492 Analyst, May, 1978, Vol. 103, p p . 492-496 Determination of Probenecid in Serum by H ig h-performance Liquid Chromatography R. K. Harle and T. Cowen International Development Laboratories, E. R. Squibb wad Sons Limited, Moreton, Merseyside, L46 1Q W The determination of probenecid in serum samples by using high-performance liquid chromatography is described. The method gives satisfactory results over the normal therapeutic range, namely up to 150 pg ml-1 of probenecid in serum, and is not affected by metabolites of the drug. The method does not require derivatisation of the drug, as in gas - liquid chromatographic procedures, and is less subject to interferences than spectrophotometric procedures. It has been used in analysis of several hundred serum samples and has given a satisfactory performance in respect of precision and accuracy.Keywords : Probenecid determination ; serum ; high-performance liquid chromatography Probenecid [4-(dipropylamino)sulphonylbenzoic acid] is a uricosuric agent that has recently been used in conjunction with penicillin derivatives in the treatment of various diseases. Probenecid reduces the excretion of penicillins by the kidneys and thus enables therapeutic levels of the penicillins to be achieved with a lower initial dose. The normal dose of pro- benecid used to obtain this effect is 1 g daily. Several methods have been described for the determination of probenecid in body fluids,l-b mainly employing spectrophotometric or gas - liquid chromatographic methods for the final quantification step.The disadvantages of these methods are that the spectrophotometric methods are not specific and the gas - liquid chiromatographic methods require derivatisation of the sample before the injection on to the column. High-performance liquid chromatography was investigated as an alternative method. The extraction of probenecid from serum , followed by injection of the non-derivatised extract into the chromatographic column, should give advantages of specificity and time compared with the other methods. Experimental Reagents All reagents were of analytical-reagent grade unless otherwise stated. Potassium chloride. Citric acid. Disodium hyd~ogen orthophosphate. Sodium hydroxide. Potassium dihydrogen orthophosfihnte. Diethyl etlzcr. Acetonitrile. Reagent grade. Bufler, pH 4.0.Dissolve 35.5 g of disodium ;hydrogen orthophosphate in 250 ml of water, adjust the pH to 4.0 with approximately 40 g of citric acid, then dilute to 500 ml with water. Bufer, PH 6.0. Dissolve 27.2 g of potassium dihydrogen orthophosphate in 500 ml of water, adjust the pH to 6.0 with sodium hydroxide solution and dilute to 1 1 with water. Mobile phase. Dilute 50 ml of pH 6.0 buffer to 700 ml with water, add 300 ml of aceto- nitrile, mix and de-gas by applying a vacuum immediately before use. Apparatus The high-performance liquid chromatograph. was assembled using the following corn- poneii ts : a constant-volume , syringe type, high-pressure pump (Metering Pumps Ltd., London ; 3 000 lb in-2), a high-pressure septurn injector head (Perkin-Elmer, Part No.0087-3015) and a suitable Bourdon-type pressure gauge. The column used, 25 cm x 4 mm i.d., was made of stainless steel, and was fitted with a stainless-steel sinter at the outlet and a 2 mm thick PTFE sinter at the inlet. InjectionsHARLE AND COWEN 493 were made into this sinter. The packing material was Partisil, particle diameter 10pm, modified by treatment with octadecyltrichlorosilane to give a reversed-phase type of chromatography. The detector used was a variable-wavelength monitor (Model 212, Cecil Instruments Ltd., Cambridge) equipped with an 8-p1 flow-through cell. The detector was operated at 252.5 nm and the signal was recorded on a flat-bed recorder (W & W, Model 1100). Injections were made using a 10-pl high-pressure syringe (SGE, Type 10BLR).The operating conditions for this system were as follows: flow-rate, 1 ml min-l (about 50 bar) ; detector range, 0.05 A full-scale deflection (f.s.d.) ; and recorder range, 10 mV f.s.d. The apparatus used in the extraction procedure was normal laboratory glassware, together with a centrifuge and a vortex mixer Procedure Preparation of standard Weigh accurately about 30 mg of probenecid, dissolve it in methanol and make the volume up to 100.0ml. This is the stock solution. Dilute a 5.0-ml aliquot of stock solution to 50.0 ml with methanol to give a 30 pg ml-l working standard solution. Pipette an appropriate volume (usually 2.0 ml) of working standard solution into a 12-ml stoppered centrifuge tube and remove the methanol by blowing with air. Add 1.0ml of distilled water to the residue and swirl in a vortex mixer to dissolve the residue.Process this standard solution as for the serum samples. The concentration of this solution will be 60 pg ml-l when 2.0 ml of working standard solution are used in its preparation. Pre$aration qf samples Pipette a 1.0-ml aliquot of serum sample into a 12-ml stoppered centrifuge tube and to it add 1.0 ml of pH 4.0 buffer and approximately 1 g of potassium chloride. Stopper the tube and vortex mix for about 15 s, then add 8.0 ml of diethyl ether from a dispensing pipette. Stopper the tube, vortex mix thoroughly for 1 min and centrifuge a t 4000 rev min-1 for 5 min. Transfer a 5.0-ml portion of the ether layer into a second tube, add an anti-bumping granule and remove the ether by evaporation on a water-bath at 40-50 “C.Final traces of ether should be removed by gentle purging of the tube with a stream of air or nitrogen. Add 1.0ml of chromatographic mobile phase to the residue and mix briefly on a vortex mixer. Chromatogra$hic fvocedztre Inject 8-pl portions of the standard solution extract until reproducible peak heights are obtained, then inject 8-p1 portions of sample extract. Inject a further portion of the standard extract after every six sample injections. Rinse the syringe at least five times with each solution prior to injection of that sample. This ensures a negligible carry-over of solutions between injections. Establish a stable solvent flow and base-line conditions. Calculation Measure the probenecid peak heights of the standard extract and of the satiple extract.Take a mean value of the peak heights for the two standard injections on eitl er side of a sample. Then, sample peak height x standard concentration standard peak height Probenecid (pg ml-l) = A typical chromatogram is shown in Fig. 1. Investigation of Experimental Variables Extraction fyom serum When extracting probenecid from serum it was found that coagulated blood proteins formed a semi-solid “plug” a t the aqueous surface. The solvent used in the extraction should494 HARLE AND COWEN : DETERMINATION OF PROBENECID IN Analyst, VoZ. 103 Fig. 1. Chromatograms of serum containing probenecid. (l), Serum blank; (2), a 4-h serum sample containing 75 pg ml-1 of probenecid; and (3), a 12-h :serum sample containing 28 p g ml-l of probenecid.The probenecid peaks are marked P. therefore, for convenience of recovery, have a density lower than that of the aqueous phase, be readily removable by evaporation and be immiscible with water. Solvents evaluated included ethyl acetate, butyl acetate and a 2: 1 mixture of l-chlorobutane with chloroform. It was found that some of these solvents did not extract probenecid completely and others were difficult to evaporate or were too miscible with the aqueous phase. Diethyl ether was finally chosen, but it was found that the reduction in volume of the solvent due to loss into the aqueous phase (diethyl ether has a so1ubi:lity in water of 6.9% at 20 "C) resulted in a bias leading to high recoveries of 103-107% when assayed against external standards. The adoption of a standard that was passed through the extraction procedure obviated the errors arising from this effect.Linearity of response Linearity of the response was established by injection of standard solutions containing from 10 to 100 pg ml-l of probenecid. The mean peak heights of three injections of each of these solutions were plotted against the concentration of the solution injected. The linear response graph was found to pass through the origin, with a slope of 2.5 mm per pg ml-1. The level of 100 pg ml-l here corresponded to 160 pg ml-l in serum because only a 5-ml volume of the 8 ml of diethyl ether extract was removed. The sensitivity of the procedure, taken as being that of a peak of height equal: to twice the base-line noise, was generally 1 pg ml-1, depending on the instrumental conditions.In order to ensure a constant absorptivity for probenecid at the detector, it was necessary to buffer the mobile phase to pH 6, thus ensuring that the same ionic form of probenecid was measured in each determina- tion. Possible interferences Pro benecid metabolites. The chromatographic behaviour of probenecid metabolites was investigated, and the results are shown in Table I. The probenecid peak was well separated from all of the metabolite peaks and hence no interference could occur. The co-administrattion of probenecid and penicillins is now relatively common so that the method should ideally be unaffected by large doses (about Co-administered epicillin.May, 1978 SERUM BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 495 TABLE I RETENTION DATA FOR PROBENECID METABOLITES Column and mobile phase as in text.Flow-rate, 1 ml min-1. Metabolite* Retention timelmin 1.67 /pr R-N ‘CH,CH,COOH R-NH, 2.00 2.20 /pr R-N ‘CH2CH,CH,0H 2.25 R-NH-‘ ?r 2.30 R-NPr, (probenecid) 3.50 * R = -SO,-Ph-COOH; Ph = C,H,; Pr = n-C,H,. 4 g) of penicillins. This was :ested in vivo by obtaining serum samples from a subject who had taken 4 g of epicillin (cc-: .mino-3,6-dihydrobenzylyenicillin). Samples were taken a t 0, 0.5, 1, 2, 3, 4, 5, 6, 9, 12, 24 and 30 h after the epicillin had been administered, extracted by using the above method and the chromatograms were examined for possible interferences. No peaks other than those d1.e to the solvent were found in samples taken up to 2 h after administration.In the 3-30-2 samples a small peak was eluted at 3.2 min (probenecid was eluted at 3.4 min). The maximum height of this peak was 2.5 mm, which occurred a t the 4-h sample, this height being aoproximately the same as that of a peak representing 1 pg ml-l of probenecid. This small pe;k was of short duration and in all instances was completely eluted by the start of the prcbenecid peak. No positive interference could thus occur. This smal peak does not correspnd to epicillin, which is not extracted from serum by this method, kut may be due to a met.1.bolite of epicillin. Some other drugs that might be found with probenecid in serum were examined by using the chromatogr; phic system described. The retention times for these drugs are given in Table 11. Other d~ugs. TABLE I1 RETENTION TIMES FOR SOME SELECTED DRUGS Column and mobile phase as in text.Flow-rate, 1.2 ml min-1. Compound Aspirin .. .. Ampicillin . . .. Epicillin . . .. Potassium penicillin G Probenecid . . .. Phenazone . . .. Niflumic acid .. Flufenamic acid . . .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. Retention timelmin 1.6 1.95 2.00 2.05 2.9 4.0 5.8-8 (tailing) 10.5-1 5 (tailing) Results and Discussion This assly method was originally developed in order to give comparative data in clinical496 HARLE AND COWEN trial studies in which probenecid was administered in different dosage forms, alone and in conjunction with epicillin. Recovery experiments were therefore carried out at probenecid concentrations that approximated to the levels expected over the initial, clinically signifi- cant, part of the trial, i e ., from 0 to 12 h. 'The recovery and precision results are given in Table 111. TAB:LE I11 RECOVERY AND PRECISION RESULTS FOR PROBENECID I N WATER AND I N SERUM Probenecid Mean Coefficient added/ recovery, of variation, Number of Matrix pg ml-l % % determinations Water . . .. 30 97.8, 98.7 * 2 60 96.8, 98.2 * 2 90 99.3, 101.1 2 * Serum . . .. 5 96.2 3.6 12 30 95.3 1 .o 8 60 97.7 1.1 6 100 100..4 0.9 10 * Both values determined are given. The coefficients of variation of probenecid peak heights from standard injections over 3-6-h periods were 1.8% (6 values), 2.4% (6 values), 2.0% (5 values) and 2.4% (11 values) on four separate days. These results indica.te that the chromatographic conditions are relatively stable, as these values represent the cumulative error due to contributions from syringe inaccuracies and to variation in inject ion technique, eluent flow-rate, detector lamp stability and peak measurement. Between 35 and 50 samples can be assayed in an 8-h period. Over 400 samples, from several subjects in a clinical trial, have been assayed for probenecid. Samples of probenecid metabolites were kindly donated by Dr. W. D. Conway of the State University of New York at Buffalo, N.Y., USA. References 1. 2. 3. 4. 5. Tillson, E. K., Pusey, N. W., and Beyer, K. H., J . Pharmac. Exp. They., 1954, 112, 252. Sabih, K., Klaassen, C. D., and Sabih, K., J. Pharm. Sci., 1971, 60, 745. Dayton, P. G., and Perel, J. M., Ann. N.Y. A w d . Sci., 1971, 179, 399. Zacchei, A. G., and Weidner, L., J . Pharm. Sci., 1973, 62, 1972. Conway, W. D., and Melethil, S., J . Chronzat., 1975, 115, 222. Received A p i l 12th, 1976 Amended January 31st, 1977 Accepted December 6th, 1977