Chiral Separation on a C18 Column? Separation of d- and l- Amphetamines Part I

Did you know that chiral chemistry was discovered by Louis Pasteur, a French chemist and biologist in 1848? However, it took about a century to find that chirality plays a key role not only in the life of plants and animals, but also in several aspects of drug design, and both pharmaceutical and illicit drug development.

As a chemist or a forensic toxicologist, you could be using chiral analysis in biological samples or some street samples to determine legal or illicit drug consumption or to identify illicit drug manufacturing locations. Whatever your field, chiral separation of drug enantiomers is essential in order to show that the active enantiomer is, in fact, present in your specimens. In the past, different techniques like chiral selector in mobile phases, GC-MS and LC-MS chiral columns were used for the chiral separation. However, most LC studies use an expensive chiral column in combination with cyclodextrin additives that can cause ion suppression and contamination in electrospray ionization, a major drawback of chiral stationary phases along with the high cost. Toxicology or forensic labs with strict budgets may not be able to adopt a method with such an expensive component.

So, do you have a C18 column in your lab? If yes, then you are all set!! A cost effective chiral separation can be achieved with a C18 column by adapting a simple pre-column derivatization technique without the need for a costly and specialized chiral column. We evaluated this achiral technique in the tech note “Analysis of Amphetamines by LC-MS/MS for High-Throughput Urine Drug Testing Labs”. Methamphetamine is an old drug with a rich history. Amphetamine and methamphetamine are psychostimulant drugs that occur as two enantiomers, dextrorotary and levorotary, as a result of their chiral center (Figure 1). The dextromethamphetamine (d-isomer) form is highly abused and typically found in illicit preparations. However, detection of abuse is complicated because consumption of over-the-counter and prescription medications containing l-isomer may yield positive results if the analytical method used cannot distinguish between the d- and l- enantiomers.

Figure 1: Structures of d- and l-Amphetamine and Methamphetamine Enantiomers.

The aforementioned enantioselective LC-ESI-MS/MS technique separated the d- and l enantiomers of methamphetamine and its metabolite, amphetamine in human urine, after pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanineamide (Marfey's reagent) using a Raptor C18 column. Marfey’s reagent is an effective derivatizing reagent for separation of d- and l- amphetamine and methamphetamine isomers by converting them to diastereomers. This method is compatible with any LC-MS/MS instruments. Accurate and reproducible analysis was achieved in 7 minutes of chromatographic analysis time, making the column, sample preparation and chromatographic method well suited for selective, low-cost, high-throughput analysis and improved methamphetamine result interpretation.

In part 2, I will discuss more about the sample preparation, derivatization and chromatographic conditions…