Journal of Chromatography A, 1364 (2014) 303–307
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Simultaneous determination of taurine, glucuronolactone and glucuronic acid in energy drinks by ultra high performance liquid chromatography–tandem mass spectrometry (triple quadrupole) Massimo Ricciutelli a , Giovanni Caprioli b , Manuela Cortese a , Antonietta Lombardozzi c , Morela Strano c , Sauro Vittori b , Gianni Sagratini b,∗ a
HPLC-MS Laboratory, University of Camerino, Via Sant’ Agostino 1, 62032, Italy School of Pharmacy, University of Camerino, Via Sant’ Agostino 1, 62032, Italy c Servizio Polizia Scientiﬁca, Gabinetto Interregionale Piemonte e Valle d’Aosta, Via Veglia 44, 10136 Torino, Italy b
a r t i c l e
i n f o
Article history: Received 14 May 2014 Received in revised form 19 August 2014 Accepted 26 August 2014 Available online 1 September 2014 Keywords: Taurine Glucuronolactone Energy drinks UHPLC–MS/MS
a b s t r a c t In this work, we present for the ﬁrst time a rapid and robust UHPLC–MS/MS method for analyzing taurine, GlcLA and GlcA in energy drinks simultaneously and without derivatization. The separation of three analytes was achieved using a Kinetex Hilic analytical column (100 mm × 4.6 mm i.d.) and a mobile phase formed by water (A) and acetonitrile (B) both with formic acid 0.1% at a ﬂow rate of 0.8 ml min−1 with isocratic elution in 3.5 min. Calibration curves were calculated using the method of standard addition in a concentration range from 2 to 6 mg/100 ml for taurine (R2 > 0.987), from 0.4 to 1.2 mg/100 ml for GlcLa (R2 > 0.997), and from 0.2 to 0.6 mg/100 ml for GlcA acid (R2 > 0.998). The validated method was applied to the analysis of nine commercial energy drinks. The level of taurine found ranged from 0.01 to 0.45 g/100 ml, and it matched with that reported in the labels of the analyzed energy drink samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Energy drinks refer to beverages that contain, besides calories, caffeine in combination with other presumed energy-enhancing ingredients such as taurine, herbal extracts, and vitamins. In recent years, beverages denominated “energy drinks” and “sports drinks” have gained popularity among students, athletes and other active people . Fully 34% of the 18–24 year olds interviewed in one study reported that they regularly consumed energy drinks, a signiﬁcant indicator of the popularity of these beverages among members of the younger generation . Another study reported that about half of the college students interviewed consumed at least 1 energy drink per month, either to increase their energy level in compensation for lack of sleep, or to mix with alcohol . The usual composition of these drinks is based on water-soluble vitamins, carbohydrates, caffeine, taurine and glucuronolactone (GlcLA). Several investigations have been published indicating the effect of this kind of beverage on the central nervous system (CNS), showing signiﬁcant improvements in mental performance
∗ Corresponding author at: School of Pharmacy, University of Camerino, Via Sant’ Agostino 1, 62032 Camerino, Italy. Tel.: +39 0737402238; fax: +39 0737637345. E-mail address: [email protected]
(G. Sagratini). http://dx.doi.org/10.1016/j.chroma.2014.08.083 0021-9673/© 2014 Elsevier B.V. All rights reserved.
(reaction time, concentration and memory) and reduction in sleepiness and sleep-related accidents, due especially to the presence of caffeine . Taurine (2-aminoethane sulphonic acid) (Fig. 1), an abundant free amino acid widely distributed throughout the body and readily found in animal-derived dietary sources, is an ingredient in many energy drinks [5–7]. Studies on caffeinated taurine drink consumption have generally observed signiﬁcantly shorter mean reaction times on attention tasks compared to placebo and control beverages [4,8,9]. However, it is not clear whether these results are due exclusively to the taurine, or whether they are also related to its interaction with other psychoactive ingredients in the beverage, such as glucose. Another ingredient commonly found in energy drinks is d-glucurono-␥-lactone (GlcLA), a normal human metabolite formed from glucose, that is in equilibrium at physiological pH with glucuronic acid (GlcA), its immediate precursor. Many pharmaceutical preparations containing either glucuronolactone (GlcLA) or glucuronic acid (GlcA) are used to treat bilirubinemia because they improve liver condition. Furthermore, when GlcA and GlcLA are analyzed by chromatographic methods, the equilibrium between GlcA and its lactone form (i.e., GlcLA in an aqueous solution) should be considered (Fig. 1). Suzuki et al.  were the only researchers that have determined GlcA and GlcLA together in pharmaceutical preparations by developing a method for the derivatization of reducing carbohydrates with
M. Ricciutelli et al. / J. Chromatogr. A 1364 (2014) 303–307
2.3. Sample preparation
2.4. UHPLC/MS/MS analysis OH
The energy drink samples were diluted with water (1:100, v/v), ﬁltered on a 0.2 m PTFE ﬁlter from Supelco (Bellofonte, PA USA) and then injected in the UHPLC–MS/MS.
Taurine Fig. 1. The molecule of glucuronolactone (GlcLa) in equilibrium with its glucuronic acid (GlcA) analogue, and the structure of taurine.
l-phenyl-3-methyl-5-pyrazolone (PMP) using high-performance liquid chromatography (HPLC) and diode array detector (DAD). On the contrary, there are many analytical methods available in literature for taurine quantiﬁcation, i.e. by using Fourier transform infrared spectroscopy (FTIR)  or nuclear magnetic resonance (NMR)  techniques. Due to absence of strong absorbing groups, taurine is quantiﬁed by means of HPLC with pre-column derivatization and ﬂuorescent detector [13–15], or with pre-column derivatization and HPLC/MS [16,17] or with HPTLC with postchromatographic derivatization . To our best knowledge, no chromatographic analytical methods are available in literature for taurine quantiﬁcation without derivatization steps. The aim of this work was to develop a sensible, rapid and robust UHPLC–MS/MS method for analyzing simultaneously taurine, GlcLA and GlcA in energy drinks without derivatization steps. The validated method was applied to the analysis of various commercial energy drinks.
UHPLC–MS/MS studies were performed using an Agilent 1290 Inﬁnity series and a Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA) equipped with an ESI source operating in negative/positive ionization mode. Optimization of the UHPLC–MS/MS conditions was carried out by varying them in ﬂow injection analysis (FIA) of the analytes (1 l of a 5 mg l−1 individual standard solutions) by using optimizer software (Agilent). The separation of taurine, GlcLA and GlcA was achieved using a Kinetex Hilic analytical column (100 mm × 4.6 mm i.d., particle size 2.6 m) from Phenomenex (Torrance, CA, USA). The mobile phase for UHPLC–MS/MS analysis was a mixture of water (A, 10%) and acetonitrile (B, 90%), both with formic acid 0.1% at a ﬂow rate of 0.8 ml min−1 with isocratic elution. The injection volume was 0.1 l. The temperature of the column was 30 ◦ C and the temperature of the drying gas in the ionization source was 300 ◦ C. The gas ﬂow was 12 l/min, the nebulizer pressure was 50 psi and the capillary voltage was 4000 V (negative and positive). Detection was performed by electrospray ionization (ESI)-MS in the “multiple reaction monitoring” (MRM) mode. The MRM peaks areas were integrated for quantiﬁcation. To enhance the sensitivity, the acquisition time was divided into two periods. The most abundant product ion was used for quantiﬁcation, and the rest of the products ions were used for qualiﬁcation. The selected ion transition and the settings of the mass analyzer are reported in Table 1. All solvents and solutions were ﬁltered through a 0.2 m nylon membrane ﬁlter from Whatman (Dassel, Germany) before use. All samples were ﬁltered before UHPLC analysis through a 0.2 m single use syringe ﬁlter from Minisart RC 4, Sartorius Stedim (Goettingen, Germany). 3. Results and discussion
2. Experimental 3.1. Chromatographic analysis and mass spectrometry 2.1. Materials and standards Standards of taurine (>99%, C2 H7 NO3 S, molecular weight 125.15, CAS no. 107-35-7), d-glucuronic acid (>98%, C6 H10 O7 , molecular weight 194.14, CAS no. 6556-12-3), and d-(+)-glucuronic acid ␥-lacton (≥99%, C6 H8 O6 , molecular weight 176.12, CAS no. 32449-92-6) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Individual stock solutions were prepared by dissolving 100 mg of each compound in 100 ml of water and stored in glass-stoppered bottles at 4 ◦ C. Standard working solutions, at various concentrations, were prepared daily by appropriate dilution of aliquots of the stock solutions in water. HPLC-grade acetonitrile >99.9% was supplied by Sigma–Aldrich (Milano, Italy), HPLC-grade formic acid by Merck (Darmstadt, Germany). Deionized water (>18 M cm resistivity) was obtained from the Milli-Q SP Reagent Water System (Millipore, Bedford, MA). All the solvents and solutions were ﬁltered through a 0.2 m PTFE ﬁlter from Supelco (Bellefonte, PA, USA) before use. 2.2. Sample collection Energy drink samples were bought from different supermarkets in Camerino, Italy.
Table 1 reports the MS/MS acquisition parameters in MRM mode, i.e. time windows, precursor ions, product ions, fragmentor, collision energy (CE), retention time and polarity used for the analysis of the compounds that present more transitions. The most intense are used for the quantitative analysis and are referred to quantiﬁer transition, while the others are employed in the identiﬁcation step. For GlcLA and GlcA the precursor ion corresponds to the deprotonated molecule [M−H]− , while for taurine the precursor ion corresponds the protonated molecule [M+H]+ . Due to the use of HPLC Kinetex Hilic column, the developed analytical methodology showed a very good performance in terms of chromatographic separation and sensitivity. Fig. 2 reports two UHPLC–MS/MS chromatograms of (a) standard mixture of taurine, GlcLA, and GlcA and (b) an energy drink sample (no. 2). In our case, hydrophilic interaction liquid chromatography (HILIC) was a perfect alternative to reverse-phase-highperformance liquid chromatography (HPLC) mode for separating polar compounds. In such a complex system as an energy drink, the HILIC proved very suitable for analyzing these polar compounds, which were instead eluted near the void in reversed-phase chromatography by using other columns reported below, including the Gemini C18,
M. Ricciutelli et al. / J. Chromatogr. A 1364 (2014) 303–307
Table 1 UHPLC–MS/MS acquisition parameters (MRM mode) used for the analysis of the target analytes. Compounds
Time window (min)
Precursor ion (m/z)
Product ion (m/z)
Retention time (min)
These product ions were used for the quantiﬁcation; the rest of the product ions were used for conﬁrmatory analysis.
Synergy Polar-RP-C18 and others. In fact, strong polar compounds such as taurine, GlcA, and GlcLA were sufﬁciently retained by the stationary phase of the Kinetex Hilic and were perfectly separated with a total time course of 3.5 min. Other columns such as the Synergi Polar-RP C18 (4.6 mm × 150 mm, 4 m), Gemini C18 110A (250 mm × 3 mm, 5 m), Gemini-NX 110A (250 mm × 4.60 mm, 5 m), Gemini C18 110A (150 mm × 4.60 mm, 5 m), and Jupiter C18 110A (250 mm × 4.60 mm, 5 m) all from Phenomenex (CA, USA) were tested, but the analytes were not retained in the column. Additionally, the use of a Kinetex Hilic (with its core–shell particles) coupled to an Inﬁnity 1290 UHPLC, which allowed us to minimize extra-column band broadening effects, provided excellent efﬁciency and sensitivity. Moreover, the use of a UHPLC system instead of a HPLC apparatus allowed us to have lower dead volumes during the chromatographic process. To avoid the tailing and irregular shape of the signals, especially of GlcA, various solvents (i.e. acetonitrile, methanol and ethanol) and ratios between them were tested, with different percentages of formic acid. The best conditions were obtained by using x103
acetonitrile and water with 0.1% formic acid, that is a right compromise between the high ionic suppression of the signal and low tailing and irregular shape of the peak (GlcA). The quantiﬁcation of taurine, GlcA, and GlcLA was then done using the standard addition method. 3.2. Method validation Linearity was tested by injecting ﬁve different concentrations of standard mixtures of three analytes studied (external calibration) at the following concentrations ranging from 0.1 to 6 mg/100 ml for GlcLA, from 0.1 to 4 mg/100 ml for GlcA, and from 0.1 to 6 mg/100 ml for taurine. These ranges were chosen taking into account the expected levels in the analyzed matrices. Calibration curves based on the peak area of the standard concentration were obtained and correlation coefﬁcients were higher than 0.995, which implies a good linearity. Five replicates for each concentration of calibration curve were performed over the course of ﬁve days, and the relative standard deviations (RSDs) ranged from 1.1 to 3.2% for run-to-run precision or intraday repeatability and from 5.3 to 19.9%
Fig. 2. UHPLC–MS/MS chromatograms of (a) standard mixture of taurine (2 mg/100 ml), GlcLA (0.5 mg/100 ml), and GlcA (0.5 mg/100 ml) and (b) an energy drink sample (no. 2). Elution time: GlcLa (1.4 min), GlcA (1.7 min) and taurine (2.9 min).
M. Ricciutelli et al. / J. Chromatogr. A 1364 (2014) 303–307
Table 2 Determination of GlcLA, GlcA and taurine in commercial samples of energy drinks. Concentration of analytes is expressed as g/100 ml. n = 3, % RSDs: 3.2–9.5 (GlcLa), 4.8–9.1 (GlcA) and 2.4–5.5 (taurine). Energy drinks
Total GlcLA + GlcA
GlcLA value indicated on the label
Taurine value indicated on the label
1 2 3 4 5 6 7 8 9
N.D.a 0.012 0.004 N.D. N.D. N.D. 0.008 0.020 0.017
N.D. 0.019 0.010 N.D. N.D. N.D. 0.014 0.012 0.011
N.D. 0.031 0.014 N.D. N.D. N.D. 0.022 0.032 0.028
Not declaredb Declared, concentration not speciﬁedc Declared, concentration not speciﬁedc Not declaredb Not declaredb Declared, concentration not speciﬁedc 0.11 0.024 0.03
0.45 0.39 0.37 0.43 0.01 0.39 0.21 0.38 0.38
0.4 0.4 0.4 0.4 0.01 0.4 0.25 0.38 0.4
a b c
Not detectable, 0.987 for taurine, >0.997 for GlcLa and >0.998 for GlcA. The injected volume in UHPLC (0.1 l) was crucial for an accurate quantiﬁcation and a good response of the three monitored analytes. Retention time stability was used to demonstrate the speciﬁcity of the method. Reproducibility of the chromatographic retention time was examined ﬁve times per day over a 5-day period (n = 25). The retention times using this method were stable with a percent RSD value of ≤1.72%.
previous one. In our experiments, we observed that energy drink samples contained both glucuronic forms, lactone and acid compounds (Table 2). Considering the sum, in sample 8, the level of GlcLA plus GlcA was 0.032 g/100 ml, exceeding the level of GlcLA declared on the label (0.024 g/100 ml), while if we consider just the concentration of GlcLA the concentration (0.020 g/100 ml) was lower than that declared (0.024 g/100 ml). Instead, in samples 7 and 9, the sum of GlcLA and GlcA was 0.022 and 0.038 g/100 ml, lower in both cases than the concentration of GlcLa speciﬁed on the label (0.11 and 0.03 g/100 ml respectively). In samples 2 and 3, the sum of GlcLA and GLcA was 0.031 and 0.014 g/100 ml, but the labels only declared the presence of GlcLA, without specifying its concentration. In conclusion, in the nine analyzed samples the level of found taurine matched with that reported in the labels, while only in sample 8 did the sum of GlcLA + GlcA exceed the level speciﬁed on the label (taking into account only the GlcLA concentration, the level was considered lower than that declared in the label).
3.3. Analysis of samples
A new UHPLC–MS/MS analytical method was developed for simultaneously analyzing GlcLA, GlcA and taurine in energy drinks. The method provided good analytical performance, due to the use of a Kinetex Hilic column and a triple quadrupole as mass analyzer. The application of the method to nine energy drinks samples showed that the levels of taurine found ranged from 0.01 to 0.45 g/100 ml, the levels of GlcLA from 0.004 to 0.02 g/100 ml and the level of GlcA from 0.010 to 0.019 g/100 ml.
Among the nine energy drink samples analyzed (Table 2), in only three samples the exact concentration of GlcLA was speciﬁed in the label. Instead, in all samples it was reported the exact concentration of taurine. The levels of taurine found ranged from 0.01 to 0.45 g/100 ml. The lower concentration was found in sample 5, while the highest one was found in sample 1. Samples 1 and 4, although the taurine levels (0.45 and 0.43 g/100 ml, respectively) are higher than those declared in the label (0.40 g/100 ml), cannot be considered out of limit because they are in the legal tolerance (20%) according to the EU regulation 1169/2011 (food labelling) . In the other samples, the level of taurine was equal to or lower than the declared values. Concerning GlcLA, the concentrations found in energy drinks ranged from 0.004 to 0.02 g/100 ml. In three cases, the levels of GlcLA found in our analyses were lower than those reported on the labels (samples 7, 8 and 9), while in two cases (samples 2 and 3), we found a concentration of 0.012 and 0.004 g/100 ml respectively, though the labels only indicated the presence of GlcLA, without specifying its exact concentration. In four samples (1, 4, 5 and 6), our analyses did not reveal GlcLA. Concerning GlcA, the concentrations found in the energy drinks ranged from 0.010 to 0.019 g/100 ml (Table 2). As reported in Fig. 1, the GlcLA molecule is in equilibrium with its GlcA analogue. In fact, in aqueous solution, the lactone ring opens and generates the acidic form that is in equilibrium with the
Conﬂict of interest statement The authors have no actual or potential conﬂict of interest. Acknowledgement The authors would like to thank Sheila Beatty for her editing of the English usage in the manuscript. References  M. Aranda, G. Morlock, Simultaneous determination of riboﬂavin, pyridoxine, nicotinamide, caffeine and taurine in energy drinks by planar chromatography-multiple detection with conﬁrmation by electrospray ionization mass spectrometry, J. Chromatogr. A 1131 (2006) 253–260.  M.A. Heckman, K. Sherry, E. Gonzalez de Mejia, Energy drinks an assessment of their market size, consumer demographics, ingredient proﬁle, functionality, and regulations in the United States, Compr. Rev. Food Sci. Food Saf. 9 (2010) 303–317.
M. Ricciutelli et al. / J. Chromatogr. A 1364 (2014) 303–307  K.E. Miller, Energy drinks, race, and problem behaviors among college students, J. Adolesc. Health 43 (2008) 490–497.  L.A. Reyner, J.A. Horne, Efﬁcacy of a ‘functional energy drink’ in counteracting driver sleepiness, Physiol. Behav. 75 (2002) 331–335.  D. Finnegan, The health effects of stimulant drinks, Nutr. Bull. 28 (2003) 147–155.  R.J. Huxtable, Physiological actions of taurine, Physiol. Rev. 72 (1992) 101–163.  Australia New Zealand Food Authority, Inquiry report. Formulated caffeinated beverages, Australia New Zealand Food Authority, 2001, pp. 1–23.  R. Seidl, A. Peyrl, R. Nicham, E. Hauser, A taurine and caffeine-containing drink stimulates cognitive performance and well-being, Amino Acids 19 (2000) 635–642.  D.M. Warburton, E. Bersellini, E. Sweeney, An evaluation of a caffeinated taurine drink on mood, memory and information processing in healthy volunteers without caffeine abstinence, Psychopharmacology 158 (2001) 322–328.  S. Suzuki, S. Hayase, M. Nakano, Y. Oda, K. Kakehi, Analysis of glucuronolactone and glucuronic acid in drug formulations by high-performance liquid chromatography, J. Chromatogr. Sci. 36 (1998) 357–360.  S. Triebel, C. Sproll, H. Reusch, R. Godelmann, D.W. Lachenmeier, Rapid analysis of taurine in energy drinks using amino acid analyzer and Fourier transform infrared (FTIR) spectroscopy as basis for toxicological evaluation, Amino Acids 33 (3) (2007) 451–457.  K. Wegert, Y.B. Monakhova, T. Kuballa, H. Reusch, G. Winkler, D.W. Lachenmeier, Regulatory control of energy drinks using 1H NMR spectroscopy, Lebensmittelchemie 66 (6) (2012) 143–145.
 X. Kang, J. Xiao, X. Huang, Z. Gu, Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples, Clin. Chim. Acta 366 (2006) 352–356.  H. Nakamura, T. Ubuka, Determination of taurine and hypotaurine in animal tissues by reversed-phase high-performance liquid chromatography after derivatization with dabsyl chloride, Exp. Med. Biol. 526 (2003) 221–228.  H. Inoue, K. Fukunaga, Y. Tsuruta, Determination of taurine in plasma by high-performance liquid chromatography using 4-(5,6-dimethoxy-2phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a ﬂuorescent labeling reagent, Anal. Biochem. 319 (2003) 138–142.  M. de Person, A. Hazotte, C. Elfakir, M. Lafosse, Development and validation of a hydrophilic interaction chromatography–mass spectrometry assay for taurine and methionine in matrices rich in carbohydrates, J. Chromatogr. A 1081 (2005) 174–181.  P. Chaimbault, P. Alberic, C. Elfakir, M. Lafosse, Development of an LC–MS–MS method for the quantiﬁcation of taurine derivatives in marine invertebrates, Anal. Biochem. 332 (2004) 215–225.  G. Indrayanto, T.K. Sia, Y.I. Wibowo, Densitometric determination of taurine and l-lysine hydrochloride in an energy drink and in multi-vitamin syrup, and validation of the method, J. Planar Chromatogr. 14 (2001) 24–27.  Regulation (EU) No. 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers. http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32011R1169& from=EN