Micro-Determination of Anthranilic Acid Derivatives of Anti-Inflammatory Drugs by High Performance Liquid Chromatography and Its Application to Forensic Chemistry

A sensitive method for the determination of four anthranilic acid derivatives (diclofenac sodium, aluminium flufenamate, mefenamic acid and tolfenamic acid) of analgesic and antiinflammatory drugs was developed by using high performance liquid chromatography (HPLC). It was found that the four drugs were converted into methylphthalimide (MPI) derivatives in a constant yield by reaction with N-chloromethylphthalimide at 60°C for 30 min. The productions of the MPI derivatives were confirmed by mass spectrometry. The MPI derivatives of the four drugs tested showed satisfactory results of separation and sensitivity by HPLC using C18 bonded phase with acetonitrile : H2O (80 : 20, v/v) as mobile phase at 282nm. The calibration curves of the MPI derivatives of the four drugs were linear from 1.0 to 5.0μg/ml. The detection limits of the four drugs were 0.5-5ng. The extraction procedure of the four anthranilic acid derivatives added in the plasma and urine was performed by using Extrelut[○!R] 1 column. Yields of column extraction of 100μl of plasma and urine samples (containing 0.5μg of anthranilic acid derivatives) with 6ml of ethyl acetate were 84-106%. The present procedure may be an easier and more convenient method for the analysis of anthranilic acid derivatives of analgesic and anti-inflammatory drugs and could be applicable for the determinations of these drugs in forensic samples.