717 Determination of Therapeutic Concentrations of Indoramin by Liquid Chromatography with Fluorimetric Detection A. J. Swaisland" Wyeth Laboratories, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 OPH Keywords: Indoramin determination; high-performance liquid chromato- graphy ; Puorimetric detection Indoramin (3- [2-(4-benzamidopiperid-l-yl)ethyl]indole 1 (Fig. 1, I) is an antihypertensive agent1 that may also have potential use in the treatment of peripheral vascular disease2 and migraine.3 In order to assess the bioavailability of the drug from various formulations it was desirable to have a method for measuring therapeutic concentrations in plasma. It was also considered that such a method might be of use to clinical pharmacologists wishing to examine the relationship between pharmacological effect and plasma concentration.Previous studies with indoramin in man used selected ion monitoring following gas - liquid chromatography4 and suggested that after a single 60-mg oral dose, plasma concentrations would reach a peak of 50 ng ml-l. As mass spectrometers are not commonplace in hospital analytical laboratories, a method was sought that made use of more readily available instru- mentation. Indoramin has relatively poor ultraviolet absorption ( E = 6500 at 273 nm) and it was found that ultraviolet detection was not sufficiently sensitive for monitoring high- performance liquid chromatography (HPLC). However, it was possible to exploit the drug's native fluorescence by using a double monochromator fluorescence detector to obtain the desired sensitivity .Experimental Indoramin was extracted from plasma using the following method. Aliquots of plasma (1 ml) were taken and the internal standard, 2-[2-(4-ben~amidopiperid-l-yl)ethyl]naphthalene * Present address : Beecham Pharmaceuticals, Bioscience Research Centre, Great Burgh, Epsom, Surrey, KT18 5XQ.718 SHORT PAPERS Analyst, Vol. 106 (Fig. 1, 11), was added as a solution in methanol (1 pg. ml-l; 100 pl). The samples. were made alkaline by the addition of sodium hydroxide solution (1 M ; 50 pl) and extracted with glass-distilled 1-chlorobutane (Rathburn Chemicals, Peeblesshire, Scotland) (3 ml) by vortex mixing for 30 s. The phases were separated by centrifugation at 1500 rev min-l for 10 min and a portion (2.5 ml) of the organic (upper) phase was removed and evaporated to dryness by heating on a block at 70 "C under a stream of nitrogen.The residues were dissolved in acetonitrile (100 pl) for analysis. Liquid chromatography was carried out on a 250 x 4.6mm i.d. column packed with 10-pm C,, silica (Partisil 10-ODs, Whatman Ltd., Maidstone, Kent) with a 50% solution of acetonitnle in aqueous ammonium acetate (0.5% m/V) as eluent. The eluent was delivered at a rate of 2 ml min-l by a constant-volume reciprocating pump (Model 750/03, Applied Chromatography Systems, Luton, Bedfordshire). Samples were applied using a loop injector (Rheodyne, Model 7 120, available through Applied Chromatography Systems), between 50 and loop1 being typical injection volumes. The retention times of indoramin and the internal standard were 3 and 5 min, respectively.The drug and internal standard were detected using a Perkin-Elmer 650-10-S fluorimetric detector fitted with a 20-4 flow cell. The excitation and emission maxima for indoramin were 283 and 345 nm, respectively, and the monochromators were set to these wavelengths. Both excitation and emission sIits were set to 10-nm band pass. Under these conditions the detector could be operated at maximum sensitivity without the problems of light scatter or background fluorescence. However, it was advantageous to use an electronic noise filter (Spectrum, Model 921, available through Magnus Scientific, Sandbach, Cheshire) in order to obtain a smooth base line. Results and Discussion Using calibration standards, the lower limit of accurate measurement for indoramin was found to be 1 ng d-* (Fig.2). The increase in peak-height ratio with concentration was linear up to 1 OOO ng ml-l. Analysis of ten replicate samples at two concentrations (10 and 50 ng ml-1) gave relative standard deviations of 7.7 and 9.0%, respectively. This variation included contributions both from the extraction procedure and from the chromatography. 'H I Fig. 1. Structural formulae of compounds referred to in the text. I, Indoramin, 3-[2-(4-benzamidopiperid-l-y1)- ethyllindole ; 11, 2-[2-(4-benzamidopiperid- 1-yl)ethyl]- naphthalene ; and 111, 6-hydroxyindoramin.June, 1981 SHORTPAPERS 7 19 I II I 4 8 12 Ti me/mi n Fig. 2. Typical trace in liquid chromatographic analysis of indoramin. I, 1 ng of indoramin; 11, 10 ng of internal standard.Injection point is arrowed. As there is a possibility that plasma samples from individuals who have taken indoramin may contain the 6-hydroxy metabolite (Fig. 1, 111), the HPLC characteristics of this com- pound were investigated. Under the conditions described, it was found to have a retention time of 1.3 min and was well separated from indoramin. Although the excitation and emission maxima for 6-hydroxyindoramin were not identical with those of indoramin, they were sufficiently close to allow detection at 60% maximum sensitivity. Analysis of plasma samples from volunteers who had ingested 100 mg of indoramin revealed, however, that the concentration of 6-hydroxyindoramin in the extracts was below the limit of detection.Further, there were no hitherto unidentified metabolites in the extracts that might interfere with the determination. As one of the major metabolites of indoramin is a conjugate that yields the parent drug on refluxing with dilute acid,5 there was a possibility that such a degradation might occur to a small extent on storage, resulting in a high value for indoramin concentration. To investigate this possibility, the samples from the volunteers were analysed before and after storage at 10 "C for 8 weeks. The results indicate that no change in indoramin concentra- tion had occurred ( p > 0.5; paired t-test). Therefore, it is reasonable to assume that storage at -20 "C for many months would result in negligible deterioration of the samples. The method described here employs a relatively unusual method of detection, i e ., Auores- cence. The technique is both sensitive and reproducible and should prove useful in the further development of the drug. One of the principal metabolites in man, 6-hydroxyindoramin, is well separated from the drug and is unlikely to interfere in the determination. As the method makes use of the native fluorescence of the indole moiety in indoramin, it provides a basis for the analysis of all indole-containing compounds that are amenable to HPLC. The author thanks Mr. Steve Boucher for technical assistance and Mr. Tom Frost and Dr. Richard Franklin for helpful comments. 1. 2. 3. 4. 5 . References Carballo, R., Conde, L., Lapelle, M., and Suarez, J., CUYY. Med. Res. Opin., 1974, 2, 437. Robson, P., Pearce, V., Antcliffe, A. C., and Hamilton, M., BY. J . Clin. Pharmacol., 1978, 6, 88. Ghose, K., Coppen, A., and Carroll, D., B r . Med. J., 1977, 1, 1191. Draffan, G. H., Lewis, P. J., Firmin, J . L., Jordan, R. W., and Dollery, C. T., BY. J . Clin. Pharmacol., Franklin, R. A., personal communication. 1976, 3, 489. Received May 2nd, 1980 Accepted November 27th, 1980