System for benzodiazepine detection

A method for analyzing a biological sample for the presence of benzodiazepines, said method comprising:

• (a) passing said sample through the following media in the sequence indicated:
• (i) a contact medium comprised of .beta.-D-glucuronide glucuronosohydrolase immobilized on an agarose gel, to cleave glucuronic acid from any species present in said sample which is conjugated with said glucuronic acid;
• (ii) a sulfonated styrene gel, under conditions permitting retention thereby of sample components comprised of any benzodiazepines present in said sample to the exclusion of a preliminary group of interfering species comprised of substantially all inorganic salts, proteins, peptides and hydrophilic compounds in said sample, thereby eliminating said interfering species from said sample prior to passage of said sample through said first separation medium;
• (iii) a styrene-divinylbenzene resin with tetramethylammonium functional sites, under conditions permitting retention of hydrophobic anions, with substantially no retention of benzodiazepines;
• (iv) a derivatized silica bearing propyl-sulfonic acid functional sites, under conditions permitting retention of basic compounds of stronger basicity than benzodiazepines, with substantially no retention of benzodiazepines;
• and
• (v) a reverse-phase medium comprised of dimethyloctadecylsilane bonded to silica, under conditions permitting the chromatographic separation of benzodiazepines;
• steps (a)(iii), (a)(iv) and (a)(v) being performed by way of a carrier liquid comprised of an aqueous solution of a member selected from the group consisting of methanol, ethanol and isopropanol, at a concentration of from about 30% to about 50% by weight and at an approximately neutral pH;
• and
• (b) detecting said benzodiazepines individually their emergence from said third separation medium.