Determination of Vitamins B and Preservatives by HPLC-UV

Dr. Ajay Kaparwan
R&D, APAC, Jigani, India

Introduction

A multivitamin is a preparation intended to serve as a dietary supplement with nutritional elements like vitamins and minerals. Such preparations are available in the form of tablets, capsules, pastilles, powders, liquids or injectable formulations. Other than injectable formulations which are typically administered under medical supervision, multivitamins are recognized by the Codex Alimentarius Commission (the United Nations' authority on food standards) as a category of food.¹

This study details the separation and quantification of water-soluble B Vitamins, methyl and propyl parabens as preservatives in a capsule formulation by HPLC using the Purospher^® STAR, RP-8 endcapped, 250 x 4.6 mm, 5 μm column.

Section Overview

• Experimental
• Results
• Conclusion
• References

Figure 1. B vitamins and its preservatives.

Components in capsule sample (indicated by the manufacturer)
Nicotinamide:	30 mg
Pyridoxine Hydrochloride:	6 mg
Riboflavin:	6 mg
Thiamine Mononitrate:	6 mg
Propyl Paraben:	0.2 mg
Methyl Paraben:	0.4 mg
Mass per capsule:	510 mg
Mass of content per capsule:	254 mg

Experimental

Standard & Sample Preparation

Diluent: Water:glacial acetic acid:acetonitrile (89:1:10 v/v)

Standard Solution for System Suitability

Weigh and transfer 6 mg of thiamine, riboflavin and pyridoxine, 15 mg of nicotinamide, 2 mg of methyl paraben and propyl paraben  each into separate 10 mL volumetric flask. Add 5 mL of glacial acetic acid to each flask and sonicate for 10 minutes. Make up to volume with diluent. Further transfer 1 mL of each solution of thiamine, riboflavin and pyridoxine, 2 mL of nicotinamide, 0.2 mL of methyl paraben and 0.1 mL of propyl paraben into a 20 mL volumetric flask. Add ~10 mL of diluent and sonicate for 10 mins. Make up to volume with diluent. Concentrations in final solution are displayed in Table 1.

Table 1.System Suitability and Repeatability Standard concentrations

Standard	Concentration
Nicotinamide	150 µg/mL
Pyridoxine	30 µg/mL
Thiamine	30 µg/mL
Riboflavin	30 µg/mL
Methyl Paraben	2 µg/mL
Propyl Paraben	1 µg/mL

Stock Solution Preparation for Accuracy Spiking

Weigh and transfer the reference standard (Table 1) to a 10 mL volumetric flask. Add about 5 mL of glacial acetic acid, sonicate for 5 minutes, and make up to volume with diluent.

Table 2.Concentrations single components stock solutions

Compound	Weight (mg)	Final Stock Solution (µg/mL)
Pyridoxine Hydrochloride	6	600
Riboflavin	6	600
Thiamine Mononitrate	6	600
Nicotinamide	10	1000
Methyl Paraben	10	1000
Propyl Paraben	10	1000

Test Sample Preparation

Open 10 capsules, pool the contents and mix well. Weigh an equivalent of five capsules into a 200 mL volumetric flask. Add 100 mL of glacial acetic acid and sonicate for about 5 mins. Make up to volume with the diluent and sonicate for another 10 minutes. Filter the solution with 0.45 μm PVDF syringe filter. Dilute 5 mL of this filtered solution to 25 mL with diluent.

Sample Preparation for Accuracy Spiking

Sample Stock (0% Spiking)

Open 10 capsules, pool the contents and mix well. Weigh an equivalent of five capsules into a 250 mL volumetric flask. Add 125 mL of glacial acetic acid and sonicate for about 5 mins. Make up to volume with the diluent and sonicate for another 10 minutes. Filter the solution with 0.45 μm PVDF syringe filter (Sample Stock for Accuracy). Dilute 2 mL of this filtered solution to 25 mL with diluent.

Sample Spiking

• Sample preparation for accuracy 50%: Transfer 2 mL from the sample stock for accuracy to a 25 mL volumetric flask and add about 625 µL of each pyridoxine, thiamine, and riboflavin stock solutions respectively. To it, then add 1875 µL nicotinamide, 250 µL methyl paraben, and 125 µL of propyl paraben and makeup to the volume with diluent and shake thoroughly.
• Sample preparation for accuracy 100%: Transfer 2 mL from the sample stock for accuracy, to 25 mL volumetric flask and add about 1250 µL of each pyridoxine, thiamine, and riboflavin stock solutions respectively. To it, then add 3750 µL nicotinamide, 500 µL methyl paraben, and 250 µL of propyl paraben and makeup to the volume with diluent and shake thoroughly.
  
• Sample preparation for accuracy 150%: Transfer 2 mL from the sample stock for accuracy to a 25 mL volumetric flask and add about 1875 µL of each pyridoxine, thiamine, and riboflavin stock solutions respectively. To it, then add 5625 µL nicotinamide, 750 µL methyl paraben, and 375 µL of propyl paraben and makeup to the volume with diluent and shake thoroughly.
  

Analysis by HPLC-UV

Standards and samples were analyzed using conditions described in Table 3.

Table 3.Chromatographic conditions for Vitamin B determination

Experimental Conditions
Column:	Purospher™ STAR RP-8 endcapped, 5 µm, 250 x 4.6 mm I.D. (1.51454)
Mobile phase:	[A] 0.01 M potassium dihydrogen phosphate and add 0.005 M sodium hexane sulphonate, adjust pH to 2.5 with 85% orthophosphoric acid; [B] acetonitrile
Gradient:	Time	A (%)	B (%)
	0	98	2
	4	85	15
	12	85	15
	19	40	60
	23	40	60
	25	98	2
	27	98	2
Flow rate:	1 mL/min
Back pressure:	187 bar (2700 psi)
Column temp.:	30°C
Detector:	UV @ 254 and 210 nm (for pyridoxine)
Injection:	5 µL
Sample Temperature:	15°C
Samples:	Test solution, Standard for system suitability, spiked test solutions

Results

System Suitability and Repeatability Summary

Table 4.Overview and summary system suitability criteria and results

Component	USP/EP Specification 2,3,4	Observed Value
% RSD Peak Area	NMT 2%	<2%
% RSD Retention Time	NMT 1%	<1%
Tailing Factor	NMT 2	<2
% Assay of Multivitamins present in capsule sample	Nicotinamide	107.6%
	Pyridoxine	101.8%
	Thiamine	103.0%
	Riboflavin	105.4%
	Methyl Paraben	94.6%
	Propyl Paraben	101.3%

Table 5.System suitability results -peak area and retention time precision

	Peak Areas of Multivitamin Standards (mAU)				Retention Time of Multivitamin Standards (min)
Compound	Mean (n=5)	Standard Deviation	% RSD		Mean (n=5)	Standard Deviation	% RSD
Nicotinamide	1411937	6216.75	0.44		6.691	0.004	0.06
Pyridoxine	584360	2621.24	0.45		7.830	0.004	0.06
Thiamine	364926	2396.42	0.66		8.984	0.003	0.03
Riboflavin	493609	1945.32	0.39		10.272	0.005	0.05
Methyl Paraben	51150	206.15	0.40		19.261	0.004	0.02
Propyl Paraben	21415	185.98	0.87		21.808	0.004	0.02

System Suitability Criteria – Standard Solution

Figure 2. Chromatogram of standards solution for nicotinamide, thiamine, riboflavin, methyl paraben, propyl paraben with detection at 254 nm

Table 6.Chromatographic Data - Standard Solution measured at 254 nm

Peaks	Compound	Conc. (µg/mL)	Retention Time (min)	Tailing Factor	Area
1	Nicotinamide	150	6.613	1.33	1422958
2	Thiamine	30	8.899	1.34	363388
3	Riboflavin	30	10.134	1.10	490871
4	Methyl Paraben	2	19.136	1.24	49389
5	Propyl Paraben	1	21.748	1.19	20758

Figure 3. Chromatogram of standards solution with detection at 210 nm for pyridoxine determination.

Table 7.Chromatographic Data – Standard Solution measured at 210 nm for Pyridoxine determination

Peaks	Compound	Conc. (µg/mL)	Retention Time (min)	Tailing Factor	Area
1	Pyridoxine	30	7.760	1.32	564248

Sample Suitability Criteria – Test Solution

Figure 4. Chromatogram Test Solution (capsules) for 5 of the analytes at 254 nm.

Table 8. Chromatographic data– Test Solution measured at 254 nm

Peaks	Compound	Conc. (µg/mL)	Retention Time (min)	Tailing Factor	Area
1	Nicotinamide	150	6.556	1.33	1535668
2	Thiamine	30	8.897	1.37	376358
3	Riboflavin	30	10.116	1.13	531748
4	Methyl Paraben	2	19.072	1.27	46794
5	Propyl Paraben	1	21.706	1.23	21036

Figure 5. Chromatogram Test Solution (capsules) at 254 nm for pyridoxine of the analytes.

Table 9.Chromatographic data – Test Solution measured at 210 nm

Peaks	Compound	Conc. (µg/mL)	Retention Time (min)	Tailing Factor	Area
1	Pyridoxine	30	7.752	1.34	607898

Table 10.LOD, LOQ and linearity of compounds for injected solutions

Compound	Calibration range (µg/mL)	No. of calibrators	R2	LOD (µg/mL)	LOQ (µg/mL)
Nicotinamide	0.15 - 255.00	12	0.9996	7.04	21.34
Pyridoxine Hydrochloride	0.03-45.00	12	0.9997	1.23	3.74
Riboflavin	0.03-45.00	12	0.9992	2.13	6.47
Thiamine Mononitrate	0.03-45.00	12	0.9999	0.95	2.89
Methyl Paraben	0.002 – 3.000	12	0.9978	0.24	0.74
Propyl Paraben	0.001 – 1.500	12	0.9994	0.06	0.19

% Recovery of Spiked B Vitamins and Preservatives over Multivitamin Capsule Sample

During accuracy study, spiking of vitamins over the sample solution led to saturation of the detector response, so while performing accuracy spiking study, it became necessary to reduce the sample concentration of the base sample (Sample Stock for Accuracy- disolved into 250 mL instead if 200 mL as for the test sample) and then proceed with and spiking of vitamin standards and preservatives at the Accuracy Levels of 50%, 100% and 150% of the actual working level over the reduced base sample solution concentration.

Table 11. Individual concentration of vitamins and preservatives present in diluted Base Samples* prepared for accuracy before spiking (* Sample Stock - disolved in 250 mL, and 2 mL further diluted to 25 mL, see above under Experimental)

Base Sample Components	Concentration in Base Sample µg/mL	Individual Peak Area Obtained in Base Sample
Nicotinamide	48	549757
Pyridoxine Hydrochloride	9.6	220258
Riboflavin	9.6	180173
Thiamine Mononitrate	9.6	123747
Methyl Paraben	0.32	15801
Propyl Paraben	0.64	6727

The results for the recovery determination using the overspiked multivitamin capsule samples are shown in Tables 12 to 17. The recoveries ranged from 96.0 to 110%.

Table 12Result for % recovery of nicotinamide

No	Sample Area Obtained for Nicotinamide	Spike Level	Area Obtained with Spike Levels	Area Difference of Sample with Spike Levels Areas	µg/mL added over Sample	µg/mL Found after Addition	% Recovery
1	549757	50%	1251485	701728	75	73.39	97.9
2		100%	1924504	1374747	150	143.98	96.0
3		150%	2716348	2166591	225	227.03	100.9

Table 13.Result for % recovery of pyridoxine

No	Sample Area Obtained for Pyridoxine	Spike Level	Area Obtained with Spike Levels	Area Difference of Sample with Spike Levels Areas	µg/mL added over Sample	µg/mL Found after Addition	% Recovery
1	220258	50%	493118	272860	15	14.97	99.8
2		100%	743966	523708	30	29.14	97.1
3		150%	1016522	796264	45	44.52	98.9

Table 14.Result for % recovery of thiamine mononitrate

No	Sample Area Obtained for Thiamine	Spike Level	Area Obtained with Spike Levels	Area Difference of Sample with Spike Levels Areas	µg/mL added over Sample	µg/mL Found after Addition	% Recovery
1	123747	50%	299852	176105	15	14.49	96.6
2		100%	472243	348496	30	28.80	96.0
3		150%	669224	545477	45	45.16	100.4

Table 15.Result for % recovery of riboflavin

No	Sample Area Obtained for Riboflavin	Spike Level	Area Obtained with Spike Levels	Area Difference of Sample with Spike Levels Areas	µg/mL added over Sample	µg/mL Found after Addition	% Recovery
1	180173	50%	408360	228187	15	14.64	97.6
2		100%	656307	476134	30	30.85	102.8
3		150%	877382	697209	45	45.31	100.7

Table 16.Result for % recovery of methyl paraben

No	Sample Area Obtained for Methyl Paraben	Spike Level	Area Obtained with Spike Levels	Area Difference of Sample with Spike Levels Areas	µg/mL added over Sample	µg/mL Found after Addition	% Recovery
1	15801	50%	38334	22533	1	0.99	99.0
2		100%	61053	45252	2	1.96	98.0
3		150%	90546	74745	3	3.22	107.3

Table 17.Result for % recovery of propyl paraben

No	Sample Area Obtained for Propyl Paraben	Spike Level	Area Obtained with Spike Levels	Area Difference of Sample with Spike Levels Areas	µg/mL added over Sample	µg/mL Found after Addition	% Recovery
1	6727	50%	15566	8839	0.5	0.48	96.0
2		100%	25099	18372	1.0	1.02	102.0
3		150%	36008	29281	1.5	1.65	110.0

Conclusion

An HPLC method for the quantification of B vitamins and preservatives using the Purospher^® STAR RP-8 endcapped (RP-8e) column was developed and evaluated. Using 2 wave lengths in the UV detection with the described conditions, all components in the capsule sample could be separated with a resolution of >2.

The method for the injected solutions was determined to be linear over a concentration range of (LOQ to highest calibrator) 2.89 to 45 µg/mL for thiamine, 3.74 to 45 µg/mL pyridoxine and 6.47 to 45 µg/mL riboflavin and, 21.34 to 225 µg/mL for nicotinamide. The methyl and propyl parabens preservatives have a linear concentration range of 0.74 µg/mL to 3 µg/mL and 0.19 to 1.5 µg/mL respectively. The % RSD for the system precision of each component was <2%.

The limit of detection (LOD) for the B vitamins ranged from 0.95 to 7.04 µg/mL and for methyl and propyl paraben, it was 0.24 and 0.06 µg/mL respectively. The limit of quantification (LOQ) for the B vitamins ranged from 2.89 to 21.34 µg/mL and for methyl and propyl paraben, it was 0.74 and 0.19 µg/mL respectively. The percentage recovery of the B vitamins and parabens ranged from 96% to 110%.

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References

1. Guidelines for Vitamin and Mineral Food Supplements (PDF). 2005. CAC/GL 55 - 2005. Reference CXG 55-2005. [Internet]. Codex Alimentarius. Available from: https://www.fao.org/fao-who-codexalimentarius/codex-texts/guidelines/en/

2. 2023. <621> Chromatography. [Internet]. USP 47 - NF 42. Available from: https://www.uspnf.com/notices/621-nitr-20230106

3. 2022. European Pharmacopoeia General notices 2.2.46 Chromatographic separation techniques. [Internet]. European Directorate for the Quality of Medicines & HealthCare. Available from: https://www.edqm.eu/en/european-pharmacopoeia-ph.-eur.-11th-edition

4. 2023. ICH Q2(R2) Validation of analytical procedures - Scientific guideline. [Internet]. European Medicines Agency. Available from: https://www.ema.europa.eu/en/ich-q2r2-validation-analytical-procedures-scientific-guideline