Instrument method development

UPLC- and UPC2-MS/MS

Multiple reaction monitoring (MRM) were set up by directly injecting 1 µL of the pure derivatized compound at a concentration of 1 ppm into a Waters Acquity UPC2 instrument coupled to a Waters Xevo TQ-S tandem mass spectrometer (MS/MS). Masslynx version 4.1 software was used for instrumental analysis. Derivatization was carried out according to Anari et al. (377). In short, the appropriate amount of each stock solution were dried with N2 and reconstituted 1:1 with 2 mg/mL DNCl in acetone and 0.1 M NaHCO3 (pH 10.5) in deionized water. The resulting solution was incubated for 10 min at 60 °C and cooled to room temperature before injection. Precursor scans were carried out between 15 and 55 V and the optimum cone voltage (CV) selected based on highest signal intensity. Using this CV, a product scan was performed between 15 and 55 V, selecting the two most prominent ions yielding the highest signal intensity at their respective collision energy (CE). The precursor CV and two product ions at their respective CE were used to construct a MRM table to monitor the different compounds. This MRM table was used for both UPC2-MS/MS analysis and UPLC-MS/MS analysis as both are connected to the same MS. Transitions for each compound yielding the strongest signal intensity was used as quantifiers, while the rest were regarded as qualifiers.

Chromatographic separation of compounds – from a 1µL injection, using the UPC2-MS/MS instrument was achieved on a Waters UPC2 BEH C18 2-EP 2.1 x 100 mm, 1.7 μm pore size column – maintained at 60°C. The mobile phase consisted of solvent A (A; super critical CO2) and solvent B (B; 1% formic acid in methanol) – supplied by a makeup pump at a rate of 0.2 ml/min. The column was equilibrated with 2% B before a linear gradient from 2 to 4.5% B, spanning 4.3 min, were applied. This gradient was

followed by a 2.5 min linear gradient to 7%B and a 1 min linear gradient to 10% B, where it was kept constant for another minute. Finally, a 30% B wash step was applied for 1 min, before the column was again equilibrated for 2 min with 2% B. In contrast, separation on the Waters Acquity UPLC instrument was achieved on a Waters UPLC BEH C18 2.1 x 100 mm, 1.7 μm pore size column – maintained at 50°C. The mobile phase consisted of A (1% formic acid in ultrapure water) and B (0.1% formic acid in acetonitrile) at a flow rate of 0.35 ml/min. Prior to a 1 µL injection, the column was equilibrated with 15% B. Following injection, a 0.1 min linear gradient, from 15% to 60% B, was applied. This was followed by a 6.8 min linear gradient from 60% to 95% B, before being kept constant for 1 min. Finally, the column was equilibrated for 2 min at 15% B.

GC-MS/MS

Like UPLC- and UPC2-MS/MS analysis, compounds and internal standards were derivatized. In short, the appropriate amount of a 10 ppm solution of each compound was dried in separate vials with N2. Compounds and internal standards were then reconstituted in a solution consisting of two thirds pyridine and one third BSTFA, before being incubated for 60 min at 60 °C. Samples were allowed to cool to room temperature before being injected on the GC-MS/MS instrument.

Method development on GC-MS/MS was carried out on a Thermo-Fisher Scientific Trace 1300 GC, fitted with a Varian FactorFour Capilliary VF-17ms (30 m x 0.25 mm ID, DF = 0.25) column, coupled to a Thermo-Fisher Scientific TQS 8000. The retention time for each compound were determined by injecting 1 μL of each compound or internal standard in a pulsed splitless w/Surge manner under the following inlet conditions: Helium was used as carrier gas at a flow rate of 1 mL/min. The inlet temperature was set to 250 °C with a split flow of 50 mL/min after 2 min. Following injection the column was kept constant at 100 °C for 3 min, ramped to 300 °C at 10 °C/min and kept constant at 300 °C for 5 min. The MS transfer line and ion source temperature were set to 280 °C and 250 °C, respectively, with MS in full-scan mode between the m/z values of 40 and 550. AutoSRM (XCalibur, version 2.2) software was used to find the two most prominent transitions of each compound at their optimal collision energy. An SRM table was constructed from this information and used for method optimization and sensitivity determination. Finally, the transitions of each compound yielding the strongest signal intensity was used as quantifiers while the rest were used as qualifiers.

3.4.4 Instrument method optimization

UPLC- and UPC2-MS/MS

A 100 ppb mixture consisting of all 12 compounds and the three internal standards were made from each compound‘s 10 ppm stock solution. Aliquots of the mixture was dried and reconstituted in the same volume as the aliquot in either 1:1 (acetone:0.1 M NaHCO3), 1:1 (acetonitrile:0.1 M NaHCO3), 3:1 (acetonitrile:0.1 M NaHCO3), 2:1:1 (acetone:acetonitrile:0.1 M NaHCO3) or 1:2:1 (acetone:acetonitrile:0.1 M NaHCO3), with the final concentration of DNCl equal to 1 mg/ml. These solutions were incubated at 60°C for 10 min, allowed to cool to room temperature and 1 µL injected on either the UPLC- or UPC2-MS/MS instruments. The best solvent composition were selected for further optimization steps based on improved peak areas, signal to noise (S/N) ratios and peak shape (b/a).

One microliter of the aforementioned was injected on the UPLC-MS/MS in triplicate, the eluent flow rate changed to either 0.3 or 0.4 ml/min and triplicate injections performed again. Peak area, peak shape and S/N were re-evaluated and the best condition selected for further optimization. Similarly, column temperature (40 or 60 °C), desolvation gas flow rate (800 or 100 L/h) and desolvation gas temperature (450 or 50 °C) were evaluated. Alike, eluent flow rate (1.7 or 1.9 mL/min), desolvation gas flow rate (700 or 900 L/h) and desolvation gas temperature (450 or 550 °C) was investigated on the UPC2-MS/MS after solvent optimization.

Results obtained from changing the different parameters were analysed with a one-way ANOVA followed by a Dunnet multiple comparisons test from the statistical package GraphPad Prism 5.00.

After the optimization of the UPC2-MS/MS method, column failure forced an investigation into an alternative column available: a Waters Acquity UPC2 Torus 2-PIC (3.0 x 100 mm, 1.7 µm pore size). Consequently, retention times for each compound and internal standard were determined as described in section 3.4.3 and incorporated into UPC2-MS/MS method. No changes were made to the gradient used in section 3.4.3 or the optimal settings found in this section.

GC-MS/MS

Similar to UPLC- and UPC2-MS/MS analysis, a 100 ppb solution of the 12 compounds and three internal standards were made and aliquots reconstituted in the same volume of either 2:1 (pyridine:BSTFA), 2:1 (Acetonitrile:BSTFA), 1:1:1 (pyridine:acetonitrile:BSTFA)

or 1:4:2.5 (Pyridine:acetonitrile:BSTFA). These samples were then incubated at 60 °C for 60 min and allowed to reach room temperature before injection. In contrast to UPLC- and UPC2-MS/MS method optimization however, two variables were investigated at the same time. First, solvent composition together with injection mode (Pulsed splitless and splitless). Second, Q1 resolution (Wide or Normal) together with emission current (50 or 75 A). Peak area, shape and S/N were evaluated in both cases and the optimal conditions selected for method sensitivity determination.