Determination of theobromine and caffeine in some Malaysian beverages by liquid chromatography-time-of-flight mass spectrometry

Purpose: To determine the concentration of theobromine (TB) and caffeine (CF) in tea and other beverages using liquid chromatography-time of flight-mass spectrometry (LC-TOF-MS). Methods: The extract of caffeine and theobromine from tea and other beverages was filtered by 0.45 μm nylon micro-syringe and then injected into a LC-ToF-MS system. Theobromine and caffeine were separated using Thermo Scientific C18-column (length 250 mm, width 2.1 mm and diameter 5 μm). Acetonitrile-methanol (ACN – MeOH, 3:1 v/v) was used as mobile phase B, while mobile phase A was 0.1 % FA in DIW. The volume injected was 30 μL at a rate of 0.3 mL/min. Results: Good linearity was obtained in the range of 0.3 – 400 and 0.2 – 200 mg/L for theobromine and caffeine, respectively (regression coefficient (R) > 0.970). The limits of detection were 0.15 and 0.05 μg/mL for theobromine and caffeine, respectively. The highest concentrations of caffeine and theobromine determined in tea samples were 159.1 and 255.8 mg/L, respectively. Conclusion: Theobromine and caffeine have been successfully analysed in tea, coffee and soft drinks. LC-TOF-MS is an accurate and promising instrument for the determination of the studied compounds in beverages.


INTRODUCTION
Methylxanthines, theobromine and caffeine are used as analgesics, diet aids, and cold/flu remedies in numerous popular carbonated drinks. They are also the main compounds found in tea, coffee, sodas, chocolate, and various energy drinks. More than 120,000 tons of caffeine is consumed worldwide annually [1,2].

Malaysia-Regular
Beverage Consumption revealed that coffee and tea are the most consumed drinks by Malaysian people [3].
ToF-MS is a highly accurate and specific detector. With this detector, data is given as a four-decimal-point m/z that responds, almost exclusively, to electrospray ionization as an ionization source [14]. The separation and extraction of TB and CF from tea, coffee, soft drinks, and other beverages has been reported in several previous studies with different extraction methods [15,16].
The aim of this study is to develop a simple and accurate LC-ToF-MS method, using an extremely limited reagent for the analysis of theobromine and caffeine in beverages. Direct injection, without any further sample preparation, was provided using LC-ToF-MS which is an accurate technique to extract caffeine and theobromine from the total ion chromatogram using a 20 mD mass window.

Standard solution preparation
Stock solutions 1 mg/mL for theobromine and caffeine were prepared by dissolving in MeOH, 0.01 g of compound was dissolved in 10 mL of MeOH, and stored at 20 for further preparation. Further solutions were prepared by diluting the stock solution (1 mg/L) with deionized water. All solutions were injected in triplicate and analysed using LC-ToF-MS. Calibration curves were built by plotting peak area against concentration.

Sample collection and preparation
All the samples were collected from different local markets in Malaysia and are as presented in Table 1.
Tea samples were extracted in hot water. A 1.0 g sample of tea was placed in 50 mL of distilled water at 95 -100 o C for 5 min as per normal preparation for a daily consumer. Tea extracts were filtered through a 0.45 m Nylon microsyringe filter. A 5 mL sample of coffee was filtered and then injected directly into the LC-MS. Dissolved gases were excluded from beverages using an ultrasonic water bath for 10 min, prior to sample analysis. Thereafter, the samples were filtered using a 0.45 μm Nylon micro-syringe filter to obtain a transparent sample ready for analysis.

LC-ToF-MS instrumentation and conditions
Separation of theobromine and caffeine was performed using a liquid chromatography instrument (Dionex Ultimate 3000/LC 09115047 (USA)) equipped with a vacuum degasser, a quaternary pump, and an auto-sampler. Sample aliquots of 30 L were injected into 5 m, 2.1 mm × 250 mm Thermo Scientific C18 column. Electrospray ionisation (ESI) was utilized as an ionization source.  9-556404-001033 18-10-2018 Theobromine was separated at 5.43 min, whereas caffeine was separated at 6.51 min. The

Evaluation of selectivity
Selectivity was evaluated by comparing the chromatograms of three different samples; real sample, spiked real sample, and deionized water. The results revealed that no interfering peaks were present at the TB and CF retention times.

Determination of linearity and sensitivity
The linearity ranged between 0.3 and 400 mg/L for theobromine and 0.2 and 200 mg/L for caffeine.
Calibration curves were generated for each compound by plotting the peak area against the concentration of each compound using the linear regression model. For the analytical chromatography instruments, LOQ was the lowest concentration corresponding to S/N ratio ≥ 10.

Determination of precision and accuracy
Precision and accuracy of the LC-ToF-MS method were evaluated at three different concentrations (1, 50, and 200 g/mL) and (5, 25 and 100 g/mL) for theobromine and caffeine, respectively. Inter-day precision was evaluated by performing three replicates of three different concentrations. Intra-day precision was assessed within three consecutive days. The precision was presented in terms of relative standard deviation percentage (RSD %). The accuracy was expressed as the ratio of mean observed concentration to nominal concentration, as a percentage.

Statistical analysis
Each assay was repeated at least three times. Means were analysed statistically by analysis of variance (ANOVA) followed by Duncan's test, using SPSS software, Version 19. The results were considered significantly different at p < 0.05.

RESULTS
The The precision of the LC-ToF-MS method was assessed by analysis of three samples replicates within the same day (inter-day) and for three different days (intra-day) (see Table 2). The precision was determined by analysing three different concentrations for theobromine (1,50 and 200 g/mL) and caffeine (5, 25 and 100 g/mL), respectively, on three different days. The results were in accordance with standard method validation [17]. RSD % values ranged from 3.11 to 13.22 % for intra-day precision and 2.52 to 11.43 % for inter-day precision. Accuracy was determined by adding known amounts of TB and CF as a mixture to the solvent (1, 50, and 200 g/mL) and (5, 25, and 100 g/mL), respectively. The accuracy results were good, ranging from 99 to 108 % for intra-day and 94 to 110 % for interday (as shown in Table 2). Figure 1 shows that no peaks interfered at the retention times of TB (5.43 min) and CF (6.51 min). Chromatogram A refers to the standard solution of a mixture of TB and CF. The tea sample was spiked with the mixture of these two compounds and then injected into LC-ToF/MS as shown in chromatogram B.  No peaks appeared TB and CF in deionized water (blank solvent); this method was therefore selective for TB and CF; as shown in chromatogram C.
Environmental samples are commonly too polluted with organic and inorganic contaminants and are therefore called complex matrices. To prevent the alteration or interference of other compounds to the target compounds, a narrow window was used in the ToF-MS analyser to reconstruct the chromatographic traces; consequently the selectivity of the instrument was increased. Extracted ion chromatograms (EICs) from the total ion chromatogram (TIC) were provided at 0.02 Da for theobromine and caffeine (as shown in Figure 2).  All 15 samples were analysed using LC-ToF-MS. Retention times were 5.43 and 6.51 min for TB and CF, respectively (as shown in Figure 2). The concentrations of theobromine and caffeine in samples are presented in Table 3.

DISCUSSION
The investigation of the LC-ToF-MS method's selectivity was discussed after the comparison of the peak chromatograms; which resulted in standard solutions, tea sample spiked with standards and deionized water without standards. The regression coefficients were 0.999 and 0.979 for TB and CF, respectively. It was observed that limit of quantification (LOQ) values of 0.15 and 0.05 g/mL for theobromine and caffeine, respectively, were more acceptable than in previous studies [18,19].
The precision and accuracy of the LC-ToF-MS method was assessed for theobromine and caffeine. Three replicates were performed for three different concentrations and then all samples were injected into the LC-ToF-MS instrument. The results showed that RSD % was ≤ 13.22; which is acceptable in this range of concentration.
It is well-known that analysis of complex matrices is a challenge; because the signals of target compounds could be impacted by organic and inorganic components that are both present in the real sample, together with analytes. The selectivity of the LC-ToF-MS method was enhanced by selecting a narrow window (0.02 Da) for analysis of theobromine and caffeine in actual samples. Improvement of S/N ratio, from twofold to fourfold, was observed after reducing the daltonic value from 0.5 to 0.02 Da [20]. The mass value (m/z) for the protonated molecular ions [M + H] + was used for quantitation analysis. The errors obtained for mass values were between 0.6 and 1.2 ppm.
All data related to accurate mass were calculated using Bruker Daltonics Data Analysis software; which provided the experimental and theoretical mass value (m/z) and elemental composition for molecular ions [M + H] + . From the results, the mass errors were less than 0.5 ppm for both CF and TB; which are more acceptable than in previous study [21].  [22].
The highest concentration of caffeine was present in Al-Kbous tea, with the concentration reaching 143 mg/L. Meanwhile, in Pepsi, the caffeine concentration was 103 mg/L; which is almost identical to that of original Cane from the market. For beverages, caffeine was highly detected in Red Bull at 136.1 mg/L. The mean caffeine content of carbonated soft drinks was lower than that of energy drinks. This finding is in line with the same result reported by Fatima et al [1]. However, the mean concentration of CF in 9 tea samples was 133 mg/L. The highest concentrations of TB were detected in Al-Wazah tea and Milo at 256 and 222 mg/L, respectively. All of these beverages are a major source of contamination of the water samples by theobromine and caffeine; as shown in previous studies [23,24].

CONCLUSION
The findings of the present study indicate that LC-ToF-MS method is accurate and sensitive enough to detect theobromine and caffeine in tea and other beverages. This study is the first to report on the analysis of theobromine and caffeine in Malaysian tea and beverages. Thus, ToF-MS is a powerful analyser that can be used to confirm the presence of theobromine and caffeine in tea and other beverages.