October, 1980 SHORT PAPERS 993 Determination of the Anthelmintic Levamisole in Plasma and Gastro-intestinal Fluids by H ig h-perf ormance Liquid Chromatography S. Marriner, E. A. Galbraith and J. A. Bogan Department of Veterinary Glasgow, G61 IQH Pharmacology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Keywords; Levaimisole determination; biological juid analysis; high- pevforwalzce liquid chromatography Levamisole (1-tetramisole, 2-[2,3,5,6-tetrahydro-6-phenylimidazo(2,l-b)thiazole] } is commonly used in many species of animal as an anthelminti~~l-~ Levamisole has been determined in cattle tissues and milk by differential cathode-ray polarography, a technique which is not available in most chemical laboratories and which lacks sensitivity. Gas - liquid chromatography has also been used for levamisole determina- tion in milk7 but this method requires a lengthy clean-up procedure using organic solvent extraction and the use of a selective alkali flame-ionisation detector.High-performance liquid chromatography (HPLC) appeared to offer a more suitable technique for the determination of levamisole and in this work a sensitive and rapid method using this technique is described. Experimental Reagents All reagents were of analytical-reagent grade. Diethyl ether. Borate buffer, $H 9. Hydrochloric acid, 0.1 N. Sodium hydroxide solution, 1.0 N. Methanol. Re-distilled before use. Ammonium carbonate soldon, 0.05 M. Solution A: dissolve 0.746 g of potassium chloride and 0.618 g of Mix boric acid in 180 ml of distilled water. solutions A and B in the approximate proportions of 9 to 1 to give a solution of pH 9.Solution B: 0.2 N sodium hydroxide solution. HPLC Apparatus Pump. Altex, Model 110. Detector. Column. Packing. ODS Hypersil (Shandon Southern). Wavelength. 220 nm. Absorbance. 0.1 a.u.f.s. Solvent. Flowrate. 1.0 ml min-l. Under these conditions levamisole had a retention time of 2.7 min. Cecil, Model CE 2012, variable-wavelength spectrophotometer. 100 x 5 mm (Shandon Southern). Methanol - ammonium carbonate solution, 0.05 M (65 + 35). Procedure add 2 ml of pH 9 borate buffer and 15 ml of diethyl ether. 10 min on a slow rotary mixer. ground glass stoppered test-tube using a 5-ml adjustable pipette. must be pre-wetted with the ether before accurate transfer can be effected.) 15 ml of ether to the first tube and shake as before for 10 min.ether layer into the second test-tube, combining it with the other 10.5 ml of ether. the aqueous layer. 10 min. the interface. To 2 ml of plasma or other body fluid contained in a 50-ml ground glass stoppered test-tube Stopptr firmly and shake for Transfer 10.5 ml of the upper ether layer into a second 50-ml (N.B., The pipette tip Add a further Transfer 15 ml of the upper, Discard Add 3 ml of hydrochloric acid, stopper tightly and shake as above for Remove and discard the upper ether layer using suction, taking care not to disturb Add a further 15 ml of ether to the aqueous layer followed by 0.5 ml of 1.0 N994 SHORT PAPERS Analyst, Vol. 105 sodium hydroxide solution and shake for 10 min as above.Transfer 12 ml of the ether layer into a 50-ml glass test-tube. Add a further 15 ml of ether to the aqueous layer and shake for 10 min as above. Remove 15 ml of the ether layer and combine it with the 12 ml of ether, Evaporate the ether to approximately 6 ml on a Dri-bath a t 50-56 "C under nitrogen and then transfer it into a 10-ml glass centrifuge tube. Wash the test-tube walls three times with 1 ml of ether, each time adding the washings to the centrifuge tube. Evaporate carefully to dryness on the Dri-bath as above, wash down the walls of the tube with 0.5 ml of ether and evaporate to dryness again. Add 100 pl of methanol to the residue and sonicate for approxi- mately 1 min whilst rotating and tilting the tube in the ultrasonic bath. Inject 5 p1 of the extract on to the HPLC column.With fresh plasma samples from sheep and cattle it was found that a shortened version of the above method could be used by evaporating the combined ether extracts from the first extraction, avoiding the need for a back-extraction. For rumeri and other gastro-intestinal samples the back-extraction was found to be necessary. The concentration of levamisole in the sample is calculated from calibration graphs pre- pared by adding known amounts (0.1-3.0 pg ml-l) of levamisole to blank plasma or gastro- intestinal fluid. The standard samples are extracted using the procedure described and the peak heights obtained for levamisole from the sample are compared with the calibration graph prepared from the standard samples. Typical chromatograms are shown in Fig.1. Using this method the concentration of levamisole in the sample is calculated as follows: By the standard method: Concentration in injected methanol solution 18.0 Concentration in sample = F By the short method: Concentration in injected methanol solution 18.6 Concentration in sample = I 1.1 I i C il ij JL G H J Time + Fig. 1. High-performance liquid chromatographic responses obtained after injection of 5 pl of: A, 5 ; B, 10; C , 15; ;and D, 20 p g ml-1 of levamisole in methanol; and 5 pl of the extracts obtained using the standard method from: E, blank plasma; F, plasma estimated to contain 0.42 p g ml-l; G, blank ruininal fluid; and H, ruminal fluid estimated to contain 0.36 pg ml-l.October, 1980 SHORT PAPERS Results 995 Recoveries of Levamisole from Plasma and Gastro-intestinal Fluids Aliquots of a solution of levamisole in methanol (20 pl) were added to samples of fresh plasma, rumen fluid and abomasal fluid to give concentrations of 1, 2, 5, 10 and 20 pg ml-I of levamisole.The plasma samples were extracted by the method outlined here and also by the short method (i.e., without back-extraction). The recoveries of levamisole from these samples after extraction were calculated from a calibration graph prepared from the peak heights of known standards of levamisole in methanol. The recoveries for plasma were 83% (73-90%, n = 12) by the standard method and 88% (81-98°/0, n = 30) by the short method, and for rumen fluid SOY0 (74-84%, n = 3) by the standard method. The limit of detection was between 0.02 and 0.05 pg ml-l, which is adequate for the con- centrations of levamisole found after normal doses (Table I). TABLE I DETERMINATION OF LEVAMISOLE IN PLASMA BY GC AND HPLC Mean concentrations of two levamisole determinations in plasma, as determined by the HPLC method and a gas-chromatographic method, after administration of levamisole (7.5 mg kg-l subcutaneously) t o two sheep.Levamisole concentration/pg ml-1 r--__-----A -- r----_h-- 1 v Sheep No. 1 Time/h HPLC GC HPLC Sheep No. 2 r---2-- 0 . . . . 0 0 0 0 1 . . . . 2.35 2.48 2.49 2.55 3 . . . . 1.02 1.18 1.56 1.69 6 . . . . 0.37 0.47 0.86 0.90 GE- Accuracy and Precision The accuracy and precision of the method for levamisole were determined by adding known amounts of levamisole to plasma. Samples of each known concentration were then assayed in triplicate by both the standard method and the short method (Table 11).I t was found that the low recoveries were due to extraction losses rather than to degradation of the levamisole. TABLE 11 LEVAMISOLE RECOVERIES Levamisole was added t o 2-ml plasma samples and assayed in triplicate by the standard procedure and by the short procedure. Allowance has been made in the levamisole measured for losses due t o not taking the total amount of extraction solvent a t each step. Amount of Levamisole Standard error Ratio of levamisole levamisole measured/ Mean & as percentage determined added/pg Method* Pg standard error/pg of the mean t o that added 0.4 A 0.34, 0.34, 0.32 0.33 i. 0.01 2.02 0.83 B 0.33, 0.30, 0.32 0.32 & 0.01 2.79 0.80 1.0 A 0.88, 0.84, 0.90 0.87 & 0.02 2.02 0.87 B 0.87, 0.80, 0.82 0.82 & 0.02 2.51 0.83 1.5 A 1.36, 1.50, 1.39 1.42 f 0.04 3.00 0.95 B 1.40, 1.28, 1.32 1.33 f 0.04 2.65 0.89 2.0 A 1.72, 1.68, 1.81 1.74 & 0.04 2.21 0.87 B 1.79, 1.67, 1.70 1.72 f 0.04 2.10 0.86 B 2.68, 2.66, 2.60 2.61 0.04 1.34 0.87 3.0 A 2.58, 2.76, 2.72 2.69 & 0.05 1.86 0.90 * A = Short procedure; B = standard procedure.996 SHORT PAPERS Analyst, Vol.105 Evaluation of the Method after Administration of Levamisole to Sheep and Com- parison with a Gas-chromatographic Method Levamisole* was administered subcutaneously at a dose of 7.5 mg; kg-l to two sheep and plasma samples were then collected at intervals. The samples w'ere nnalysed in duplicate by the standard method and also by an unpublished gas-chromatographic method. 1 The results obtained are shown in Table I. There was good agreement between the results obtained by the two methods. References 1. 2. 3. 4. 5. 6. 7. Forsyth, B. A,, J . S. Afr. Vet. Med. Assoc., 1966, 37, 403. Reid, J . F. S., Armour, J., Jennings, F. W., and Urquhart, G . M., Vet. Ilec., 1968, 83, 14. Lyons, E. T., Drudge, J . H., and Tolliver, S. C., Am. J . Vet Res., 1968, 29, 2157. Forsyth, B. A,, Aust. Vet. J., 1968, 44, 395. Ciordia, H., and Baird, D. M., Am. J . Vet. Res., 1969, 30, 1145. Holbrook, A,, and Scales, B., Anal. Biochem., 1967, 18, 46. Smith, J. E., Pasarela, R., and Wycroff, J. C., J. Assoc. O f f . Anal. Chem., 1976, 59, 954. Received March 28th, 1980 Accepted May lst, 1980 * Nilverm, Imperial Chemical Industries. t The gas-chromatographic analyses were kindly undertaken by Dr. A Featherstone of the Safety of Medicines Section, Imperial Chemical Industries.