11


Table 1: HPLC and Mass Spectrometer conditions used for analysis of bisphenols using Multiple Reaction Monitoring (MRM).


HPLC-MS/MS Method: HPLC: ACQUITY Premier UPLC


Solvent A: H2 O Solvent B: MeOH + 0.01% ammonia Gradient


Time (min) A (%) 0.00 0.25 0.75 3.50 3.75 3.76 4.75 4.76 5.75


94.0 94.0 65.0 30.0 30.0 5.0 5.0


94.0 94.0


Flow Rate 400 µL/min Seal Wash On (5 min) Sample Temperature 10°C


MRM Transitions: Analyte


B (%) 6.0 6.0


35.0 70.0 70.0 95.0 95.0 6.0 6.0


To validate recovery, three concentrations of the bisphenols were used – 10 ng/mL (low), 40 ng/mL (medium) and 80 ng/mL (high). These three concentrations validated recovery across the calibration range - near the LOQ, the midpoint of the calibration curve and the upper part of the calibration curve.


Mass Spectrometer: Xevo TQ-S Micro Needle Wash MeOH (3 sec)


Syringe Draw Rate 30 µL/min Needle Placement 4mm Injection Volume 2 µL


Column Waters ACQUITY UPLC-BEH C18 (2.1 x 50, 1.7 µm)


Column Temperature 40°C Source Type ESI Negative Capillary Voltage 2500 V Desolvation Temperature 350°C Source Temperature 150°C Desolvation Gas Flow 1000 L/hr Cone Gas Flow 0 L/hr Detector Gain 1.00 Minimum Points Per Peak 15


Figure 2: Chromatogram of the lowest calibration standard for each bisphenol. Peaks: (1) bisphenol S (0.5 ng/mL), (2) bisphenol F, (3) bisphenol E, (4) bisphenol A, (5) bisphenol AP, and (6) bisphenol Z (all 1.5 ng/mL).


Testing commercial e-liquids


The commercially available e-liquids were diluted and tested using the same SPE method that was validated with the blank matrix. Calibration standards were prepared as matrix matched standards, using post-spiked 70% VG 30% PG solutions.


Results and Discussion Table 2 shows the results of the validation of the method for all six of the bisphenols.


Polarity Bisphenol A Negative


Precursor ion (m/z)


226.82


Product ion (m/z)


211.94 (Quan)


132.99 Bisphenol AP Negative 288.84


273.97 (Quan)


210.97 Bisphenol E Negative 212.75


197.92 (Quan)


118.92 Bisphenol F Negative 198.72


92.91 (Quan)


104.91 Bisphenol S Positive 250.79


92.94 (Quan)


156.91 Bisphenol Z Negative Method validation


To validate the method for suitability in processing e-liquids with SPE, a blank matrix of 70% VG 30% PG (v/v) was processed through the SPE plate (following the previously outlined method), then spiked at concentrations of 0.1, 1.5, 5, 10, 100, 250 and 500 ng/mL. This was then compared to standards prepared in ultrapure water, spiked to the same concentrations, to test for matrix effects and linearity.


Alongside this, ten replicates of the blank solution were injected to confi rm the limit of detection (LOD) and limit of quantifi cation (LOQ) of each bisphenol compound. This was calculated using equation 1.


Equation 2: Calculation of MDL or MQL (Mₓ), where Lₓ is the LOD (for MDL) or LOQ (for MQL), recovery is the value at 10 ng/mL (as a decimal), ME is the matrix effect (as a decimal), and the concentration factor is 5


Matrix effect was classed as signifi cant if it was >±20%, this was only the case for bisphenol AP and Z. Therefore, to correct for this matrix effect, matrix match calibration was used for the recovery and repeatability validation and sample testing.


Equation 1: Calculation of LOD or LOQ (Lx), where meanblank is the average of 10 blank replicates, � is the standard deviation of the blanks, slope is the gradient of the calibration curve and k is a constant of 3.3 (LOD) or 10 (LOQ).


Recovery and repeatability validation results for the three concentrations (10, 40 and 80 ng/mL, n=8) are in table 3. All compounds met recovery criteria of 80 – 120% at all three concentrations. All repeatability values were <20%RSD at the lowest concentration and <15%RSD at the other two concentrations, ranging from 1.5 – 18.6%RSD. This showed that the SPE method was repeatable at the three different concentrations.


266.85


173.03 (Quan)


144.96 50 55 38 33 38 Cone voltage (V) 43


Collision energy (V)


17 24 20 24 17 26 Bisphenol F 1.5 – 500 0.9900 0.42 0.87 0.10 0.20 20 18 24 15 26 34


Validation of the method showed that all six compounds were linear. Bisphenol AP and Z had R2


values slightly below 0.99, however the residuals at each calibration level


were within ±20%. LOQ values ranged from 0.05 ng/mL (bisphenol S) to 2.10 ng/mL (bisphenol A). The method detection limit (MDL) and method quantifi cation limit (MQL) were calculated using recovery at a low concentration (10 ng/mL results in table 3), matrix effect and concentration factor (equation 2) to give the true limits of detection and quantifi cation in a sample processed via the SPE method.


Bisphenol S 0.5 - 500 0.9950 0.02 0.05 0.0055 0.012 Bisphenol Z 1.5 – 500 0.9860 0.47 1.01 0.22 0.47 -16.6 -42.3 -6.6 Bisphenol A 1.5 – 500 0.9906 1.20 2.10 0.34 0.60 Bisphenol AP 1.5 – 500 0.9866 0.50 1.23 0.28 0.70 Bisphenol E 1.5 – 500 0.9933 0.23 0.54 0.06 0.14 -13.0 -59.0 -10.0


Table 2: Validation results for six bisphenols in 70% VG / 30% PG using SPE–LC–MS/MS. R² = coeffi cient of determination, MDL = method detection limit, MQL = method quantifi cation limit (calculated using Equation 2), ME = matrix effect


Analyte Linearity R2 LOD (ng/mL) LOQ (ng/mL) MDL (ng/mL) MQL (ng/mL)


ME (%)


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