Jordan L. Cohenl and Roberf 1. Koda School of Pharmacy University of Southern California Los Angeles, 90007
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Gas Chromatographic Determination - of Glutethimide in Biological Fluids An analytical chemistry experiment
One of the most significant areas of application of analytical chemistry today is the development of analytical methods for drues or metabolites in bioloeical fluids:. i.e.. . blood, urine, e t c B o t h the correlation of d ~ activity g and toxicitv with dose and the eventual clinical evaluation of drugs from pharmacokinetic data require prior development of extremely sensitive assays specific for the parent drug molecule and/or its metabolites. Because of the extreme sensitivitv. often reauirina a determination of nanograms of compound per milliliter of plasma, and the specificity required in a biologic system due to large numbers of interferences, we strongly feel that courses in organic analysis and instrumentation should utilize these kinds of problems as required laboratory experiences. The difficulty and time involved in the experiment could also be modified in order to meet the needs of students in undergraduate analytical courses. The approaches in solving problems of this type would prove valuable to analysts in general and essential to students with research interests in pharmacology, biopharmaceutics, and clinical pbarmacology. The application of instrumental methods of analysis to significant problems would also satisfy the students' concern for relevant laboratory experiences in courses of this type. This paper describes the development of a gas-liquid chromatographic method of analysis for glutethimide, a widely used, and abused, sedative-hypnotic, in blood or urine. The assay as developed was used as an elective project in a n advanced analysis course and was modified for use in an undergraduate course in pharmaceutical analysis. Experimental Apparatus Varian Model 1440 Gas Chromatograph equipped with a flame ionization detector. Ten microliter Hamilton Syringe. Reagents All chemicals and solvents were of analytical reagent grade. Water was glass double-distilled. Internal Standard Solution: p-dimethylaminobenzaldehyde in dichlammethane, 100rg/ml. 0.001NNaOH. 0.WlNHCI Procedure Procedure for Biological Samples. Venous blood samples were taken from an in-dwelling intravenous catheter, collected in Citrated Vacutainers (Beeton, Dickinson and Company) to prevent coagulation, and centrifuged to allow colledion of the plasma. Plasma was stored at 4'C until ready for analysis. Urine samples were adjusted to pH 7 and either directly frozen or lyophilized and stared in a similar fashion. One milliliter of plasma was pipetted into a screw-cap tuhe and extracted with 5.0 ml of dichloromethane for 30 min using an aliquot mixer to avoid emulsification. The layers were separated by
centrifugation and 4 0 ml of the organic layer was pipetted intn a secund tube while the plasma layer nas drsrarded. The dirhlommethane laver was washed bv extracting with 1.0 ml of 0.001 N NaOH for -15 min, aspirating off and-discarding the aqueous layer, and then re-extracting the organic laver with 4.0 ml of 0.001 N HCI for 15 min. Two milliliters of the washed dichlommethane layer was pipetted into a centrifuge tuhe, 0.1 ml of the internal standard stock solution in dichloromethane was added and the solvent was removed under a gentle stream of dry nitmgen in the hood. The residue was redissolved in 10-15 drops of dichloromethane and 1-2 ul was iniected into the -gas ehmmatograph. Gas Chromatography. A glass coiled column, 6 ft by 0.25 in. 0.d. was packed with 3% 0V-225 on Chromosorh W (Applied Science Laboratories), conditioned at 250°C for 72 hr and silylated with periodic injections of Silyl 8 (Pierce Chemical Co.). Nitrogen, as a carrier gas, was maintained at 40 ml/min, while the flow rates for air and hydrogen were 250 ml/min and 30 ml/min, respectively. The detector and injector were both maintained at 250T throughout. The dichloromethane sample was injected at a column oven temperature of 200T and the glutethimide eluted as a symmetrical peak after 5 min while the p-dimethylaminebenzaldehyde appeared as a sharp peak after 2 min. Generally the column operated tmuhle-free. After heavy use, the column was reconditioned at 250°C overnight and silylated with Silyl 8 to remove non-volatile materials present in the biological mixture which accumulated after repeated injections. Prepomtion of a Standard Curve. Standard solutions were prepared to contain fmm 25 to 200 *g/ml of glutethimide in dichlommethane. An appropriate volume of the stock internal standard solution was added so each solution also contained 100 pgjml of p-dimethylaminohenzaldehyde.One microliter of each sol~tion was injected into the gas chromatograph and an average standard curve of peak-height ratios (glutethimide: p-dimethylaminohenzaldehyde) versus ng of glutethimide injected was constructed (Table 1). A linear relationship was found over the concentration range investigated (Figure 1). Calculations The peak heights of the two peaks in each sample were measured, the peak-height ratios calculated and the amount of glutethimide injected determined directly from the ~reviouslydetermined standard curve. The amount of glut&imide-injected can then be related to the original drug concentration in the sample as follows Nanograms of drug injected in sample
X
5 x (1/201 = Micrograms of drug
where the nanograms of drug is determined from the standard curve, the factor of five results from the dilution upon extraction and the factor of 1/20 results from the difference in concentration of the internal standard in soTable l. Standard Curve Data tor Glutethirnide
1 Present address: Virginia Commonwealth University School of Pharmacv. MCV Station. Box 666.Richmond. Virginia 23298. To whom al
