Qualitative and Quantitative Analysis of Water-Soluble B Vitamins by HPLC PDA in Various Multivitamin Tablets

A P P L I C AT I O N N O T E
Liquid Chromatography
Authors:
Andrew P. Zdzieblo
Wilhad M. Reuter
PerkinElmer, Inc.
USA
The Qualitative and Quantitative
Analysis of Water-Soluble B
Vitamins by HPLC-PDA in
Various Multivitamin Tablets
Introduction
Vitamins are essential
nutritional elements
in a human diet.
Though we are able
to synthesize certain
vitamins, such as Vitamin D when exposed to sunlight, we mainly depend on
our diet to supply our vitamins.
Since Ancient Egypt, humans have recognized that eating certain foods guarded
us from certain diseases. But only in 1912 did Polish biochemist Kazimierz Funk
establish the term “Vitamine”, derived from the compound words Vital and
Amine, together meaning amine of life. Today, the term “Vitamin” is described
as an organic compound serving as a vital nutrient that an organism requires in
limited amounts.1
The food industry routinely fortifies many foods with vitamins to enhance their
nutritional value and to help with deficiencies in dietary requirements. However,
to meet legal requirements, the manufacturers must label their products in
accordance to the specific regulations pertaining to the country in which the
products are sold and consumed.
In 2009, the United States Pharmacopeial Convention introduced
the USP Dietary Supplements Compendium (DSC) – an industrydirected resource, including regulatory guidance and reference
tools. In the U.S., the Food and Drug Administration (FDA)
regulates all dietary supplement products and manufacturing
practices under 21 CFR (Code of Federal Regulation), Part 111.2,3
In Europe, the main EU legislation for vitamins and minerals in
food supplements is Directive 2002/46/EC.4
Considering the above, this application focused on providing a
robust chromatographic method for the separation of eight
water-soluble B vitamins in vitamin supplement tablets using
HPLC with photodiode array (PDA) detection. Due to the diverse
absorptivity range amongst these vitamins, multiple analytical
wavelengths were used to provide the best overall response.
Method conditions and performance data, including linearity
and repeatability, are presented.
Vitamin B standards were obtained from Sigma-Aldrich Inc®
(St Louis, MO). These included: thiamine HCl (B1), riboflavin (B2),
niacin (nicotinic acid; B3), niacinamide (B3; labeled “B3′” to
distinguish it from niacin), pyridoxine (B6), biotin (B7), folic acid
(B9) and cyanocobalamin (B12). The structures of these vitamins
are shown in Figure 1.
Thiamine – B1
Riboflavin – B2
Experimental
Hardware/Software
For chromatographic separations, a PerkinElmer Altus™ HPLC
System was used, including the Altus A-10 solvent/sample module,
integrated vacuum degasser, A-10 column module and Altus A-10
PDA detector. All instrument control, analysis and data processing
was performed using the Waters® Empower® 3 chromatography
data software (CDS) platform.
Niacin – B3
Niacinamide – B3′
Method Parameters
The HPLC method parameters are shown in Table 1.
Table 1. HPLC Method Parameters.
Pyridoxine – B6
HPLC Conditions
Column:
Biotin – B7
PerkinElmer Brownlee™ SPP C18 2.7 µm,
3.0 x 100 mm (Part# N9308410)
Mobile Phase A: 5 mM ammonium formate (NH4
formate) buffer; pH 4.8 – 4.9
Mobile Phase B: Acetonitrile (ACN)
Solvent program:
Mobile Phase:
Time
1
Flow
%A
%B
%C
%D
0.60
100.00
0.0
0.0
0.0
Curve
2
2.00
0.60
100.00
0.0
0.0
0.0
6
3
8.00
0.60
70.00
30.0
0.0
0.0
6
4
10.00
0.60
70.00
30.0
0.0
0.0
6
5
10.01
0.60
100.00
0.0
0.0
0.0
6
Analysis Time:
10 min.; 6-minute injection delay time between injections
Flow Rate:
0.6 mL/min. (max. pressure: ~2300 psi/~153 bar)
Oven Temp.:
40 ºC
Detection:
Altus A-10 PDA;
Wavelengths used for calibration/quantitation:
214 nm: for B3, B3', B6, B7, B9, and B12
267 nm: for vitamins B1 and B2
Injection Volume:
3 µL
Sampling (Data) Rate:
10 pts./sec
Solvents, Standards and Samples
All solvents and diluents used were HPLC-grade and filtered via
0.45-µm filters.
2
Folic Acid – B9
Cyanocobalamin – B12
Figure 1. Chemical structures of B vitamins.
As the concentration of individual B vitamins in dietary supplements
varies widely from one vitamin to another, two sets of standards
were prepared. The first, working standard 1 (WS1), was used for
demonstrating chromatographic separation and retention time
(RT) reproducibility. It was prepared by weighing out 80 - 320 µg/
ml of each of the eight vitamins, depending on the vitamin’s
relative absorptivity. The second, working standard 2 (WS2),
was used for calibration/linearity. It was prepared by weighing
individually selected amounts for each vitamin, depending on its
expected concentration range in typical dietary supplements.
For both WS1 and WS2, the eight vitamins were weighed out
and transferred into individual 50-mL volumetric flasks, to which
30 mL of water was added. Additionally, for vitamins B9, B2 and
B7, to improve solubility, 0.5 mL of 0.1N NaOH was added to the
corresponding flasks. All flasks were sonicated for five minutes
and then filled to the mark with water. Equal volumes of the eight
respective stock standards were then added together to produce
each of the two WS solutions. For WS1, the resulting concentration
for the eight vitamins was between 10 - 40 µg/ml. For WS2,
the vitamins ranged in concentration from 0.9 to 525 µg/mL,
depending on the vitamin. For calibration, all subsequent
concentration levels were prepared from WS2.
All individual stock solutions and working standards were stored
at 4.0 °C when not in use.
The multivitamin tablet samples, Multivitamin X and Multivitamin Y,
were purchased from a local store. For each sample, a multivitamin
tablet was transferred into an individual 50-mL volumetric flask and
30 mL of water was then added. The flask was then stoppered and
placed on a heater/stirrer and heated at 40 °C while stirring for
15 min. Once the tablet was completely dissolved, each solution
was sonicated for 10 min and then filled to the mark with HPLCgrade water. To remove any small particles, all standards and
samples were filtered through 0.45 µm filters prior to injection.
Results and Discussion
Using the optimized chromatographic conditions described above,
Figure 2 shows the HPLC separation of the eight B vitamins in WS1,
at both 214 and 267 nm. All eight B vitamins are well resolved.
The unknown peak at about 1.5 minutes did not interfere with the
separation of the B vitamins and was not further characterized.
P roject Name: Water S oluble Vit.B
0.20
B3
B3′
0.16
a) 214 nm
B2
0.18
0.14
0.12
B12
0.06
B6
unknown
0.08
B9
AU
0.10
0.04
B7
B1
0.02
0.00
-0.02
0.00
0.35
1.00
2.00
3.00
4.00
5.00
Minutes
4.00
5.00
Minutes
6.00
P roject Name: Water S oluble Vit.B
7.00
9.00
10.00
B2
b) 267 nm
8.00
0.30
0.25
AU
0.20
0.15
B9
B3
B3′
0.05
unknown
0.10
B12
B1
B6
0.00
S ampleName
All1.0
Vitamin
B
's ~40ug/ml
F eb13
0.00
0
2.00
3.00
Current Date: 4/28/2015
S ample S et Id 5377
Channel
6
.00
2998
Ch3 214nm@
1.2nmMB F10.00
7.00
8.00
9.00
S ample S et Name
VitaminB _ S S uit_ F eb18_ S S
Figure 2. Chromatogram of the vitamin B working standard 1 (WS1); wavelength: a) 214 nm and b) 267 nm.
3
Figure 3 shows the overlay of 20 replicate WS1 injections, demonstrating exceptional reproducibility. The retention time % RSDs for all
eight vitamins were < 0.3% (0.142% for riboflavin).
ProjectNam
e: WaterSoluble Vit.B
0.30
0.25
AU
0.20
0.15
0.10
0.05
0.00
0.00
1.00
2.00
3.00
4.00
Minutes
5.00
6.00
7.00
8.00
9.00
Figure 3. Overlay of 20 replicates of the WS1 vitamin B standard; wavelength: 267 nm.
Table 2 shows the concentration at each calibration level for each of the eight vitamins. Figure 4 shows the calibration results for four
example vitamins. All B vitamins displayed linear fits, with most resulting in R2 values > 0.999 (n = 3 at each level).
Table 2. Concentration (µg/mL) of B vitamins in WS2, for calibration/linearity.
Vitamins
B1
Level 1
Level 2
Level 3
Level 4
Level 5
Level 6
Level 7
2.64
8.79
17.58
26.38
35.17
43.96
52.75
8.88
17.75
26.63
Nam
e B12, B2, B3
', B7, B9, B1, B6, B335.50
44.38
53.25
@1.2nm
B2Channel 2998Ch2267nm
2.66
4
Table 1. HPLC
Method Parameters.17.67
8.83
B3
2.65
26.50
35.33
44.17
53.00
B3′
26.23
87.42
174.83
262.25
349.67
437.08
524.50
B6
3.13
10.42
20.83
31.25
41.67
52.08
62.50
B7
B9
0.54
1.79
3.58
5.38
7.17
8.96
10.75
0.91
3.04
6.08
9.13
12.17
15.21
18.25
B12
0.04
0.15
0.29
0.44
0.58
0.73
0.88
50000
8000.0
0
Channel 2998 Ch1 214nm@ 1.2nm
6000.0
-50000
0.00
2.00
4.00
6.00
8.00
10.00
Amount
12.00
14.00
16.00
0.00
18.00
0.10
0.20
0.30
0.40
Amount
0.50
0.60
0.70
0.80
0.90
Name:
Name: B9; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11588;
A: B12; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order);
6.662744e+003; B: 9.783058e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.995405
-1.051709e+004; B: 1.239506e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.996478
Cal Curve Id: 11590;
A:
Name: B3'; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11586;
-6.838097e+004; B: 1.707714e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999349
A:
ProjectName: WaterSoluble Vit.B
800000
1.0x10 7
B3
700000
8.0x10 6
600000
500000
B3′
6.0x10 6
Area
A r ea
400000
4.0x10 6
300000
200000
2.0x10 6
100000
Channel 0
2998 Ch1 214nm@ 1.2nm
0.00
5.00
10.00
0.0
Channel
2998 Ch1 214nm@ 1.2nm
R² = 0.999216
100000
15.00
20.00
25.00
Amount
30.00
35.00
40.00
45.00
R² = 0.999349
-2.0x10 6
50.00
0.00
Name: B3; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11584; A:
-6.208800e+003; B: 1.362139e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999216
50.00
100.00
150.00
200.00
250.00
300.00
Amount
350.00
400.00
450.00
500.00
700000
B7
10000.0
600000
B2
500000
8000.0
400000
6000.0
A r ea
A r ea
300000
4000.0
200000
Channel [email protected]
2000.0
100000
0.0
0
Channel 2998
Ch1 214nm@ 1.2nm
R² = 0.998639
-2000.0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Amount
7.00
8.00
9.00
10.00
R² = 0.999052
-100000
0.00
11.00
5.00
10.00
15.00
20.00
25.00
30.00
Amount
35.00
40.00
45.00
50.00
Name:
Name: B7; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11589;
A: B2; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11591;
-6.887704e+003; B: 1.101062e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999052
-1.638886e+002; B: 9.109663e+002; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998639
900000
A:
Figure 4. Calibration plots of 7-level calibration set for the vitamins B3, B3′, B7 and B2.
800000
700000
180000.0
600000
160000.0
500000
140000.0
400000
120000.0
300000
100000.0
200000
80000.0
100000
0
-100000
0.00Ch1 214nm@
10.00
Channel 2998
1.2nm
20.00
30.00
Amount
40.00
50.00
60.00
For both samples, the large initial peak eluting very near the void
volume was expected to be primarily vitamin C (ascorbic acid).
As vitamin C was not the focus of this application, this peak was
not further characterized.
A r ea
A r ea
Using the same chromatographic conditions, the two
multivitamin tablets, Multivitamin X and Multivitamin Y, were
then analyzed. The chromatographic results are shown in Figures
5 and 7-9, displayed at 267 and 214 nm for each of the two
samples. Comparing the chromatograms of both of these
samples with the WS2 standard mix, it appears that both
samples have a similar vitamin B profile. Their presence was
further confimed by UV spectral matching, except for vitamins
B7 and B9, due to their very low concentration in the samples.
60000.0
40000.0
Per Figure 5, for Multivitamin X, although a peak eluted at the
expected retention time for vitamin B9 (folic acid), the UV
spectra for that peak did not match the expected spectra
for vitamin B9 (Figure 6). As there was no positive spectral
confirmation of vitamin B9 in either sample, the amount of
vitamin B9 could not be calculated.
20000.0
0.0
Channel 2998 Ch1 214nm@ 1.2nm
-20000.0
Name: B6; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11585;
-1.383973e+004; B: 1.216932e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998618
16000.0
A r ea
10000.0
A:
5.00
10.00
15.00
20.00
25.00
30.00
Amount
35.00
40.00
45.00
50.00
Name: B1; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11587;
-3.589116e+003; B: 2.890673e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999492
A:
P roject Name: Water S oluble Vit.B
14000.0
12000.0
0.00
0.45
0.40
Channel 2998 Ch1 214nm@ 1.2nm
8000.0
0.35
Channel 2998 Ch1 214nm@ 1.2nm
6000.0
Project Name: Water Soluble Vit.B
0.00
0.10
0.20
0.30
0.50
0.60
0.70
0.80
0.90
B1
0.40
Amount
Name: B12; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order);
6.662744e+003; B: 9.783058e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.995405
0.25
B2
0.30
Cal Curve Id: 11590;
A:
AU
1.0x10 7
0.20
8.0x10 6
250000
6.0x10 6
200000
150000
0.05
0.0
0.00
A r ea
100000
50000
-2.0x10 6
B9
unknown
0.10
2.0x10 6
unknown
excipient
A r ea
0.15
4.0x10 6
0
0.00
50.00
100.00
Channel 2998
Ch1 214nm@
1.2nm
150.00
200.00
250.00
300.00
Amount
350.00
400.00
Report Method Name: Overlay sample for450.00
App Notes
Sample Set Id 7974
Current Date: 3/17/2015
500.00
Injection Id 8011, 8075
SampleName MultiVitamin Sample X2, Vit B
WS High
B3'; Processing
Assay_Feb24_PM;
Linear
(1st
Curve
.00 Id: 11586;
4.0
0 Order); Cal 5
3.00FitChannelType:
2.00
1.00 Method: WS VitB
-50000
Sample Set Name MultiVitAssay_Feb23_SS
2998 Ch2 [email protected]
Page: 1 of 1
-6.838097e+004; B: 1.707714e+004; C: 0.000000e+000; D: 0.000000e+000; R^2:
0.999349 Minutes
0Name:
.00
0.00
2.00
4.00
6.00
8.00
10.00
Amount
10.00
9.00
8.00
7.00
A:
6.00
12.00
14.00
16.00
18.00
Name: B9; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11588;
-1.051709e+004; B: 1.239506e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.996478
Figure 5. Overlaid chromatograms of Multivitamin X (red) with WS2 (black); wavelength: 267 nm. For optimal sensitivity, only vitamins B1 and B2 were quantitated at 267 nm.
700000
600000
500000
400000
A r ea
300000
200000
Channel 2998 Ch1 214nm@ 1.2nm
100000
Channel 2998 Ch1 214nm@ 1.2nm
0
5
-100000
0.00
5.00
10.00
15.00
20.00
25.00
30.00
Amount
35.00
40.00
45.00
50.00
B2; Processing
Method:
WS VitB Assay_Feb24_PM;
Fit Type:
Linear
(1st Order);
R eportName:
Method
Name:
Overlay
sample for
App
Notes
Cal Curve Id: 11591;
A:
A:
B1
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
AU
234.8
S ampleName MultiVitamin S ample X1,
MultiVitamin S ample Y1, Vit B W S Low
263.3
0.001
0.030
0.020
395.8
0.000
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
197.1
194.8
B9
a) WS2
282.3
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
P roject Name: W ater S oluble Vit.B
S ampleName: Vit B W S Low Name: B 1
S ampleName: MultiVitamin S ample Y1 Name: B 1
B9
b) Multivitamin X
0.030
0.010
0.0010
279.9
AU
AU
AU
266.8
0.0002
343.2
0.0000
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: Vit B W S Low Name: B 9
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: MultiVitamin S ample Y1 Name: B 9
S ampleName: MultiVitamin S ample X1 Name: B 9
B7
B7
0.006
0.003
Figure 6. UV spectra captured at the expected elution time forPvitamin
B9:Name:
a) WS2, b)
Multivitamin
X and Vit.B
c) Multivitamin Y.
roject
Water
S oluble
Injection Id 7976, 8043, 8107
0.004
AU
AU
0.002
0.001
B3′
1.40
381.3
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: Vit B W S Low Name: B 7
207.7
0.005
B 12
0.003
360.4
277.5
0.001
S ampleName: Vit B W S Low Name: B 12
266.8
0.00
AU
B2
1.00
5.80
6.00
6.20
0.000
Minutes
6.40
3.00
5.00
Minutes
S ample S et Id 7974
7.00
S ampleName: MultiVitamin S ample Y1 Name: B 12
6.00
265.6
B2
223.0
0.040
Injection Id 8055
7.00
8.00
9.00
S ampleName MultiVitamin S ample X 1
Current Date: 3/12/2015
0.030
S ample S et Name MultiV itAssay_ F eb23_ S S
Channel 2998 Ch1 214nm@ 1.2nm
0.10
6.80
0.050
B2
R eport Method Name: Overlay sample for App Notes
4.00
0.15
6.60
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
266.8
223.0
0.25
364.4
0.010
5.60
0.20
2.00
360.4
0.001
P roject Name: W ater S oluble Vit.B
AU
223.0
257.3
0.002
0.000
B2
B6
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
0.003
B 12
-0.010
unknown
excipients
0.20
0.00
Channel 2998
Current Date: 3/10/2015
0.005
-0.005
0.000
0.030
0.010
0.004
AU
S ampleName MultiVitamin S ample X1,
MultiVitamin S ample Y1, Vit B W S Low
AU
0.003
0.015
207.7
B12
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
S ampleName: MultiVitamin S ample Y1 Name: B 7
0.005
P roject Name: Water S oluble V it.B
AU
AU
0.020
0.40
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
unknown
excipients
unknown
excipient
0.025
Injection Id 7976, 8043, 8107
0.002
-0.0010
S ampleName: MultiVitamin S ample X1 Name: B 12
B 12
386.1
-0.0005
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
P roject Name: W ater S oluble Vit.B
0.004
312.1
AU
359.4
0.000
0.60
207.7
B7
0.0000
0.002
0.001
0.005
191.3
0.0005
AU
Channel 2998
Current Date: 3/10/2015
0.80
0.020
0.0010
0.004
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
MultiVitamin S ample Y1, Vit B W S Low
P roject Name: W ater S oluble Vit.B
S ampleName: MultiVitamin S ample X1 Name: B 7
1.20
S ampleName MultiVitamin S ample X1,
1.00
Channel 2998
Current Date: 3/10/2015
0.000
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
Injection Id 7976, 8043, 8107
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
S ampleName MultiVitamin S ample X1,
MultiVitamin S ample Y1, Vit B W S Low
303.7
0.002
352.7
268.0287.0302.5316.8
0.000
AU
B9
0.0004
0.000
196.0
c) Multivitamin Y
0.0006
0.010
346.7
0.000
192.4
0.0008
0.020
0.005
0.004
Current Date: 3/10/2015
0.010
333.6 355.1
0.000
0.015
Channel 2998
AU
Injection Id 7976, 8043, 8107
0.002
B1
0.040
0.003
P age: 1 of 1
10.00
0.020
364.4
Figure 7. Single chromatogram of Multivitamin X; wavelength: 214 nm. For optimal sensitivity, only vitamins B6, B3’ and B12 were quantitated at 214 nm. Vitamins B3364.4
and B7 were
not detected..
0.000
0.05
0.010
0.00
0.000
200.00 220.00
240.00 260.00 Water
280.00 300.00 S
320.00
340.00 360.00Vit.B
380.00
P roject
Name:
oluble
nm
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: Vit B W S Low Name: B 2
0.002
0.22
212.4
0.000
0.004
B3
B3
Injection Id 7976, 8043, 8107
0.002
AU
0.18
0.16
255.0
298.9
S ampleName: MultiVitamin S ample Y1 Name: B 3
194.8
B6
B1
0.15
0.10
AU
Channel 2998
Current Date: 3/10/2015
B2
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
S ampleName MultiVitamin S ample X1,
S ample Y1, Vit B W S Low
0MultiVitamin
.08
290.6
0.05
256.1
Report Method Name: Overlay sample for App Notes
0.06
200.00 220.00 240.00 260.00
Current Date: 3/
12/2015
P roject Name: W ater S oluble Vit.B
S ample S et Name MultiVitAssay_ Feb23_ S S
194.8
0
.02
B6
0.00
Injection Id 7976, 8043, 8107
AU
290.6
1.0
0
324.0
3.00
2.00
Report Method Name: Overlay sample for App Notes
Sample Set Id 7974
0.00
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
S ampleName MultiVitamin S ample X1,
MultiVitamin S ample Y1, Vit B W S Low
0.10
256.1
Injection Id 8055
S ampleName MultiVitamin S ample X 1
280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: MultiVitamin S ample Y1 Name: B 6
Channel
2998 Ch1 214nm@ 1.2nm
0.20
0.00
324.0
0.00
S ample S et Id 7974
0.05
398.2
Project Name: Water Soluble Vit.B
0.12
0.04
Current Date: 3/17/2015
Channel 2998 Ch2 [email protected]
Injection Id 8139
5.00
Minutes
4.00
6.00
7.00
8.00
SampleName MultiVitamin Sample Y2
Sample Set Name MultiVitAssay_Feb23_SS
Page: 1 of 1
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
P roject Name: W ater S oluble Vit.B
S ampleName: MultiVitamin S ample X1 Name: B 6
196.0213.6
B 3'
0.80
0.60
262.1
0.20
0.00
0.00
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: MultiVitamin S ample X1 Name: B 3'
0.06
B 3'
213.6
0.40
262.1
0.50
6
194.8
AU
AU
1.00
194.8
B1
0.050
0.040
S ampleName: MultiVitamin S ample Y1 Name: B 3'
196.0
P age: 1 of 1
Channel 2998
Current Date: 3/10/2015
Figure 8. Single chromatogram of Multivitamin Y; wavelength: 267 nm. For optimal sensitivity, only vitamins B1 and B2 were quantitated at 267 nm.
1.50
Channel 2998
Current Date: 3/10/2015
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: MultiVitamin S ample X1 Name: B 3
AU
300.1
0.000
0.14
.108107
Injection Id 7976, 0
8043,
Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3
S ampleName MultiVitamin S ample X1,
MultiVitamin S ample Y1, Vit B W S Low
0.001
398.2
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
0.15
S ampleName: MultiVitamin S ample Y1 Name: B 2
211.2
0.003
0.20
AU
0.001
200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm
S ampleName: MultiVitamin S ample X1 Name: B 2
B1
9.00
10.00
P roject Name: Water S oluble Vit.B
B3′
0.70
0.60
B9
0.070
unknown
excipients
0.50
P roject Na me: Wa te r S oluble V it.B
0.060
0.40
0.050
8a)
0.040
AU
B12
AU
0.030
0.020
0.010
0.30
0.000
-0.010
-0.020
5.60
5.80
6.00
6.20
Minutes
6.40
6.60
6.80
7.00
0.20
unknown
excipients
B6
0.10
R e port Me thod Na me : Overla y sa mple for App Notes
S a mple S et Id 7974
Injection Id 8011, 8151
S a mpleNa me MultiV ita min S a mple Y 2, V it B
WS High
S a mple S et Na me MultiV itAssa y_ F eb23_ S S
P age: 1 of 1
Current Da te: 3/
12/
2015
Cha nnel 2998 Ch1 214nm@ 1.2nm
0.00
0.00
1.00
3.00
2.00
4.00
5.00
Minutes
6.00
7.00
8.00
9.00
10.00
Figure 9. Chromatogram of Multivitamin Y; wavelength: 214 nm. For optimal sensitivity, only vitamins B6, B3’ and B12 were quantitated at 214 nm Vitamins B3 and B7 were not
detected. The insert (8a) highlights the region in which vitamins B9 and B12 were expected. Multivitamin Y (red), and WS2 (black) were overlaid to show the expected elution times
of vitamins B9 and B12. In this region, only vitamin B12 was positively identified and quantitated.
Table 3. Quantitative results for Mutivitamin X and Y.
Multivitamin X:
Label claim per serving (µg)
(serving size: 1 tablet)
Vitamins
Actual results (µg) (based on avg. of 2 indivdual tablets)
(3 replicate injections per tablet)
B1 – Thiamine HCl
1,500
2,237
B2 - Ribolfavin
1,700
1,680
0
0
B3 – Niacin (Nicotinic Acid)
B3′ – Niacinamide
20,000
B6ample
- Pyridoxine
S
S et Id
2,000
R eport Method Name: Overlay sample for App Notes
7974
B7 - Biotin
Current Date: 3/12/2015
Channel 2998 Ch1 214nm@ 1.2nm
B9 – Folic Acid
B12 - Cyanocobalamin
30
400
23,625
Injection Id 8151
2,738
S ampleName MultiVitamin S ample Y 2
ND*
S ample S et Name MultiVitAssay_ F eb23_ S S
ND*
P age: 1 of 1
6
7
Amount per serving (µg)
(serving size: 1 tablet)
Actual results (µg) (based on avg. of 2 indivdual tablets)
(3 replicate injections per tablet)
B1 – Thiamine HCl
1,500
1,218
B2 - Ribolfavin
1,700
518
0
0
B3′ – Niacinamide
10,000
10,711
B6 - Pyridoxine
2,000
2,341
B7 - Biotin
30
ND*
B9 – Folic Acid
400
ND*
6
11
Multivitamin Y:
Vitamins
B3 – Niacin (Nicotinic Acid)
B12 - Cyanocobalamin
* Not detected: as vitamins B7 and B9 were not spectrally confirmed, identification was not established and, therefore, amounts could not be calculated.
Table 3 shows the quantitative results for each multivitamin
sample as compared to the label claim. This was based on the
average of two analyzed tablets per sample and three replicates
per injection. The daily recommended dose for each of these
products is one tablet.
For both tablets, the calculated amounts for vitamins B3′and B6
were all close to label claim. Vitamin B3 was shown to be absent
in both tablets, also as per label claim. For vitamins B1, though
the amount found in Multivitamin Y was closer to label claim,
the amount found in Multivitamin X was about 1.5-fold higher
than label claim. For vitamin B2, the amount found in Multivitamin
X was close to label claim; however, the amount found in
Multivitamin Y was about 3-fold lower than label claim. For
vitamins B12, though the amount found in Multivitamin X was
close to label claim, the amount found in Multivitamin Y was
about 2-fold higher than label claim.
7
The descrepencies from the label claim noted above were certainly
of interest but could not be explained. As the sample preparation
of these two tablet samples involved merely solubilizing in water
followed by filtering through a 0.45 µm filter, there is of course
the distinct possibility that some of the vitamin B content in these
tablets was indeed other than claimed.
Regarding vitamin B7 (biotin), due to the combination of the low
concentration and low-level excipient interference, the amounts
could not be calculated.
Conclusion
References
1.“Vitamins”, Wikipedia , The Free Encyclopedia,
www.wikipedia.org
2.U.S. Pharmacopeial Convention – USP 38, NF 33 Edition, 2015,
USP Dietary Supplements Compendium (DSC), 2012
www.usp.org
3.U.S. Food and Drug Administration (FDA), U.S. Department
of Health and Human Services, www.fda.gov
4.European Food Safety Authority (EFSA) – European Food
Safety Authority, www.efsa.europa.eu
The results obtained confirm the applicability of this method
for the effective and robust analysis of B vitamins in dietary
supplement tablets. All eight vitamins are well separated in
under eight minutes.
Except for vitamins B7 and B9, the amount of each vitamin B
found in two analyzed multivitamin tablets was easily quantitated
and any excipients present did not interfere with the peaks of
interest. Other than the noted exceptions, the determined
amounts were close to the label claim.
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