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From: Food Quality & Safety magazine, June/July 2011

Soda Showdown

Simultaneous quantitation of 2- and 4-methylimidazoles in carbonated drinks containing caramel coloring by ultrahigh-performance liquid chromatography tandem mass spectrometry

by Jinyuan (Leo) Wang, Xiaodong Liu, Christopher A. Pohl, William C. Schnute

Soda Showdown

Caramel coloring is often used to darken food products such as carbonated beverages and soy sauces. Recently, two byproducts of its manufacture—2- and 4-methylimidazoles (2-MI and 4-MI)—have come under scrutiny. Studies from the National Toxicology Program (NTP) and other researchers concluded that there is clear evidence of the carcinogenicity of both chemicals.1–3 California’s Office of Environmental Health Hazard Assessment (OEHHA) listed 4-MI as a carcinogen in January with a calculated no significant risk level (NSRL) of 16 µg per person per day.4 In February, a group of scientists from the Center for Science in the Public Interest filed a petition with the U.S. Food and Drug Administration (FDA) to bar the use of caramel coloring containing 2-MI and 4-MI, which “serve purely cosmetic purposes.”

Conventional methods for identification of 2-MI and 4-MI in caramel color include gas chromatography methods that involve labor-intensive procedures such as hot solvent extraction and acetyl derivatization and are thus not suitable for high-throughput analysis.5–6 Several liquid chromatography methods were also reported for 4-MI analysis.7–8 However, to the authors’ best knowledge, no previous study has reported the simultaneous quantification of 2-MI and 4-MI in beverages and other food products.

This study describes an ultrahigh-performance liquid chromatography tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantification of 2-MI and 4-MI in various soda products. In this method, soda samples were degassed by sonication, diluted, and directly analyzed. Chromatography was performed on an UltiMate 3000 Rapid Separation (RS) UHPLC system (Dionex Corporation, Sunnyvale, Calif.), and separation was achieved using an Acclaim Trinity P1 Mixed-Mode column. The TSQ Quantum MS/MS instrument (Thermo Fisher Scientific, San Jose, Calif.) was operated in selected-reaction-monitoring (SRM) mode for the best sensitivity and selectivity. In the dark soda drinks selected in this study, 4-MI was quantified at hundreds of parts-per-billion (ppb) levels. For comparison, a colorless lemon-lime flavored soda drink was also analyzed by this method, and no quantifiable levels of target analytes were found.

Figure 1. The chemical standards used in this study.
Figure 1. The chemical standards used in this study.

Chemical standards used in this study were purchased from Sigma-Aldrich (2-MI, M50850; 4-MI, 199885, structures shown in Figure 1). Methanol and acetonitrile were obtained from Burdick & Jackson (HPLC/UV grade). Ammonium formate and formic acid were purchased from Sigma-Aldrich for mobile phase preparation. Stock solutions of 2-MI and 4-MI were prepared by dissolving each pure chemical in deionized (DI) water to the concentration of 1 mg/mL (1000 parts-per-million, ppm). Working solutions were prepared by mixing 2-MI and 4-MI in DI water at 10 ppm and then diluting to 1 ppm and 100 ppb. Calibration standards were prepared from working solutions at seven levels: 1 ppb, 5 ppb, 10 ppb, 50 ppb, 100 ppb, 200 ppb, and 500 ppb.

Bottled dark-colored soda drink samples were purchased from a local retail grocery store, including regular, diet, and zero calorie varieties where possible. A bottled lemon-lime flavored colorless soda sample was also purchased for comparison. All samples were stored at room temperature until opened for analysis. A 20 mL aliquot of each sample was poured into a 150 mL specimen cup and degassed for 30 seconds in a sonication bath. A 100 µL aliquot of each degassed sample was then pipetted into a 1.5 mL autosampler vial and vortex mixed with 900 µL DI water; 10 µL of each prepared sample was injected for UHPLC-MS/MS analysis.

Figure 2. Optimized SRM chromatograms of a mixed standard containing 50 ppb of each target analyte.
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Figure 2. Optimized SRM chromatograms of a mixed standard containing 50 ppb of each target analyte.

Chromatography was performed on the UltiMate 3000 RS UHPLC system, with separation of 2-MI and 4-MI achieved on an Acclaim Trinity P1 column with isocratic elution. Mobile phase consisted of 10% methanol, 5% acetate buffer (100 mM, pH 5.7), and 85% DI water and was delivered at a flow rate of 0.5 mL/min. The column temperature was set at 15 degrees C. A TSQ Quantum Access MS/MS instrument was selected as the detector and was operated in SRM mode to achieve the best selectivity and sensitivity. A heated electrospray ionization (HESI) source was used to interface the UHPLC and MS/MS systems. The detailed source parameters are listed in Figure 2. Two SRM transitions were used for the quantitation (Q-SRM) and confirmation (C-SRM) for each analyte, with collision energy set at 22 V. Details of the SRM transitions are listed in Figure 2.

Method Development

The objective of this study was to develop a quantitative method for simultaneous determination of 2-MI and 4-MI in soda products. These two target analytes are positional isomers with identical molecular weight.

Our initial investigation into using MS/MS detection for analytical method development showed the same fragmentation paths for both compounds, i.e., same precursor and product ions. Therefore, chromatographic separation is essential for the accurate quantitation of each individual analyte. Both analytes are very hydrophilic and difficult to retain on a reversed-phase column.

Table 1. Studied Compounds and SRM Scans
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Table 1. Studied Compounds and SRM Scans
Table 2. Precision and Accuracy
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Table 2. Precision and Accuracy

Reported methods used basic mobile phases to retain the analyte, which are not desirable when using silica-based LC columns due to chemical instability. The column that we used in this study features simultaneous cation-exchange, anion-exchange, and reversed-phase retention mechanisms.9 To analyze 2-MI and 4-MI, both cation-exchange and reversed-phase mechanisms were used.

During the development of the method, we found that although both isomers were adequately retained on the column, their resolution was highly dependent upon buffer pH and organic solvent content. Because the resolution between the two isomers is essential for accurate quantification, a mobile phase containing 10% methanol and 85% ammonium acetate buffer (pH 5.7) was selected. To obtain baseline separation, the retention factors for both isomers were greater than 20 (or 6.1 min and 7.5 min retention times, respectively), which at the same time minimized potential interferences from sample matrices. The optimized SRM chromatograms of a mixed standard containing 50 ppb of each target analyte are shown in Figure 2.

Total chromatographic separation was achieved with either methanol or acetonitrile as a mobile phase organic modifier; however, strong interference was observed when using acetonitrile. The interference can be explained by the acetonitrile solvent cluster ion [2M+H]+, which has the same mass-to-charge ratio (m/z) at 83 as the analytes. Given this interference, we chose to use methanol as the mobile phase organic modifier in this study.

Both analytes exhibited strong molecular ion ([M+H]+) MS response at 83 m/z, which was used as the precursor ion for both. The two most intensive fragment ions, 42 and 56 m/z, were observed for both analytes and were selected as product ions for detection, as shown in Table 1.

Figure 3. SRM chromatograms of an original soda sample, left, and one that is spiked.
Figure 3. SRM chromatograms of an original soda sample, left, and one that is spiked.

Although both SRM transitions (83‡42 and 83‡56 m/z) were observed and used for the quantitation and confirmation of target analytes, a significant difference was observed in the relative intensities of the selected SRMs for each individual analyte. As seen in Figure 2, for 2-MI, a relatively stronger response was observed with SRM 83‡42, which was used as Q-SRM, and SRM 83‡56, which was used as C-SRM. Because 4-MI demonstrated the opposite relative intensity, SRM 83‡56 was used as the Q-SRM for 4-MI, and the SRM 83‡42 was used as C-SRM. The difference in relative intensity of the two SRM transitions can be monitored, and the ratio can be used as additional identity confirmation in conjunction with chromatographic retention time.

Method Performance

This method was evaluated against performance parameters such as specificity, carryover, calibration, correlation of determination (R2), detection limit, precision, accuracy, and recovery. Method specificity was confirmed by the absence of quantifiable peaks when injecting sample blanks and a positive detected peak response at the specific analyte retention times when injecting each individual standard. Carryover was evaluated by injecting two blanks immediately after the assay of a standard with the highest concentration in the calibration range (500 ppb). No quantifiable peaks were observed in the subsequently injected blank samples, indicating the absence of system carryover.

Calibration curves were generated from calibration standards from the low limit of quantitation (LLOQ) to 500 ppb. A quadratic fit was used for the experimental data and 1/x weighting was used to achieve better quantitation accuracy at lower levels. Excellent R2 values were observed, with 0.9994 calculated for 2-MI and 0.9995 for 4-MI. LLOQ was determined as the lowest concentration in the calibration standards, with observed Q-SRM signal-to-noise (S/N) greater than 10 and C-SRM S/N greater than three. In this study, the LLOQs were determined to be 5 ppb for both analytes.

To keep consumer exposure to 4-MI under limits in the California NSRL, only a small amount of soda drinks containing 4-MI can be consumed each day.

Precision and accuracy were evaluated by replicate assays of standards at 5 ppb and 200 ppb presented as %relative standard deviation (RSD) and %Accuracy (calculated as observed amount/specified amount × 100%). As shown in Table 2, excellent precision was observed for both analytes with %RSD less than 6% for the performed experiments; %Accuracy was observed from 93.8% (4-MI at 5 ppb) to 99.0% (4-MI at 200 ppb), indicating that accurate measurements could be achieved using this method. Recovery was evaluated by spiking both target analytes in three matrices. A blank matrix, consisting of a colorless lemon-lime flavored soda drink (Matrix A), which was assayed and showed no quantifiable target analytes, was spiked at two concentrations (20 and 200 ppb). A regular cola drink (Matrix B) and a zero-calorie cola drink (Matrix C) were each spiked with 100 ppb. Quantifiable 4-MI was detected in Matrix B and Matrix C, and the original observed 4-MI was subtracted from total observed amount for recovery calculation, i.e., %Recovery = (total observed amount – original observed amount)/spiked amount × 100%. The results are shown in Table 3. Observed recovery ranged from 79.4% (4-MI in Matrix B, 100 ppb) to 103% (4-MI in Matrix A, 20 ppb).

Table 3. Recovery of 2-MI and 4-MI in Three Matrices
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Table 3. Recovery of 2-MI and 4-MI in Three Matrices
Table 4. 4-MI in Tested Dark-Colored Carbonated Drinks
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Table 4. 4-MI in Tested Dark-Colored Carbonated Drinks

Analysis of Carbonated Drinks

As mentioned earlier, dark-colored carbonated drinks (n=10) were prepared following described procedures and analyzed for target analytes. A colorless lemon-lime flavored soda drink was also analyzed, for comparison, and neither of the target analytes was detected in the colorless sample.

No quantifiable 2-MI was detected in any of the tested samples, but 4-MI was observed in each of the tested dark-colored samples, with concentrations ranging from 280 ppb to 793 ppb in the original (undiluted) sample. Results are listed in Table 4. Daily limit of 4-MI intake (in mL) for each sample was calculated based on California OEHHA NSRL at 16 µg per person per day. It is obvious that, in order to maintain the exposure to 4-MI under the NSRL, only a small amount (less than 60 ­mL) of these 4-MI-containing soda drinks can be consumed each day.

A UHPLC-MS/MS method was developed and demonstrated successfully for the quantification of 2-MI and 4-MI in carbonated drinks. A colorless lemon-lime flavored carbonated drink was assayed for comparison and was found to contain no quantifiable target analytes. Among the tested dark-colored soda drinks, 4-MI was observed in each of them in the range of 280 to 793 ppb, and all samples appeared to be free of 2-MI. The detected concentrations of 4-MI were so high that consumption of a single serving would exceed the NRSL at 16 µg per person per day set up by California’s OEHHA, posing potentially adverse health effects to consumers.

Dr. William Schnute is the senior manager for the Mass Spectrometry Technical Center at Dionex Corp. in Sunnyvale, Calif.; Jinyuan (Leo) Wang is senior applications chemist for the center. Christopher Pohl is senior vice president and chief science and technology officer of Dionex’s Research and Development Division; Dr. Xiaodong Liu the manager for HPLC in the division.

To keep consumer exposure to 4-MI under limits in the California NSRL, only a small amount of soda drinks containing 4-MI can be consumed each day.

References

  1. National Toxicology Program (NTP). Toxicology and Carcinogenesis Studies of 2-Methylimidazole (CAS No. 693-98-1) in F344/N Rats and B6C3F1 Mice (Feed Studies). Research Triangle Park, N.C.: U.S. Dept. of Health and Human Services; 2004. NTP Technical Report 516, NIH Publication 05-4456.
  2. National Toxicology Program (NTP). Toxicology and Carcinogenesis Studies of 4-Methylimidazole (CAS No. 822-36-6) in F344/N Rats and B6C3F1 Mice (Feed Studies). Research Triangle Park, N.C.: U.S. Dept. of Health and Human Services; 2007. NTP Technical Report 535, NIH Publication 07-4471.
  3. Chan PC, Hills GD, Kissling GE, et al. Toxicity and carcinogenicity studies of 4-methylimidazole in F344/N rats and B6C3F1 mice. Arch. Toxicol. 2008;82(1):45-53.
  4. California Environmental Protection Agency. Office of Environmental Health Hazard Assessment. No significant risk level (NSRL) for the proposition 65 carcinogen 4-methylimidazole. Available online at: http://oehha.ca.gov/prop65/law/pdf_zip/010711NSRLrisk4EI.pdf. Accessed May 10, 2011.
  5. Fuchs G, Sundell S. Quantitative determination of 4-methylimidazole as 1-acetyl derivative in caramel color by gas-liquid chromatography. J Agric Food Chem. 1975;23(1):120-122.
  6. Wilks RA, Johnson MW, Shingler AJ. An improved method for the determination of 4-methylimidazole in caramel color. J Agric Food Chem. 1977;25(3):605-608.
  7. Moretten C, Crétier, G, Nigay H, et al. Quantification of 4-methylimidazole in class III and IV caramel colours: validation of a new method based on heart-cutting two-dimensional liquid chromatography (LC-LC). J Agric Food Chem. 2011;59(8):3544-3550.
  8. Klejdus B, Moravcová J, KubáňV. Reversed-phase high-performance liquid chromatographic/mass spectrometric method for separation of 4-methylimidazole and 2-acetyl-4-(1,2,3,4- tetrahydroxybutyl)imidazole at pg levels. Anal Chim Acta. 2003;477(1):49-58.
  9. Liu X, Pohl CA. HILIC behavior of a reversed-phase/cation-exchange/anion-exchange trimode column. J Sep Sci. 2010;33(6-7):779-786.

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