Soda Showdown

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.

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.

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.

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).