B Vitamins Quantified Using HPLC

Scientists from the University of Isfahan in Iran developed a new system for detecting and quantifying B vitamins in human samples. Their findings were published in the Journal of Chromatography A (1).

Vitamin B. Capsules B1 B2 B6 B12 on white isolated background. | Image Credit: © Maksym Yemelyanov - stock.adobe.com

In this study, a novel azo-linked porous organic polymer (AL-POP) was synthesized from caffeic acid and benzidine via an azo-coupling reaction and characterized by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), Brunauer-Emmet-Teller (BET), thermogravimetric analysis (TGA), X-ray diffraction analysis (XRD), and zeta potential analysis. AL-POPs were then incorporated into melamine sponges and used for pipette tip micro solid-phase extraction (PT-MSPE) of six types of B vitamins: thiamine, riboflavin, nicotinamide, pyridoxine, folic acid, and cyanocobalamin. After extraction, the samples were analyzed with a high-performance sliquid chromatography (HPLC)-diode array detection (DAD) system.

Vitamins are organic micronutrients that aid in carrying out various natural activities in the human organism. A proper diet can supply the vitamins the body needs, but occasionally, nutritional supplements are also recommended. Vitamins can be classified into two categories: water-soluble vitamins (WSVs) and fat-soluble vitamins (FSVs); vitamin C and B vitamins are considered water-soluble vitamins. B vitamins, consisting of eight essential nutrients, are vital for ensuring the body’s cells are properly functioning, helping the body convert food into energy, create new blood cells, and maintain the health of various body tissues (2).

Low intake of B vitamins can cause anemia, weaken the immune system, and suppress the nervous and metabolic systems; however, excess intake can cause skin changes, intestinal ulcers, indigestion, and more health injuries. Precisely quantifying vitamins in foods, nutritional supplements, and pharmaceuticals is important. HPLC is the most preferred method for detecting vitamins due to its high selectivity, sensitivity, and ease of use; additionally, it can be coupled with various detectors, including ultraviolet (UV), electrochemical, fluorescence, and mass spectrometry (MS).

In this study, the effect of AL-POP composition on the extraction efficiency (EE) of vitamins was investigated, with benzidine to caffeic acid mol ratio of 1.5, 3.35 mmol of NaNO2, and reaction time of 8 h was selected as optimum conditions. From there, the efficiency of the extraction process was improved by optimizing various parameters, such as the amount of sorbent, pH and ionic strength of the sample, sample volume, number of sorption and desorption cycles, type of wash solvent, and type and volume of eluent solvent. Using calibration curves plotted under optimum conditions, linearity (R² ≥ 0.9987), Limit of detection (LOD) (11.88–18.97 ng/mL), limit of quantification (LOQ) (39.62–63.23 ng/mL), and enrichment factor (EF) (1.27–4.31) values were obtained. Recovery values of these six B vitamins in spiked multivitamin syrup samples varied from 80.01% to 108.35%, with a relative standard deviation (RSD) below 5.44%. From there, the optimized method was used to successfully extract and quantify B vitamins in multivitamin syrup and non-alcoholic beer.

According to the authors, this study is the first to show the successful synthesis of AL-POP from caffeic acid in aqueous solution using an azo-coupling reaction (1). A synergic effect was observed by incorporating the AL-POP as an adsorbent into the cavities of the melamine sponge. The pipette tip micro solid phase extraction (PT-MSPE) was used as a compact, fast, flexible, and feasible method that requires only a small sample volume, a small amount of adsorbent, and solvent to achieve high-efficiency extraction. Six B vitamins were efficiently extracted using this method, with the evaluated statistical parameters all confirming the capability of this method in simultaneous extraction and determination of B vitamins with appropriate precision and accuracy. Altogether, B vitamin content from multivitamin syrups and non-alcoholic beer were effectively extracted and measured using AL-POP-IMS as an adsorbent in PT-MSPE.

References

(1) Khoshoei-Darki, F.; Momenbeik, F. Melamine Sponges Incorporated Azo-Linked Porous Organic Polymer as Adsorbent for Extraction and Determination of Six B Vitamins Using Pipette Tip Micro Solid-Phase Extraction. J. Chromatogr. A 2024, 1727, 464978. DOI: 10.1016/j.chroma.2024.464978

(2) Richter, A. A Complete Guide to B Vitamins. https://www.medicalnewstoday.com/articles/325292#overview (accessed 2024-6-17)