Food Anal. Methods DOI 10.1007/s12161-014-9880-0 Simultaneous Determination of Caffeine and Water-Soluble Vitamins in Energy Drinks by HPLC with Photodiode Array and Fluorescence Detection Anna Gliszczyńska-Świgło & Iga Rybicka Received: 11 March 2014 /Accepted: 15 April 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com Abstract The high-performance liquid chromatography Introduction (HPLC) method with photodiode array (PDA) and fluores- cence (FL) detection for the simultaneous separation and Energy drinks are one of the fastest growing soft drink mar- determination of caffeine and water-soluble vitamins is de- kets around the world (Euromonitor 2013). They have been scribed. The method is relatively rapid: 13 compounds can be recently consumed in different societies, especially by youn- analyzed within 30 min: caffeine; ascorbic acid (vitamin C); ger consumers, because they give power throughout the day thiamine (vitamin B1); riboflavin 5-phosphate (FMN, flavin due to caffeine, vitamins, well-absorbed monosaccharides, mononucleotide) and riboflavin (vitamin B2); nicotinic acid and other ingredients such as taurine (Euromonitor 2013; and nicotinamide (vitamin B3); pantothenic acid (vitamin B5); Sather and Vernig 2011). The results of a European survey pyridoxal, pyridoxamine, and pyridoxine (vitamin B6); folic of 52,000 respondents from 16 countries showed that approx- acid (vitamin B9); and cyanocobalamin (vitamin B12). This imately 30 % of adults are regularly drinking caffeine- HPLC method is designated for quantification of caffeine and enriched beverages (Higdon and Frei 2006). Vitamins and water-soluble vitamins in liquid and tablet energy drinks as dietary supplements are also a relevant category in the field well as for determination of vitamins in vitamin supplements. of consumer’s health. In 2013, their worldwide value was The method was validated in terms of linearity, limit of estimated at US$84.4 billion (Euromonitor 2014). detection (LOD) and quantification (LOQ), accuracy, and Caffeine is a xanthine alkaloid acting as a central nervous instrument precision. The LODs determined using HPLC- system stimulant. It temporarily increases blood pressure and PDA ranged from 13 to 121 ng mL−1 and using HPLC-FL eliminates drowsiness. Caffeine is classified by the Food and were between 8 and 61 ng mL−1. Intra- and interday instru- Drug Administration as generally recognized as safe (GRAS), ment precisions for liquid energy drink, expressed as relative and its common consumption, below 400 mg per day standard deviation (RSD), were less than 1.87 and 1.73 %, (Zucconi et al. 2013), has low health risk or even its protective respectively, and less than 1.44 and 1.83 %, respectively, for effects against some diseases, including Parkinson disease tablet energy drink. The method could be useful in the quan- (Prediger 2010), and certain types of cancer have been report- titative analysis of caffeine and of water-soluble vitamins in ed (Uccella et al. 2013; Miura et al. 2014). However, the energy drinks and vitamin supplements. overconsumption of this drug could potentially be harmful (Jiang et al. 2013). Water-soluble vitamins include nine water-soluble groups Keywords Water-soluble vitamin . Vitamin B . Caffeine . of compounds (eight B vitamins and vitamin C) showing HPLC . Energy drink . Vitamin supplement diverse biochemical functions. Each “vitamin” refers to a number of vitamin derivatives that all show the biological activity associated with a particular vitamin. Although the Recommended Dietary Allowances (RDAs) for vitamins are relatively small, even the mild deficiency of one of them can ń Ś ł * : A. Gliszczy ska- wig o( ) I. Rybicka lead to serious health problems. Nutritional deficit of vitamin Faculty of Commodity Science, Poznań University of Economics, al. Niepodległości 10, 61-875 Poznań,Poland B1 (thiamine) can lead to beriberi disease manifested by e-mail: [email protected] cardiovascular disorders and impairment of digestive and Food Anal. Methods nervous systems (Sriram et al. 2012). Deficiencies of vitamin Materials and Methods B2 (riboflavin and its co-enzymes: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)), vitamin B3 Materials (niacin), vitamin B6 (pyridoxine, pyridoxamine, pyridoxal), and vitamin B7 (biotin) mainly occur as skin inflammation and Thiamine hydrochloride (vitamin B1) and pyridoxine hydro- discomfort from the gastrointestinal tract (Powers 2003;Ball chloride (vitamin B6) were purchased from Fluka (Buchs, 2006). Since vitamin B5 (pantothenic acid) is found in most Switzerland). Pyridoxamine dihydrochloride, pyridoxal hy- food products, its deficiency is relatively rare (Ball 2006). drochloride (vitamin B6), cyanocobalamin (vitamin B12), ri- Several methods exist for the determination of caffeine as a boflavin and riboflavin 5′-monophosphate sodium salt hydrate single component or in combination with other drugs in (FMN, vitamin B2), nicotinic acid and nicotinamide (vitamin pharmaceutical formulations or biological matrix of food B3), folic acid (vitamin B9), pantothenic acid sodium salt (Patil 2012). The most popular include chromatographic tech- (vitamin B5), and caffeine were purchased from Sigma (St. niques such as high-performance liquid chromatography Louis, MO, USA). Ascorbic acid (vitamin C) was from Merck (HPLC), high-performance thin layer chromatography (Darmstadt, Germany). Methanol of HPLC grade was from (HPTLC), or capillary electrophoresis with ultraviolet (UV) Chempur (Piekary Śląskie, Poland). or mass spectrometry (MS) detection. They allow the deter- Certified reference materials (CRM; Cerilliant, TX, USA), mination of caffeine as a single compound in herbal products caffeine solution (1 mg mL−1 in methanol), pyridoxine hydro- −1 and energy drinks or its separation from other alkaloids in chloride (vitamin B6)solution(1mgmL in methanol), and −1 chocolate, tea, coffee, urine, or serum (Abourashed and Mossa nicotinamide (vitamin B3)solution(1mgmL in methanol), 2004; Tokusoglu and Kemal 2002;Jafarietal.2011; Perrone were purchased from Sigma-Aldrich (Munich, Germany). et al. 2008; Peri-Okonny et al. 2005; Zhao and Lunte 1997). CRMs of other vitamins were not commercially available or Caffeine can be separated from polyphenols in tea and coffee were withdrawn from the market. (Hadad et al. 2012; Samanidou et al. 2012; Poerner and Demineralized water was obtained from a Hydrolab Bragagnolo 2013) or from other drugs in pharmaceutical System (Hydrolab, Wiślina, Poland) and filtered by preparations (Gámiz-Gracia and Luque de Castro 1997;Zen Millipore system using 0.45 μm nylon filters (Waters, and Ting 1997; Sultan et al. 2013). Milford, MA, USA). Energy drinks and vitamin supplements For vitamin-enriched energy drinks, a micellar electroki- were purchased in local supermarkets and pharmacies. netic chromatography method was proposed to determine 2- aminoethanesulfonic acid, nicotinamide, pyridoxine, caffeine, Sample Preparation riboflavin, and thiamine (Okamoto et al. 2002). Sather and Vernig (2011) and Leacock et al. (2011) described the HPLC Liquid energy drinks were analyzed directly after ultrasonic method for the determination of caffeine and vitamin B6 degassing for 10 min. Energy drinks and vitamin supplements (pyridoxine). The HPTLC for determination of riboflavin, in tablets were weighted, dissolved in 150 mL of pyridoxine, nicotinamide, caffeine, and taurine in energy demineralized water, and sonicated for 10 min. drinks was developed by Aranda and Morlock (2006), but Subsequently, the sample was transferred to a 200-mL volu- HPTLC is a less commonly used technique than HPLC. metric flask and filled up with water to the mark. Samples In the present study, a new HPLC method with were protected from light by aluminum foil and analyzed photodiode array (PDA) detection was proposed for using HPLC. Before the HPLC analysis, the samples were determination of caffeine and water-soluble vitamins. centrifuged for 5 min at 14,000g (microcentrifuge Eppendorf The method with fluorescence (FL) detection for deter- MiniSpin plus, Warsaw, Poland). No supernatant for the tested mination of vitamins B2 (in the form of FMN and products was observed; thus, the recovery studies were not riboflavin) and B6 (as pyridoxal, pyridoxamine, and necessary. For each type of product, three independent sam- pyridoxine) was also described. According to the au- ples were prepared. thors’ knowledge, there is no chromatographic method that allows separation and determination of caffeine in HPLC Determination of Caffeine and Water-Soluble Vitamins combination with most of the water-soluble vitamins. Moreover, simultaneous PDA and FL detection of these A Waters 600 high-performance liquid chromatograph compounds cannot be found in the literature. The pro- (Waters, Milford, MA, USA) equipped with Nova-Pak C18 posed HPLC-PDA-FL method is dedicated for the de- column (150 mm×3.9 mm, 5 μm) fitted with μBondapak C18 termination of caffeine and water-soluble vitamins in cartridge guard column (Waters, Milford, MA, USA) was energy drinks. The method can be also an alternative used. A gradient of mobile phase consisting of methanol for existing methods of water-soluble vitamin determi- (solvent A) and 0.05 M NaH2PO4 containing 0.005 M nations in pharmaceutical preparations. hexanesulfonic acid, pH 3.0 (solvent B) was developed and Food Anal. Methods 1 used according to the following program: linear increment 2 3 starting with 10 % A to 40 % A for 20 min and the return to 8 13 [a.u.] 6 the initial conditions within the next 10 min with the flow rate 9 10 1112 −1 254 nm 5 7 of 1 mL min . The injection volume was 20 μL. The eluate A was detected using a Waters 996 PDA detector set at the 0 2 4 6 8 1012141618202224 wavelength characteristic for appropriate vitamin or caffeine. Moreover, a Waters 474 scanning fluorescence detector set at [a.u.] 3 an excitation wavelength of 290 nm and emission at 390 nm 202 nm 6 8 1011 12 13 A 9 1 2 4 5 7 for vitamin B6 or 450 nm excitation and 530 nm emission wavelengths for vitamin B2 was used. Emission slit width was 0 2 4 6 8 10 12 14 16 18 20 22 24 10 nm, fluorometer gain was 10, and attenuation was 1.