Determination of taurine in soft drinks by an ultrahigh-performance liquid chromatography-mass spectrometry method

Liquid chromatography (LC)-MS experiments were performed with the use of Acquity UPLC H-Class (Waters, Prague, Czech Republic). The UHPLC system was fitted with a 2.1 mm × 100 mm, 1.6 μm particle size Cortecs UPLC C18 column. The mobile phase was acetonitrile and water in LC-MS grade, and both contained 0.1% formic acid. The flow rate was 0.5 mL/min, and the column temperature was 55 °C. The injection volume was 1 μL and was constant for all samples and the taurine standard. The separation was performed under gradient elution conditions summarized in Table 1.

The detection was performed with the use of a single quadrupole mass spectrometry detector – QDa detector (Waters) – equipped with an electrospray (ESI) ionization source working in positive (ESI+) mode. The ion source setup is as follows: capillary voltage – 0.8 kV, probe temperature – 600 °C, source temperature – 150 °C, cone voltage – 30 V, and desolvation gas – nitrogen.

Analytical-grade taurine was obtained from Sigma Aldrich (Steinheim, Germany) and a deuterated internal standard (IS) of D5-L-glutamine was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). LC-MS–grade formic acid (HFo) and acetonitrile (ACN) were purchased from Sigma Aldrich, and LC-MS–grade water was purchased from Merck (Darmstadt, Germany). Also, 0.1 mol/L hydrochloric acid (HCl) p.a. quality was obtained from Sigma Aldrich. The derivatization reagent and diluent AccQ•Tag Ultra and borate buffer (pH 8.6) were obtained from Waters.

The stock solution of taurine reference substance was prepared by dissolving its powder in 0.1 mol/L HCl to the desired concentration of 1 mol/L. Working solutions of taurine were obtained by proper dilution of the stock solution with 0.1 mol/L HCl. The taurine concentration levels were in the range of 10–500 μmol/L (10, 20, 50, 100, 250, and 500 μmol/L). A volume of 5 μL of each calibration level was taken into the derivatization process.

The stock solution of the IS (D5-L-glutamine) was prepared in 0.1 mol/L HCl to the desired concentration of 1 mol/L. Working solutions of D5-L-glutamine were made by proper dilution of the stock solution with 0.1 mol/L HCl to the desired concentration of 250 μmol/L.

Four commercial energy drinks (i.e., Red Bull, Hell, Tiger, Maxx) were obtained from a local store. In all beverages, the content of taurine was declared by the manufacturer to be 4 mg/mL. Before the analysis, 10 mL of each beverage was transferred into 10 mL volumetric flask and then the samples were sonicated in three intervals for 15 min in an ultrasonic bath. After the sonication procedure, the samples were 1000 times diluted with the LC-MS water and then 5 μL of each sample was taken into the derivatization process.

The derivatization procedure of the calibration standards and samples of taurine was carried out according to the following protocol. At first, the derivatization solution was prepared by mixing the AccQ•Tag Ultra (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate [AQC] reagent with 1 mL of the AccQ•Tag Ultra diluent and heating at 55 °C for 15 min. Then, 70 μL of borate buffer (pH 8.6), 5 μL of calibration standard or sample, and 5 μL of IS were transferred into a 1.5 mL Eppendorf vial. The sample was vortexed and then 20 μL of prepared derivatization solution was added. Further, the sample was heated at 55 °C for 10 min. The mechanism of the derivatization procedure of taurine is illustrated in Figure 1b. When the derivatization process was finished, the samples were centrifuged at 30,000 ×g for 10 min and then directly transferred to injection vials.

The UHPLC-MS method, including sample preparation (derivatization) and analysis conditions, was applied based on our previously developed method for analysis of 20 proteinogenic amino acids (Piestansky et al., 2019). It was found that the selected method was fully implementable for the analysis of nontypical amino acid taurine in soft drinks. The quantitative analytical approach was based on the IS method. The ideal IS in LC-MS approaches is an isotopically labeled version of the analyte (Hansen, 2015). The addition of an IS minimizes errors during the analysis, mainly the loss of analyte during sample preparation/treatment, and prevents the influence of matrix effects, which, in most cases, negatively affect the analysis. Due to the lack of isotopically labeled taurine in our laboratory, we decided to use isotopically labeled D5-L-glutamine as a suitable IS. This selection was made due to the very similar elution properties of both substances. Moreover, previous papers also indicated the use of isotopically labeled L-glutamine as an IS for taurine determination (Gamagedara et al., 2012).

During MS step optimization, it was necessary to identify appropriate m/z of taurine and IS derivative (D5-L-glutamine) ions for their unequivocal quantification in selected ion monitoring (SIM) regime of the single quadrupole mass spectrometer. The following m/z values of [M+H]⁺ ions were identified and used in further experiments: 296.1 (taurine), 322.2 (D5-L-glutamine).

The adapted UHPLC-MS method for taurine determination was validated according to the International Conference on Harmonisation (ICH) Q2(R1) guideline (ICH 2005). Validation parameters such as specificity, linearity, accuracy, precision, detection limit (LOD), quantitation limit (LOQ), and robustness were investigated. All resulting validation and performance parameters are summarized in Tables 2–4. The calibration curves in the water matrix were calculated using TargetLynx XS software (Waters). As a result of the evaluation, a calibration curve was constructed based on the ratio of analyzed taurine to IS D5-L-glutamine. The coefficient of determination (r²) of taurine was higher than 0.99. It reflects appropriate linearity in the selected concentration range (10–500 μmol/L). The predicted LOD and LOQ values calculated from the calibration lines were 4.29 and 10 μmol/L, respectively.

The precision and accuracy of the method were determined using a pooled sample prepared by mixing 1 mL of each energy drink. The pooled sample was diluted and spiked with an appropriate amount of taurine standard solution to obtain final spiked concentrations of 10, 100, and 500 μmol/L. The precision of the developed method was determined as intra- and inter-day precision (Table 3). The relative standard deviation (RSD) values were in the range of 0.1%–2.62% for the intra-day assay and 0.57%–3.95% for the inter-day assay. Accuracy of the method was measured as the percent of analyte recovered by the assay (expressed as mean bias from nominal concentration, %Nom, for the measurements of three spiked levels of taurine). The values determined during the accuracy test were in the range of 91.5%–108% (Table 4). All observed results were within the acceptance criteria of the ICH guideline.

The small, but deliberate variation of the operational parameters (changes in the separation column temperature ±1ºC and mobile phase A composition ±0.01%) in the robustness test resulted in standardized peak area changes not higher than 1.5% when compared to the results obtained under standard conditions. The presented results clearly show the applicability of the developed method in the field of quality control of commercial products with taurine content.

Finally, the validated UHPLC-MS approach was applied to determine the concentration of taurine in four commercially available energy drinks – Red Bull, Hell, Tiger, and Maxx. The manufacturers declared a taurine concentration of 4 mg/mL in each investigated energy drink. . The overview of determined taurine concentrations in such beverages is presented in Table 5. In three samples, the content of taurine was in relatively good agreement with the declared content, but in one case, the content of taurine was only 70% of the declared value. One of the reasons why so low content of taurine was observed in the tested beverage could be degradation of taurine and formation of some degradation products. This hypothesis is supported by Figure 5a, where the presence of another peak with retention time at 1.91 min with the same m/z ratio was observed. However, a peak at the same retention position (1.91 min) was observed in all remaining samples (Figure 2b–d). The intensities of these peaks were significantly lower in comparison to those observed in case of Red Bull sample. Therefore, it can be stated that such a low content of taurine could be a result of unsuitable storage of the tested beverage.

Figure 2.

Energy drinks can currently be sold in all EU Member States, although some national legislators have decided to take a more specific regulatory approach. In Canada, on energy drinks, the labels contain the maximum daily consumption and include warnings about mixing energy drinks with alcohol (BFR, 2008). The manufacturers of products in Australia and New Zealand have bypassed regulations by classifying them as “dietary supplements” to avoid the 80 mg/250 mL limit for caffeine (Oddy & O’Sullivan, 2009). In 2012, Hungary adopted a “public health tax” that applies to caffeinated energy drinks and other products. Tax for the drinks containing >100 mg of taurine per 100 mL is calculated at the rate of approximately 0.81 €/L (Zámbó et al., 2020). Due to the long-term and widespread consumption of beverages such as coffee and tea that contain caffeine and taurine naturally, energy drinks in developed countries remain largely unregulated (Buxton & Hagan, 2012; Reissig et al., 2009). May be the Hungarian tax or no regulations is the main reason why the manufacturers are not required to meet taurine-declared quantities in soft drinks. An example of the unregulated and uncontrolled amount of taurine in soft drinks can be found in a product sold on the Czech market, which contained 96.1% of the declared amount (Vochyánová et al., 2014), while the Slovak one contained only 70% (see Table 5).