Determination of B-vitamins in Energy Drinks by CE/MS/MS

Transcription

1 Application ote Food Determination of B-vitamins in Energy Drinks by E/MS/MS Authors laudimir Lucio do Lago and Zuzana ieslarová Department of Fundamental hemistry, Institute of hemistry, University of São Paulo, Brazil Daniela Daniel Agilent Technologies, Inc. Abstract Vitamins are essential nutrients found in foods, and are vital to maintaining optimal health. This Application ote describes a sensitive and reliable method for the determination of seven B-vitamins in energy drinks, using E/MS/MS. The best separation of B1, B2, B3a, B3b, B5, B6, B12, and cetirizine (used as internal standard) was obtained using mm formic acid (ph 2.5) as the background electrolyte (BGE). With a 6 cm fused silica capillary, it was possible to achieve a run time of under eight minutes. The proposed method is simple, fast, and presented a linear calibration with correlation coefficients greater than.995. Limits of detection (LDs) and limits of quantification (LQs), based on the signal-to-noise ratio (S/), were in the range of.1.19 and.4.63 ppm, respectively. The method was successfully used to determine B-vitamins in commercial samples of energy drinks.

2 Introduction Vitamins are indispensable compounds for the human organism. However, not all vitamins can be synthesized by the organism itself, so they need to be supplemented 1. Vitamins are so crucial for good health that there is a wide range of fortified foods and supplements on the market. However, some vitamins, such as B vitamins, are sensitive to temperature and light; therefore, there are concerns about the real amounts of vitamins present in fortified foods. These concerns have led to the need for specific techniques to analyze these vitamins. Traditionally, methods for vitamin analysis were based on microbiological assays and immunoassays. These assays are usually labor-intensive, time-consuming, and generally do not allow simultaneous determination of multivitamins. Liquid chromatography (L) is also a common technique for determining vitamins in many food products. However, water soluble vitamins, such B-vitamins, are very polar and have relatively poor retention on reversed-phase columns. apillary electrophoresis (E) is useful for the separation and determination of ions or ionizable compounds such as B-vitamins, which are fully ionic at low ph. Therefore, E (compared to L) offers advantages, including short analysis time, low reagent cost, and minimal sample requirements. It is also considered an environmentally friendly analytical method because it produces less waste 2,3. Analytical separation using E coupled to mass H icotinic acid (B3a) pka 2.8 H H 2 S Thiamine (B1) pka 15.5 H H H Riboflavin (B2) pka 5.9 H H 2 icotinamide (B3b) pka 13.4 H H H 2 H 2 H H H H H 3 spectrometry (MS) detection can provide significant advantages by combining the high separation efficiency of E with the identification power of MS. The aim of this study was to develop and validate a method for the determination of B-vitamins in energy drinks using E/MS/MS 4. Figure 1 shows the molecular structures of the B-vitamins analyzed in this study, along with their strongest basic pka values. H H Pyridoxine (B6) pka 9.4 Pantothenic acid (B5) pka 4.3 H 2 H o + H H H H 2 H 2 H 2 yanocobalamin (B12) pka 1.8 Figure 1. Molecular structures of the B-vitamins analyzed and their strongest basic pka values. The pka values were calculated at (accessed April 8). 2

3 All separations were performed at 25 using mm formic acid, ph 2.5, as the BGE. ew fused silica capillaries were preconditioned by flushing.1 M ah solution (5 minutes), de ionized water (1 minutes), and BGE (1 minutes). An extra flushing step with BGE for 6 seconds with de-ionized water and 9 seconds with BGE was included between the runs. Samples were introduced hydrodynamically in five seconds at mbar and analyzed with an applied voltage of 29 kv. The mass spectrometer was operated in positive ionization mode, using multiple reaction monitoring (MRM) mode for two specific transitions. Table 1 lists the migration time (t M ), monitored ions, and other MS/MS acquisition parameters used for the identification and quantification of the targeted B-vitamins in energy drinks. Sample preparation Samples of energy drinks were bought from local supermarkets. They were then degassed for three minutes in an ultrasonic bath and filtered through a.2 µm polyvinyldifluoride and polypropylene membrane (Agilent aptiva filter cartridges, p/n A532). ext, they were diluted 1 times before being transferred to a 2 µl polypropylene vial for E/MS/MS analysis (p/n ). Experimental E conditions Instrument Parameter MS conditions Agilent 7 E system Value Background electrolyte mm formic acid, ph 2.5 Applied voltage apillary Injection 29 kv Temperature 25 Instrument Ion mode Parameter Sheath liquid Flow rate apillary voltage Drying gas flow ( 2 ) Silica capillary µm id with 6 cm total length (p/n , cm length, cut to 6 cm) 5 seconds at mbar Value Agilent 643 triple quadrupole L/MS ESI, positive ionization.1 % Formic acid/methanol (: v/v) 5. µl/min 2, V 7 L/min Drying gas temperature 3 ebulizer pressure 9 psi Table 1. Migration time (t M ) and MS/MS acquisition parameters used for the identification and quantification of B-vitamins in energy drinks. Vitamins t M (min) Q1 a (m/z) Q3 b (m/z) E c (V) FE d (V) Thiamine B icotinamide B3b Pyridoxine B eritizine (IS) * * * 77.2.* icotinic acid B3a * yanocobalamin B * Riboflavin B * Pantothenic acid B * a = precursor ion (Q1); b = fragment ion (Q3); c = collision energy; d = fragmentor energy 3

4 Results and discussion The BGE, sheath liquid composition, applied potential, and hydrodynamic injection were optimized for separation efficiency and sensitivity. Figure 2 shows a representative MRM electropherogram obtained under optimum conditions for the B-vitamin standards and internal standard (IS) in the BGE. The t M was shorter than 8. minutes. The methodology for B-vitamins analysis using E/MS/MS was validated in terms of selectivity, linearity, sensitivity, precision, and accuracy. alibration curves were constructed with standard solutions at 11 levels of concentration using cetirizine as the IS. The correlation coefficients (R 2 ) of calibration curves were greater than.995. Each level of concentration was analyzed in quintuplicate, and the related standard deviations (RSDs) ranged from.2 to 6.6 % for run-to run precision. The LDs and LQs were determined considering the corresponding concentration to three and ten times, respectively, the baseline noise in a time close to the migration time measured around each vitamin. The proposed method allows B-vitamins to be determined with LDs in the range of.1.19 ppm using a very low injection volume (calculated approximately 4.5 nl). Table 2 shows the regression equations and other characteristic parameters for the developed method. Standard deviations of residuals were obtained by analysis of variance (AVA). ounts (%) ounts (%) ounts (%) ounts (%) ounts (%) ounts (%) ounts (%) ounts (%) B1 B3b B6 etirizine (IS) Figure 2. ormalized MRM electropherogram under optimum conditions for the B-vitamins and internal standard in BGE at 1.8 ppm for B1, B2, B6, and IS, at 18 ppm for B3a and B3b, and at 45 ppm for B5 and B12. Table 2. Figures of merit for the B-vitamin analysis in energy drinks by E/MS/MS. Vitamin Linear range (ppm) y = ax+b R 2 S x/y LD (ppm) LQ (ppm) B y =.19x B3a B Acquisition time (min) B2 B5 B3a y =.925x B y =.18x B3b y =.17x B y =.95x B y =.351x B y =.254x a = Intercept; b = slope; R 2 = determination coefficient; S y/x = standard deviation of residuals; LD = limit of detection; LQ = limit of quantitation 4

5 The consistency of the proposed method was evaluated by applying it to real samples to determine B-vitamins in 11 energy drinks sold at a Brazilian market, using standard additions to avoid any matrix effects. The RSD was lower than 8.3 %. o significant difference was observed between the values found and those values declared by the manufactures on the labels. Table 3 summarizes these results, where the values of vitamins B3a and B3b were added together and expressed as total vitamin B3. ote that even in samples where levels of vitamins B1 and B3 were declared as zero by the manufacturer, small concentrations of these vitamins were found. This happens because guarana extract is used in the formulation of many of these samples, and is a natural source of vitamins B1 and B3. Table 3. oncentration (mg/l) of B-vitamins in energy drink samples (n = 5) as well the RSD (%) values. <LD = lower than detection limit. ontinued on next page. Sample Vitamin oncentration informed (mg/l) oncentration found (mg/l) RSD (%) B ± B ± B ± B ±.3.8 B ±.1.8 B µg/l <LD B1.4.9 ± B ± B ± B ± B ± B12 18 µg/l <LD B1.4.5 ± B ± B ± B ± B ± B12 18 µg/l <LD B1.4.3 ± B ± B ± B ± B ± B12 18 µg/l <LD B1.6 ±. 1. B2.9 ±..8 B3 6.5 ± B5 2.2 ± B6 1.1 ±. 2.2 B12 <LD B1.2 ±..4 B ± B ± B ± B ± B µg/l <LD B1.2 ±. 4.3 B ± B ± B ±.2.8 B ±..1 B µg/l < LD 5

6 B1.3 ±. 3.6 B ±. 3.9 B ± B ± B ±.3 4. B µg/l <LD B1.2 ±. 2.2 B ±..9 B ±.3.4 B ±..2 B ±.1.8 B12.2 ±. 2.2 B1.2 ±. 3.9 B2 1.4 ± B ±.8 1. B ± B ± B µg/l <LD B1.2 ±..7 B ± B ± B ± B ± B12 1. µg/l <LD onclusions We have been able to show that E/MS/MS is well suited for the analysis of B-vitamins in energy drinks. The proposed method presented a linear response with excellent precision data for replicate injections and LDs lower than.19 ppm. In addition, the method is simple and fast, spending less than eight minutes per sample, using a small amount of sample with low reagent consumption. References 1. Modern hromatographic Analysis of Vitamins. 3rd Edition,. Revised and Expanded. Edited by Andre P. De Leenheer, Willy. E. Lambert, Jan F. Van Bocxlaer (University of Ghent,. Belgium). Marcel Dekker: ew York Simó,.; Barbas,.; ifuentes, A. apillary electrophoresis mass spectrometry in food analysis. Electrophoresis 25, 26, Wang, X.; et al. Recent advances in vitamins analysis by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis 8, 147, Fotsing, L.; et al. Determination of six water-soluble vitamins in a pharmaceutical formulation by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis 1997,15, This information is subject to change without notice. Agilent Technologies, Inc. 8 Printed in the USA, August 8, E