Analysis of Bvitamins

APPLICATION NOTE

Liquid Chromatography

Authors: Andrew P. Zdzieblo Wilhad M. Reuter PerkinElmer, Inc. USA

The Qualitative and Quantitative Analysis of Water-Soluble B Introduction Vitamins are essential Vitamins by HPLC-PDA in nutritional elements in a human diet. Various Multivitamin Tablets 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 Vitamin B standards were obtained from Sigma-Aldrich Inc® the USP Dietary Supplements Compendium (DSC) – an industry- (St Louis, MO). These included: thiamine HCl (B1), riboflavin (B2), directed resource, including regulatory guidance and reference niacin (nicotinic acid; B3), niacinamide (B3; labeled “B3′” to tools. In the U.S., the Food and Drug Administration (FDA) distinguish it from niacin), pyridoxine (B6), biotin (B7), folic acid regulates all dietary supplement products and manufacturing (B9) and cyanocobalamin (B12). The structures of these vitamins practices under 21 CFR (Code of Federal Regulation), Part 111.2,3 are shown in Figure 1. 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 Thiamine – B1 Riboflavin – B2 and repeatability, are presented.

Experimental Hardware/Software For chromatographic separations, a PerkinElmer Altus™ HPLC System was used, including the Altus A-10 solvent/sample module, Niacin – B3 Niacinamide – B3′ 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. Method Parameters The HPLC method parameters are shown in Table 1. Table 1. HPLC Method Parameters. HPLC Conditions Pyridoxine – B6 Biotin – B7 PerkinElmer Brownlee™ SPP C18 2.7 µm, Column: 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:

Time Flow %A %B %C %D Curve Mobile Phase: 1 0.60 100.00 0.0 0.0 0.0 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 Folic Acid – B9 Cyanocobalamin – B12 Flow Rate: 0.6 mL/min. (max. pressure: ~2300 psi/~153 bar) Figure 1. Chemical structures of B vitamins. Oven Temp.: 40 ºC Altus A-10 PDA; As the concentration of individual B vitamins in dietary supplements Wavelengths used for calibration/quantitation: Detection: 214 nm: for B3, B3', B6, B7, B9, and B12 varies widely from one vitamin to another, two sets of standards 267 nm: for vitamins B1 and B2 were prepared. The first, working standard 1 (WS1), was used for Injection Volume: 3 µL demonstrating chromatographic separation and retention time Sampling (Data) Rate: 10 pts./sec (RT) reproducibility. It was prepared by weighing out 80 - 320 µg/ ml of each of the eight vitamins, depending on the vitamin’s Solvents, Standards and Samples relative absorptivity. The second, working standard 2 (WS2), All solvents and diluents used were HPLC-grade and filtered via was used for calibration/linearity. It was prepared by weighing 0.45-µm filters. individually selected amounts for each vitamin, depending on its expected concentration range in typical dietary supplements.

2 For both WS1 and WS2, the eight vitamins were weighed out The multivitamin tablet samples, Multivitamin X and Multivitamin Y, and transferred into individual 50-mL volumetric flasks, to which were purchased from a local store. For each sample, a multivitamin 30 mL of water was added. Additionally, for vitamins B9, B2 and tablet was transferred into an individual 50-mL volumetric flask and B7, to improve solubility, 0.5 mL of 0.1N NaOH was added to the 30 mL of water was then added. The flask was then stoppered and corresponding flasks. All flasks were sonicated for five minutes placed on a heater/stirrer and heated at 40 °C while stirring for and then filled to the mark with water. Equal volumes of the eight 15 min. Once the tablet was completely dissolved, each solution respective stock standards were then added together to produce was sonicated for 10 min and then filled to the mark with HPLC- each of the two WS solutions. For WS1, the resulting concentration grade water. To remove any small particles, all standards and for the eight vitamins was between 10 - 40 µg/ml. For WS2, samples were filtered through 0.45 µm filters prior to injection. the vitamins ranged in concentration from 0.9 to 525 µg/mL, depending on the vitamin. For calibration, all subsequent Results and Discussion concentration levels were prepared from WS2. Using the optimized chromatographic conditions described above, All individual stock solutions and working standards were stored Figure 2 shows the HPLC separation of the eight B vitamins in WS1, at 4.0 °C when not in use. 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.

Project Name: Water Soluble Vit.B

0.20 B2 a) 214 nm 0.18 B3

0.16 B3 ′

0.14

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Current Date: 4/28/2015 S ample S et Id 5377 S ample S et Name VitaminB_S S uit_Feb18_S S Figure 2. Chromatogram of the vitamin B working standard 1 (WS1); wavelength: a) 214 nm and b) 267 nm.

S ampleName All Vitamin B's ~40ug/ml Feb13 Channel 2998 Ch2 [email protected]

Current Date: 5/1/2015 S ample S et Id 5377 S ample S et Name VitaminB_S S uit_Feb18_S S 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). Project Name: Water Soluble Vit.B

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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 Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Level 7 B1 2.64 8.79 17.58 26.38 35.17 43.96 52.75

B2Channel 2998 Ch2 [email protected] 8.88 17.75 26.63 35.50 44.38 53.25 Name B 12, B 2, B 3', B 7, B 9, B 1, B 6, B 3 B3 2.65 Table 1. HPLC8.83 Method Parameters.17.67 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 0.54 1.79 3.58 5.38 7.17 8.96 10.75 B9 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

4 Project Name: Water Soluble Vit.B

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0. 00 5. 00 10. 00 15. 00 20. 00 25. 00 30. 00 35. 00 40. 00 45. 00 50. 00 Amount 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

Channel 2998 Ch1 [email protected]

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0.00 10.00 20.00 30.00 40.00 50.00 60.00 Amount Name: B6; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11585; A: -1.383973e+004; B: 1.216932e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998618

Channel 2998 Ch1 214nm@ 1.2nm

Project Name: Water Soluble Vit.B 1.0x10 7

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-2.0x10 6 0 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 Amount -100000 Name: B3'; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11586; A: -6.838097e+004; B: 1.707714e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999349

Channel 2998 Ch1 [email protected] Channel 2998 Ch1 214nm@ 1.2nm

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0.00 10.00 20.00 30.00 40.00 50.00 60.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Amount Amount Name: B6; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11585;Name: A: B1; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11587; A: -1.383973e+004; B: 1.216932e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998618 -3.589116e+003; B: 2.890673e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999492

Channel 2998 Ch1 214nm@ 1.2nm Channel 2998 Ch1 214nm@ 1.2nm

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Project Name: Water Soluble Vit.B

800000

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0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 Amount Amount Name: B1; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11587;Name: A: B7; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11589; A: -3.589116e+003; B: 2.890673e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999492 -1.638886e+002; B: 9.109663e+002; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998639

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0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Amount Amount Name: B9; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11588;Name: A: B12; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11590; A: -1.051709e+004; B: 1.239506e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.996478 6.662744e+003; B: 9.783058e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.995405

Project Name: Water Soluble Vit.B

7 800000 1.0x10 B3 B3′ 700000 8.0x10 6 600000

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-100000 R² = 0.999216 R² = 0.999349 -2.0x10 6 0. 00 5. 00 10. 00 15. 00 20. 00 25. 00 30. 00 35. 00 40. 00 45. 00 50. 00 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 Amount Amount Name: B3; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11584; A: Name: B3'; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11586; A: -6.208800e+003; B: 1.362139e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999216 -6.838097e+004; B: 1.707714e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999349

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0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Amount Amount Name: B7; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11589;Name: A: B2; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11591; A: -1.638886e+002; B: 9.109663e+002; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998639 -6.887704e+003; B: 1.101062e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999052 900000

Figure800000 4. Calibration plots of 7-level calibration set for the vitamins B3, B3′, B7 and B2.

700000 180000.0 Using600000 the same chromatographic conditions, the two 160000.0For both samples, the large initial peak eluting very near the void 500000 140000.0 multivitamin400000 tablets, Multivitamin X and Multivitamin Y, were 120000.0volume was expected to be primarily vitamin C (ascorbic acid). Ar ea 300000 100000.0 then200000 analyzed. The chromatographic results are shown in Figures 80000.0As vitamin C was not the focus of this application, this peak was Ar ea 5 100000and 7-9, displayed at 267 and 214 nm for each of the two 60000.0not further characterized. 0 40000.0 samples.-100000 Comparing the chromatograms of both of these 20000.0 Per Figure 5, for Multivitamin X, although a peak eluted at the 0.0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Channelsamples 2998 Ch1with 214nm@ the 1.2nm WS2 standard mix,Amount it appears that both Channel 2998 Ch1 214nm@ 1.2nm -20000.0 Name: B6; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curveexpected Id: 11585; A:retention time for vitamin B9 (folic acid), the UV samples have-1.383973e+004; a similar B: 1.216932e+004; vitamin B C: profile. 0.000000e+000; Their D: 0.000000e+000;presence was R^2: 0.998618 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 spectra for that peak did not matchAmount the expected spectra Name: B1; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11587; A: further confimed by UV spectral matching, except for vitamins -3.589116e+003; B: 2.890673e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999492 for vitamin B9 (Figure 6). As there was no positive spectral B7 and B9, due to their very low concentration in the samples. confirmation of vitamin B9 in either sample, the amount of

16000.0 vitamin B9 could not be calculated.

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Project Name: Water Soluble Vit.B B2 0.30 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Amount B1 Name: B12; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11590; A: 6.662744e+003; B: 9.783058e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.995405 0.25 1.0x10 7 AU

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0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00Report Method Name: Overlay sample for450.00 App Notes 500.00 Channel 2998 Ch1 214nm@ 1.2nm Amount Injection Id 8011, 8075 S ample S et Id 7974 Current Date: 3/17/2015 S ampleName MultiVitamin S ample X2, Vit B Name: B3'; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: LinearWS High (1st Order); Cal Curve Id: 11586; A: 0.00 1.00 2.00 3.00 Channel 2998 Ch2 [email protected] Sample Set Name MultiVitAssay_Feb23_SS4.00 5.00-50000 6.00 7.00 8.00 9.00 10.00 -6.838097e+004; B: 1.707714e+004; C: 0.000000e+000; D: 0.000000e+000; R^2:P age: 1 of 1 0.999349 M inutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 Amount Name: B9; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11588; A: Figure 5. Overlaid chromatograms of Multivitamin X (red) with WS2 (black); wavelength: 267 nm. For-1.051709e+004; optimal sensitivity, B: 1.239506e+004; only vitamins C: 0.000000e+000; B1 and B2 were D: 0.000000e+000; quantitated at R^2: 267 0.996478 nm.

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0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Amount Name: B2; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11591; A: Report-6.887704e+003; Method Name: B: 1.101062e+004; Overlay C: 0.000000e+000; sample D: 0.000000e+000;for App Notes R^2: 0.999052 180000.0 Injection Id 8011, 8075 S ample S et Id 7974 160000.0 Current Date: 3/11/2015 S ampleName MultiVitamin S ample X2, Vit B

140000.0 WS High Channel 2998 Ch2 [email protected] Sample Set Name MultiVitAssay_Feb23_SS 120000.0 10000.0 P age: 1 of 1

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0.0 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Channel 2998 Ch1 214nm@ 1.2nm Amount Name: B1; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11587; A: -3.589116e+003; B: 2.890673e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999492 -2000.0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 Amount Name: B7; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11589; A: -1.638886e+002; B: 9.109663e+002; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998639

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0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 Amount 6000.0 Name: B9; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11588; A: -1.051709e+004; B: 1.239506e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.996478 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Amount Name: B12; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11590; A: 6.662744e+003; B: 9.783058e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.995405

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Name: B7; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal -100000Curve Id: 11589; A: -1.638886e+002; B: 9.109663e+002; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.998639 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Amount Name: B2; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11591; A: -6.887704e+003; B: 1.101062e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999052

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0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 Amount Name: B12; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11590; A: 6.662744e+003; B: 9.783058e+003; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.995405

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0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Amount Name: B2; Processing Method: WS VitB Assay_Feb24_PM; Fit Type: Linear (1st Order); Cal Curve Id: 11591; A: -6.887704e+003; B: 1.101062e+004; C: 0.000000e+000; D: 0.000000e+000; R^2: 0.999052

Channel 2998 Ch1 214nm@ 1.2nm Project Name: Water Soluble Vit.B

0.030 193.6 B3

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0.05 262.1

0.00

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 WS Low Name: B 3'

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 PP age: age: 1 2583467 of of 8 8 S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y 1, Vit B WS L ow Current Date: 3/10/2015

Project Name: Water Soluble Vit.B

0.004 194.8 B1

0.003

0.002 AU 234.8 263.3

0.001

0.000 333.6 355.1 395.8

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm

SampleName: Vit B WS Low Name: B1

197.1 B9 0.015 282.3

0.010 AU

0.005 346.7

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm

SampleName: Vit B WS Low Name: B9 0.004 196.0 B7

0.003

0.002 AU

0.001

0.000 268.0287.0302.5316.8 352.7 381.3

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm

SampleName: Vit B WS Low Name: B7

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015

Project Name: Water Soluble Vit.B

0.005 207.7 B 12

0.004

0.003 AU

0.002 360.4

0.001 277.5

0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm

SampleName: Vit B WS Low Name: B12

0.030 223.0 266.8 B2

0.020 AU

0.010 364.4

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B SampleName: Vit B WS Low Name: B2

212.4 0.002 B3 0.030 193.6 B3

0.001

0.020 AU AU 298.9 0.000 398.2 0.010 262.1 255.0

0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 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

S ampleName: Vit B WS Low Name: B 3

193.6 B6 0.015 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 S ampleName MultiVitamin S ample X1, 0.010 Current Date: 3/10/2015

AU MultiVitamin S ample Y1, Vit B W S Low

291.8 0.005 324.0 256.1

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B

S ampleName: Vit B WS Low Name: B 6 0.20 194.8 B6 193.6 B 3' 0.20 0.15

0.15 213.6

0.10AU

0.10AU 290.6 0.05 324.0 256.1 0.05 262.1 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 6 S ampleName: Vit B WS Low Name: B 3' 1.50 196.0213.6 B 3'

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 1.00

PP age: age: 1 2345678 of of 8 8 S ampleName MultiVitamin S ample X1, AU MultiVitamin S ample Y 1, Vit B WS L ow Current Date: 3/10/2015 0.50 262.1

0.00

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 3' 0.004 194.8 B1 0.06 194.8 B1

0.003

0.04 0.002 AU AU 234.8 234.8 263.3 264.4 0.02 0.001

0.000 333.6 355.1 395.8 0.00

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm

SampleName: Vit B WS Low Name: B1 S ampleName: MultiVitamin S ample X1 Name: B 1

197.1 B9 0.015 282.3

Name B12, B2, B3', B7, B9, B1, B6, B3 0.010 Injection Id 7976, 8043, 8107 Channel 2998 AU S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.005 346.7

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm

SampleName: Vit B WS Low Name: B9 Project Name: Water Soluble Vit.B 0.004 196.0 B7 194.8 B9

0.003 0.030

0.002 AU 0.020 AU

0.001 0.010 266.8

0.000 268.0287.0302.5316.8 352.7 381.3 0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm SampleName: Vit B WS Low Name: B7 S ampleName: MultiVitamin S ample X1 Name: B 9

0.006

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.004

S ampleName MultiVitamin S ample X1, AU MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.002 303.7

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 7

0.005 207.7 B 12 207.7 B 12 0.005

0.004 0.004

0.003 0.003 AU AU

0.002 360.4 0.002 359.4 0.001 277.5 0.001

0.000 0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm

SampleName: Vit B WS Low Name: B12 S ampleName: MultiVitamin S ample X1 Name: B 12

0.030 223.0 266.8 B2

0.020 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 AU S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.010 364.4

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm

SampleName: Vit B WS Low Name: B2 Project Name: Water Soluble Vit.B

212.4 B3 0.002 266.8 0.25 223.0 B2

0.20 0.001

AU 0.15 AU

298.9 0.10 0.000 398.2 364.4 255.0 0.05

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 Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 3 S ampleName: MultiVitamin S ample X1 Name: B 2

0.004 211.2 B3

0.030 193.6 B3 0.003 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.002

0.020 S ampleName MultiVitamin S ample X1, AU MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 AU 0.001

0.010 262.1 300.1 0.000 398.2

0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B 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 3

S ampleName: Vit B WS Low Name: B 3 0.20 194.8 B6 194.8 B6 0.15 193.6 B6 0.15 0.015 0.10 AU 0.10AU 0.010

AU 290.6 0.05 290.6 0.05 324.0 324.0 291.8 256.1 256.1 0.005 324.0 256.1 0.00 0.00

0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm 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 6 S ampleName: MultiVitamin S ample Y1 Name: B 6

S ampleName: Vit B WS Low Name: B 6 1.50 196.0213.6 B 3'

193.6 B 3' 0.20 1.00 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.15 213.6 AU S ampleName MultiVitamin S ample X1, Current Date: 3/10/2015 AU 0.50 262.1 MultiVitamin S ample Y1, Vit B W S Low 0.10

0.05 262.1 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' Project Name: Water Soluble Vit.B

S ampleName: Vit B WS Low Name: B 3' 0.06 194.8 B1 194.8 B 3' 0.80 213.6 0.04 0.60 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 AU

PP age: age: 1 3245678 of of 8 8 S ampleName MultiVitamin S ample X1, 234.8 AU 264.4 0.40 MultiVitamin S ample Y 1, Vit B WS L ow Current Date: 3/10/2015 0.02 262.1 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 Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 1 S ampleName: MultiVitamin S ample Y1 Name: B 3' 0.004 0.050 194.8 B1 196.0 B1 0.040 0.003

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.030 0.002 AU 234.8 S ampleName MultiVitamin S ample X1, AU 263.3 MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.020 0.001 0.010

0.000 333.6 355.1 395.8 0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm Project Name: Water Soluble Vit.B SampleName: Vit B WS Low Name: B1 S ampleName: MultiVitamin S ample Y1 Name: B 1

197.1 B9 194.8 B9 0.0010 192.4 B9 0.015 282.3 a) WS2 0.030 b) Multivitamin X c) Multivitamin Y 0.0008

0.010 0.020 0.0006 279.9 AU AU AU

0.0004 0.005 0.010 266.8 346.7 0.0002 343.2

0.000 0.000 0.0000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm nm

SampleName: Vit B WS Low Name: B9 S ampleName: MultiVitamin S ample X1 Name: B 9 S ampleName: MultiVitamin S ample Y1 Name: B 9 0.004 196.0 B7 B7

0.006 Figure0.003 6. UV spectra captured at the expected elution time for vitamin B9: a) WS2, b) Multivitamin X and c) Multivitamin Y. Project Name: Water Soluble Vit.B Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.002 0.004 AU AU S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.001 0.002 303.7 B3 ′

0.000 268.0287.0302.5316.8 352.7 381.3 1.40 0.000 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm

SampleName: Vit B WS Low Name: B7 S ampleName: MultiVitamin S ample X1 Name: B 7 Project Name: Water Soluble Vit.B

1.20 207.7 B 12 0.005 0.0010 191.3 B7

0.004 0.0005 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.003

AU 312.1

1.00S ampleName MultiVitamin S ample X1, AU 0.0000 386.1 MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.002 359.4 0.001 -0.0005

0.000 0.80 -0.0010 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

AU Project Name: Water Soluble Vit.B

S ampleName: MultiVitamin S ample X1 Name: B 12 excipients unknown unknown unknown unknown S ampleName: MultiVitamin S ample Y1 Name: B 7 excipient Project Name: Water 0.005Soluble Vit.B 0.005 0.60207.7 B 12 0.025 207.7 B 12

0.020 0.004 0.004 B12 0.015

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3

Channel 2998 0.003 0.010 0.003 257.3 AU AU S ampleName MultiVitamin S ample X1, AU 0.40 0.005 Current Date: 3/10/2015 MultiVitamin S ample Y1, Vit B W S Low 0.002 0.002 0.000 360.4 360.4 -0.005 0.001 277.5 0.001

excipients -0.010 unknown unknown

5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 M inutes B2 B6 0.000 0.0000.20 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm Project Name: Water Soluble Vit.B SampleName: Vit B WS Low Name: B12 S ampleName: MultiVitamin S ample Y1 Name: B 12

0.050 0.030 266.8 B2 266.8 265.6 0.00 223.0 0.25 223.0 B2 B2 0.040 223.0 Report Method Name: Overlay sample for App Notes 0.20 Injection Id 8055 S ample S et Id 7974 Current Date: 3/12/2015 S ampleName MultiVitamin S ample X1

0.020 Channel 2998 Ch1 [email protected] Sample Set Name MultiVitAssay_Feb23_SS 0.00 1.00 2.00 3.00 4.00 5.00 6.00 0.0307.00 P8.00 age: 1 of 1 9.00 10.00 0.15 M inutes AU AU AU 0.020 0.10 0.010 364.4 364.4 364.4 Figure 7. Single chromatogram of Multivitamin X; wavelength: 2140.05 nm. For optimal sensitivity, only vitamins B6, B3’ and B120.010 were quantitated at 214 nm. Vitamins B3 and B7 were

not detected..0.000 0.00 0.000

SampleName: Vit B WS Low Name: B2 S ampleName: MultiVitamin S ample X1 Name: B 2 S ampleName: MultiVitamin S ample Y1 Name: B 2

0.22212.4 0.004 0.002 B3 211.2 B3

0.003 0.20 0.001 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.002 AU AU S ampleName MultiVitamin S ample X1, 0.18 MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 298.9 0.001 0.000 398.2 255.0 300.1 0.16 0.000 398.2

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm 0.14 Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 3 S ampleName: MultiVitamin S ample Y1 Name: B 3

194.8 B6 0.12 B1 0.15 B2 0.10AU Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.10 S ampleName MultiVitamin S ample X1, AU Current Date: 3/10/2015 MultiVitamin S ample Y1, Vit B W S Low 290.6 0.08 0.05 324.0 256.1 Report Method Name: Overlay sample for App Notes 0.06 0.00 Injection Id 8055

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 S ample S et Id 7974 nm Current Date: 3/12/2015 S ampleName MultiVitamin S ample X1 0.04 Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample Y1 Name: B 6

Channel0.20 2998 Ch1 [email protected] Sample Set Name MultiVitAssay_Feb23_SS 0.02194.8 B6 P age: 1 of 1

0.15 0.00 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998

0.10AU S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.00 1.00290.6 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 324.0 Report Method Name: Overlay sa mple for App Notes 0.05 Injection Id 8139 M inutes Sample Set Id 7974 256.1 Current Date: 3/17/2015 SampleName MultiVitamin Sample Y2

Channel 2998 Ch2 [email protected] Sample Set Name MultiVitAssa y_Feb23_SS Page: 1 of 1 Figure0.00 8. Single chromatogram of Multivitamin Y; wavelength: 267 nm. For optimal sensitivity, only vitamins B1 and B2 were quantitated at 267 nm. 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 6

1.50 196.0213.6 B 3' 194.8 B 3' 0.80 213.6 1.00 0.60 AU 0.40AU 0.50 262.1 262.1 0.20

0.00 0.00

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm 6 S ampleName: MultiVitamin S ample X1 Name: B 3' S ampleName: MultiVitamin S ample Y1 Name: B 3' 0.050 0.06 194.8 B1 196.0 B1 0.040

0.04 0.030 AU AU 234.8 0.020 264.4 0.02 Report Method Name: Overlay sample for0.010 App Notes Injection Id 8139 S0.00 ample S et Id 7974 0.000 Current Date: 3/12/2015 S ampleName MultiVitamin S ample Y2 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm Channel 2998 Ch2 [email protected] Sample Set Name MultiVitAssay_Feb23_SS S ampleName: MultiVitamin S ample X1 Name: B 1 S ampleName: MultiVitamin S ample Y1 Name: B 1 P age: 1 of 1

0.0010 192.4 B9

0.0008 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.0006 279.9 S ampleName MultiVitamin S ample X1, AU MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.0004

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 Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample Y1 Name: B 9

194.8 B9

0.030

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.020

AU S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.010 266.8

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample X1 Name: B 9

B7 0.0010 191.3 B7 0.006 0.0005

0.004 312.1 AU AU 0.0000 386.1

0.002 303.7 -0.0005

0.000 -0.0010 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm

S ampleName: MultiVitamin S ample X1 Name: B 7 S ampleName: MultiVitamin S ample Y1 Name: B 7

0.005 207.7 B 12 207.7 B 12 0.005 0.004 0.004

0.003 0.003 257.3 AU AU 0.002 0.002 359.4 360.4 0.001 0.001

0.000 0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm nm

S ampleName: MultiVitamin S ample X1 Name: B 12 S ampleName: MultiVitamin S ample Y1 Name: B 12

0.050 265.6 B2

0.040 223.0

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.030 S ampleName MultiVitamin S ample X1, AU 0.020 MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 364.4 0.010

0.000

200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm Project Name: Water Soluble Vit.B S ampleName: MultiVitamin S ample Y1 Name: B 2

266.8 0.25 223.0 B2

0.20 Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 0.15

AU S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 0.10 364.4 0.05

0.00

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

0.004 211.2 B3

0.003

0.002 AU

0.001

300.1 0.000 398.2

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 3

194.8 B6 0.15

0.10 AU

290.6 0.05 324.0 256.1

0.00

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 6

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015

Project Name: Water Soluble Vit.B

194.8 B 3' 0.80 213.6 0.60

0.40AU

262.1 0.20

0.00

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 3' 0.050 196.0 B1 0.040

0.030 AU 0.020

0.010

0.000

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 1

0.0010 192.4 B9

0.0008

0.0006 279.9 AU

0.0004

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

S ampleName: MultiVitamin S ample Y1 Name: B 9

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015

Project Name: Water Soluble Vit.B

0.0010 191.3 B7

0.0005

312.1

AU 0.0000 386.1

-0.0005

-0.0010 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 7

0.005 207.7 B 12

0.004

0.003 257.3 AU 0.002 360.4 0.001

0.000

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 12

0.050 265.6 B2

0.040 223.0

0.030 AU 0.020

364.4 0.010

0.000

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 2

Injection Id 7976, 8043, 8107 Name B12, B2, B3', B7, B9, B1, B6, B3 Channel 2998 S ampleName MultiVitamin S ample X1, MultiVitamin S ample Y1, Vit B W S Low Current Date: 3/10/2015 Project Name: Water Soluble Vit.B B3 ′ 0.70

0.60 excipients unknown unknown 0.50 B9

Project Name: Water Soluble Vit.B

0.070

0.060

0.050 8a)

0.40 0.040

0.030 B12 AU

AU 0.020

0.010 0.30 0.000 -0.010

-0.020 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 M inutes

0.20 B6 excipients unknown unknown

0.10 Report Method Name: Overlay sample for App Notes Injection Id 8011, 8151 S ample S et Id 7974 Current Date: 3/12/2015 S ampleName MultiVitamin S ample Y2, Vit B WS High Channel 2998 Ch1 [email protected] Sample Set Name MultiVitAssay_Feb23_SS P age: 1 of 1

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 M inutes

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) Actual results (µg) (based on avg. of 2 indivdual tablets) Vitamins (serving size: 1 tablet) (3 replicate injections per tablet) B1 – Thiamine HCl 1,500 2,237 B2 - Ribolfavin 1,700 1,680 B3 – Niacin (Nicotinic Acid) 0 0 B3′ – Niacinamide 20,000 23,625 Report Method Name: Overlay sample for App Notes B6 - Pyridoxine 2,000 Injection2,738 Id 8151 S ample S et Id 7974 Current Date: 3/12/2015 S ampleName MultiVitamin S ample Y2 B7 - Biotin 30 ND* Channel 2998 Ch1 [email protected] Sample Set Name MultiVitAssay_Feb23_SS B9 – Folic Acid 400 ND* P age: 1 of 1 B12 - Cyanocobalamin 6 7

Multivitamin Y: Amount per serving (µg) Actual results (µg) (based on avg. of 2 indivdual tablets) Vitamins (serving size: 1 tablet) (3 replicate injections per tablet) B1 – Thiamine HCl 1,500 1,218 B2 - Ribolfavin 1,700 518 B3 – Niacin (Nicotinic Acid) 0 0 B3′ – Niacinamide 10,000 10,711 B6 - Pyridoxine 2,000 2,341 B7 - Biotin 30 ND* B9 – Folic Acid 400 ND* B12 - Cyanocobalamin 6 11

* 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 the amount found in Multivitamin Y was closer to label claim, sample as compared to the label claim. This was based on the the amount found in Multivitamin X was about 1.5-fold higher average of two analyzed tablets per sample and three replicates than label claim. For vitamin B2, the amount found in Multivitamin per injection. The daily recommended dose for each of these X was close to label claim; however, the amount found in products is one tablet. Multivitamin Y was about 3-fold lower than label claim. For vitamins B12, though the amount found in Multivitamin X was For both tablets, the calculated amounts for vitamins B3′and B6 were all close to label claim. Vitamin B3 was shown to be absent close to label claim, the amount found in Multivitamin Y was in both tablets, also as per label claim. For vitamins B1, though about 2-fold higher than label claim.

7 The descrepencies from the label claim noted above were certainly References of interest but could not be explained. As the sample preparation 1. “Vitamins”, Wikipedia , The Free Encyclopedia, of these two tablet samples involved merely solubilizing in water www.wikipedia.org 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 2. U.S. Pharmacopeial Convention – USP 38, NF 33 Edition, 2015, tablets was indeed other than claimed. USP Dietary Supplements Compendium (DSC), 2012 www.usp.org Regarding vitamin B7 (biotin), due to the combination of the low concentration and low-level excipient interference, the amounts 3. U.S. Food and Drug Administration (FDA), U.S. Department could not be calculated. of Health and Human Services, www.fda.gov 4. European Food Safety Authority (EFSA) – European Food Conclusion 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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