The HPLC system was a Perkin-Elmer Series 200 autosampler and micro pumps. A Keystone Scientific Inertsil ODS2 column (150 X 4.6 mm) was used with a water-methanol gradient increasing from 80% methanol to 90% methanol over 5 minutes at a flow rate of 1.0 mL/min. Both standards and samples were prepared in methanol. Injection volumes were 20 μL. The MRM optimization program of the Analyst software was utilized to automatically determine optimal product ions, as well as correct settings for lens voltages and collision energies for each MRM transition. These values are shown in Table 1.
Figure 1 shows the XIC of the 1 ppb nitrosamine standard mixture. The signal to noise (S/N) ratio varies according to the analyte. The NDMA had the lowest S/N with a value of 3.0. The highest S/N was obtained by the NMEA with a value of 37.8. Other S/N values observed were NPYR (6.0), NDEA (8.6), NPIP (13.8), and NMOR (13.6). The S/N calculation within the Analyst software requires two regions to be selected in the chromatogram; a region containing the noise and a region containing the peak of interest. The algorithm first subtracts the average backgroundsignal intensity from the background and then divides the subtracted signal by 3 times the standard deviation of the noise region.
signal intensity from the background and then divides the subtracted signal by 3 times the standard deviation of the noise region. Standards containing 1, 5, 10, 50, 100, 500, 1000, and 2000 ppb (ng/mL) of the ni