Analysis of Drugs in Urine

1. Sample Preparation: Most of the drugs gets metabolized to their respective glucuronide conjugates, and both of free drug and their metabolites will be excreted in the urine. So, it is necessary to deconjugate these metabolites to free base prior to extraction by hydrolyzing with the help of -glucoronidase enzyme (Ref 1). Enzymatic deconjugation procedure is as follows Mix 1 mL of urine, 1.0 mL of 1M KH2PO4 (PH 4) buffer and 100 L of glucoronidase enzyme. Vortex and incubate for atleast 2 hours or overnight at 37oC.

2. Solid Phase Extraction (SPE): Oasis MCX cartridges has to be placed on Rapid trace SPE workstation and conditioned initially with 2 mL of methanol, followed by 2 mL of water and phosphate buffer each at a rate of 12 mL/min. 2 mLSample supernatant are applied on to the column at a rate of 1.2 mL/min. Columns were subsequently washed with water (3 mL), 0.1 M Hydrochloric acid (3 mL), and finally with 5% methanol in water (2 mL) each at a rate of 9 mL/min. Acidic and neutral compounds have to be eluted with acetone/chloroform (4 mL) (1:1, 1.2 mL/min flow rate). Basic compounds have to be eluted with

ethylacetate/ammonium hydroxide (2 mL) (98:2, 1.2 mL/min flow rate) and Dichloromethane/Isopropanol/ammonium hydroxide (78:20:2) (2 mL) (98:2, 1.2 mL/min flow rate). The elution fractions have to be evaporated to dryness and to residue add 100 ul of BSTFA for derivatization and heat at 60oC for 30 minutes.

After derivatization evaporate the BSTFA under the stream of Nitrogen. And reconstituted in ethylacetate prior to GC injection (1 uL).