A thin-layer-chromatographic procedure for the separation of pethidine and its metabolites in urine [proceedings].

Pethidine, first used in 1940, has retained its position as the most widely used obstetrical analgesic (Burt, 1971 ; Jaffe & Martin, 1975; ODonoghue, 1971 ; O'Driscoll etal., 1973; Rosen, 1971). The metabolismof pethidine in the pregnant woman gives rise to norpethidine, pethidinic acid and norpethidinic acid, and conjugated pethidinic acid and norpethidinic acid respectively. A simple t.1.c. procedure for the separation of this drug and its metabolites in urine is described. The procedure should be of use in biochemical and pharmacokinetic studies on pethidine and its metabolites, which are becoming increasingly important in modern therapeutics. The experimental procedure is as follows: a 20ml sample of urine is adjusted to pH9.2 with SM-NaOH, and buffered with 2ml of satd. NaHCOJ (adjusted to pH9.2 with satd. Na2C03). The solution is poured on to an Amberlite XAD-2 ready-packed column (Drug-Skreen; Brinkmann Instruments, Westbury, NY 11 590, U.S.A.). Pethidine and its metabolites are retained on the column. These can be eluted completely with 1,2-dichloroethane/acetone/methanol (2:2:1, by vol.), after removing a filter plug which retains sediment. The eluate is dried by filtering through Whatman SP1 paper and rotary-evaporated to dryness under reduced pressure at 40°C. The residue is dissolved in l O m l of 0.1 M-HCI and the pH is adjusted to 9.2 with 1 M-NaOH; this solution is extracted twice with equal volumes of 1,2-dichloroethane. The pooled extract is concentrated almost to dryness, again under reduced pressure at 40°C, and transferred to a 5 m l tapered tube with three washings of acetone/methanol (1 :1, v/v). The volume of this solution is then decreased to approx. 5 0 ~ 1 under N2 at 40°C. The resultant extract, which contains the unconjugated compounds, is hereinafter denoted extract I. The aqueous phase remaining after the above extraction contains the conjugated metabolites. This phase is adjusted to pH4.5 with 0.1 M-HCI and buffered with 5ml of 0.5 M-sodium acetate (adjusted to pH4.5 with ~M-HC~) . Then 8-glucuronidase is added [from Helix pomatia; 5000units/ml of original urine; Sigma (London) Chemical Co., London S.W.6, U.K.]; this preparation is particularly suitable as it contains not only B-glucuronidase, which hydrolyses the glucuronides, but also arylsulphatase, which hydrolyses the sulphates. Propan-I ,2-diol(2ml) is added to enhance enzyme activity, and 0.2ml of chloroform is added as a preservative. The solution is incubated at 37°C for 48h. The solution is then adjusted to pH9.2 and treated as above for the unconjugated compounds. The resultant extract, which contains pethidinic acid and norpethidinic acid that were originally conjugated, is denoted extract 11. Pure reference compounds of pethidine, norpethidine, pethidinic acid and norpethidinic acid (supplied by courtesy of Dr. H. T. Openshaw and Dr. E. Frisch, Wellcome Research Laboratories) were added to urine in the range I-10,ug and processed in the same way to give the standard extracts. A control urine sample with no additions should also be processed to give control extracts I and 11 respectively. In clinical trials this control should be an extract from a urine specimen collected before drug administration. The drug and its metabolites in the various extracts are then separated by using the following t.1.c. procedure. Silica-gel G plates (20cmx 20cm; Merck, Darmstadt, Germany) are predeveloped with pursed methanol so that any possible interfering contaminants (which form a yellow band at the solvent front) can be removed with a spatula after drying. The plates are then divided into 1 cm lanes to permit concurrent