Question

1. As an NIH lab scientist you are required to determine benzodiazines and related metabolites in urine samples provide by the public health department monitoring the abuse of these drugs in teenage population. Describe the steps to your analysis using HPLC.

Answer 1

The wide variety of equipment (HPLC instruments), columns (stationary phases, which is heart of HPLC), eluent (mobile phases or solvents) and operational parameters (temperature, pressure, pH etc.) involved may make HPLC method development complex. The process is influenced by the nature of the analytes (in this case, benzodiazines and related metabolites in urine samples), and generally follows the following steps:

• Selection of the HPLC method and the initial system
• Selection of the initial conditions
• Selectivity optimization
• System optimization
• Method validation

Since benzodiazines are relatively non-polar, and its metabolites are relatively polar, in a reversed-phase chromatography (column is non-polar, and eluent is polar), metabolites have relatively less retention time in the column, and therefore, in the chromatograph, metabolites will appear left to benzodiazines.

The above is just a synopsis of an HPLC method development. Every step in this process is important, and exhaustive, which is beyond the scope here.