Reversed-phase liquid chromatography for separation of illicit drugs utilizing water-rich mobile phases

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely accepted method in drug and toxicological analyses to detect and quantify drugs and metabolites. However, current RP-HPLC methods use mobile phases that contain significant amounts of organic modifiers such as acetonitrile. Although these organic modifiers are effective, they have been linked to negative environmental effects. The focus of this research is to develop a water-rich mobile phase system composed of >90% water and a smaller amount of a nonpolar organic modifier. Long-chain alcohols such as 1-butanol have proved to be suitable organic modifiers for water-rich RP-HPLC analysis. The use of 1-butanol as an organic modifier is studied in the current research to analyze drugs belonging to amphetamine-related drug classes. A series of differing gradients and elution tests were run to observe which method produced the best separation and resolution among the compounds. The preliminary samples include quinine caffeine, vanillin, and acetylsalicylic acid. An isocratic elution using mobile phases containing 0.1% formic acid and butanol in water was able to separate the preliminary sample. The retention times are 1.775, 4.96, 9.769, and 13.373, respectively; additionally, the resolution values are 1.13, 2.53, 2.72, and 1.86. The illicit substances include amphetamine, methamphetamine, and MDEA. An isocratic elution using mobile phases containing a phosphate buffer, pH-adjusted to 7.0 with a 3.5% butanol in water has produced the efficient peak resolution in an illicit drug sample. The retention times are 7.208, 7.932, and 10.053, respectively. A Waters: Atlantis T3 column helped to achieve the separation but could not fully prevent peak tailing. The peak tailing was caused by silanol effects that occur in the stationary phase, but efforts have been made to correct those effects. However, with the minimal peak tailing, the peaks still achieved a resolution of 1.52, 1.74, and 4.50 which meets the HPLC drug analysis requirement. Overall, this method was able to utilize a water-rich mobile phase with a small amount of organic modifier to separate the amphetamine-related compounds in a mixed sample