
3.24
20
Niacinamide
Positive
123.06
53
Unit/Unit
3.24
36
Positive
250.1
126
Unit/Unit
4.06
28
Biotin
Positive
245.1
123
Unit/Unit
4.07
28
Biotin
Positive
245.1
97.1
Unit/Unit
4.07
32
Biotin
Positive
245.1
227.1
Unit/Unit
4.07
8
Folic Acid
Positive
442.15
295
Unit/Unit
4.12
20
Folic Acid
Positive
442.15
176
Unit/Unit
4.12
44
Folic Acid
Positive
442.15
120
Unit/Unit
4.12
44
Positive
447.15
295.1
Unit/Unit
4.12
20
Biotin
13C
Folic Acid
5
13C
5
Riboflavin
Positive
377.15
243
Unit/Unit
4.28
16
5
85
Riboflavin
Positive
377.15
198.1
Unit/Unit
4.28
44
5.1
1
Riboflavin
Positive
377.15
172
Unit/Unit
4.28
16
1
Riboflavin
13C 15N
4
2
Positive
383.15
249
Unit/Unit
4.28
16
Note: dMRM analysis was used with a max dwell time of 27ms and a
maximum of 19 concurrent MRM.
NIST Range
(mg/kg)
Assay mean
(mg/kg)
NIST Range
Assay mean
%RSD
Thiamine HCl
94-118 mg/g
116 mg/g
2.9
99-119
111
3.0
Pyridoxine HCl
1.47-1.98 mg/g
1.91 mg/g
4.6
12.53-14.39
13.2
2.7
(Ca)Pantothenic acid
66.3-70.1
68.6
3.4
Niacinamide
Niacinamide
10
Presented at AOAC INTERNATIONAL 2014
Table 3. NIST 3280 Multivitamin Tablet Results
Table 2. NIST 1849a Infant Formula Results
0.6
7.5
Accuracy was demonstrated by analyzing NIST 1849a and NIST 3280 over 7 days of analysis. Results for both
CRMs were compared to the mean and certified range of the NIST certificate. Spike recoveries were performed
on all other matrices validated.
Pyridoxine
Pantothenic acid
Thiamine
Analyte
%RSD
6.94-8.26 mg/g
7.46 mg/g
4.6
13.87-14.33 mg/g
14.88 mg/g
3.8
6.2
11.59-13.55
13.1
3.2
Biotin
2.02-2.66 μg/g
2.34 μg/g
1.86-2.12
1.98
2.7
Folic acid
37.2-41.6 μg/g
38.8 μg/g
3.8
Riboflavin
19.85-20.89
21.4
2.9
Riboflavin
1.15-1.49 mg/g
1.39 mg/g
3.6
Folic acid
2.23-2.36
2.31
2.8
Biotin
Precision was determined by calculating the %RSD for the various analyte/matrix combinations and comparing to the
maximum predicted reproducibility using the Horwitz equation (%RSD ≤2C^-.0150). All matrices and compounds fell within
acceptable range.
Table 4. Precision for All Matrices Analyzed
Thiamine HCL
% RSD
Pyridoxine HCL
% RSD
Pantothenic acid
% RSD
Niacin
% RSD
Biotin
% RSD
Folic acid
% RSD
Riboflavin
% RSD
Milk Based
2.4
1.8
2.6
1.9
2.9
2.5
2.7
Soy Based
2.4
1.4
2.5
4.1
2.1
1.2
1.7
Ready to Feed
3.2
2.8
1.4
3.5
3.0
7.9
3.3
Hypoallergenic
(protein sensitivity)
2.5
2.3
1.9
6.4
3.2
2.9
2.5
3.7
5.5
1.4
3.4
4.7
Infant Formula
Dietary
Supplements
Gelatin Gummies
3.2
Pectin Gummies
1.7
2.9
4.5
2.6
Tablets/Capsules
Energy Drinks
3.8
4.2
4.4
4.2
5.5
4.6
6.2
Adult ready to feed
nutritional
2.3
1.7
2.8
2.7
2.1
2.3
2.4
Discussion
This method was developed to make testing for B vitamins more accurate and much faster than traditional methods. It has
proven to be simple, robust and easily transferable from lab to lab, as it is now being run at three different Covance sites on
three different continents around the world (USA, Singapore and United Kingdom). Method development is currently underway
to incorporate food matrices, other forms of B vitamins, as well as many other special requests by our clients.

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