Both methods were rapid with all compounds eluting in under 4.5 minutes. These separations were also highly efficient. Average peak widths for ACN and MeOH were 2.41 and 3.00 seconds, respectively. In each case, baseline resolution was achieved for all isobaric compounds, such as morphine and hydromorphone, as well as structural isomers with the same molecular mass, such as methamphetamine and phentermine. Differential selectivity can be seen for several compounds. For example, benzoylecgonine (compound 22) demonstrates a significant shift to a later retention time relative to other compounds when methanol is used vs. acetonitrile (see Figures 1A and 1B). A selectivity difference can also be seen with methamphetamine and phentermine, which have the same molecular mass and share an MRM transition. While baseline separated using acetonitrile (1.47 vs 1.52 min.), the use of methanol significantly improves their separation from 3.0 to 9.6 seconds, which translates to a resolution increase from 1.88 to 4.85, a significant enhancement for two compounds than can interfere with each other. The benzodiazepines also demonstrate selectivity differences between the two mobile phases. Flunitrazepam and temazepam coelute with ACN but are baseline separated when using MeOH. A similar change is seen with oxazepam and nitrazepam, whose retention time difference changes from .02 minutes to .08 minutes.
