A gasliquid chromatographic method for the determination of balsamic acids and esters in crude drugs is described. Methylation of free acids is followed by a single-stage separation and quantitative determination of methyl and benzyl esters of benzoic and cinnamic acids. Results are presented for the column parameters and reproducibility of the method. The analysis of a commercial sample of tolu balsam is reported; the interpretation of the results may offer additional information to that obtained from official standards.OFFICIAL methods for the evaluation of balsamic drugs rely upon the determination of total balsamic acids, together with acid, ester and saponification values, which are normally calculated with reference to the dry alcohol-soluble matter.lS2 These standards yield limited information on the drug and do not enable sophisticated products to be readily detected. Additionally, the determination of total balsamic acids presents practical difficulties owing to the resinous nature of these drugs,394 and the analysis of individual acids can be achieved only after bromination of the cinnamic acid fraction.More recent procedures based on thin-layer chromatography do not permit the satisfactory resolution of acids and esters on a single system and are difficult to make quantit a t i ~e . ~-' Gasliquid chromatography is a convenient method for the evaluation of the free acids and esters, but the separation of free benzoic and cinnamic acids is difficult owing to tailing, as found by Monard and Grenier,' although more efficient separations can be achieved by esterification of these acids.*s9This paper describes the development of a gasliquid chromatographic method for the determination of free balsamic acids as their methyl esters together with the benzyl esters in a commercial sample of tolu balsam. EXPERIMENTAL REAGENTS AND MATERIALS-verified by gasliquid chromatography on the system described below. was used.All reagents and standards were of general laboratory grade, and their purities were Diazomethane-A solution in ethyl acetate was prepared from nitrosomethylurea.lO Internal standard solution-A 0.6 per cent. mlV solution of n-hexadecane in ethyl acetate APPARATUS-Gas chromatograph-A Pye, Model 104, gas chromatograph was used, with a flameionisation detector maintained at the temperature of the column.GZass column-The column, of dimensions 2-7 m x 3 mm i d . , packed with 10 per cent.E301 on Diatomite C (acid washed and dimethyldichlorosilanised, 90 to 100 mesh, supplied by J. J. Chromatography Ltd.), was conditioned at 300 "C for 24 hours before use. In order to prevent blockage of the column by resin from the balsam, the injection port was lightly plugged with glass-wool, which was replaced daily.Integrator-Peak areas were determined by means of a Honeywell digital integrator.Operating coutditions-The initial column temperature was 175 "C for 1 minute, programmed at the rate of 9 "C min-l to a final temperature of 240 "C for 8 minutes. The initial injection block temperature was 220 "C and the final t...

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The generic name Euphrasia is derived from the Greek Euphrosyne (gladness) due to its use in folk medicine for the treatment of failing eyesight. The use in eye medicine probably originated in the Doctrine of Signatures as the flower bears a resemblance to an eye, the latter gave rise to the vernacular name Eyebright.One of the first records of the use of Eyebright occurred in a 15th century herbal (D a w s o n , 1934). G e r a r d ( W o o d w a r d , 1927) extolls its virtues in the treatment of eye disorders. L e c 1 e r c (1936) stated that an infusion had astringent properties and was soothing in conjunctivitis. The British Herbal Pharmacopoeia (1971) lists Euphrasia as a specific treatment for conjunctivitis. Other uses for Eyebright are for the treatment of catarrh ( P o t t e r , 18971, hay fever and bronchial congestion ( G r i e v e , 1931; B e r g e r , 1954). This use is probably due to homeopathy, large doses of the herb causing irritation of the bronchial mucosa promoting copious secretion. The herb is also an ingredient of herbal tobacco ( G r i e v e , 1931). Phytochemical investigations have established the presence of a tannin, euphrastic acid ( P o t t e r , 1897), a 0-glycoside, aucubin ( B r a e k e , 1924; N a t h e r o v a and F i a 1 a , 1960; K r o 1 i k o w s k a , 1960, 1967) together with volatile and fixed * oils, reducing sugars, gallotailnins (M el t o n and S a y r e , 1925; S r e p e l , 1950). B r o d a , J a r o n i e w s k i and S w i a t e k (1960) detected 0.6% caffeic acid in E. .officinalis which they found to have a bacteriostatic action and to inhibit seed germination. M e 1 t o n and S a y r e (1925) attributed the medicinal properties of E. officinalis to aromatic, resinous compounds. Early tests for alkaloids were inconclusive ( M e l t o n and Sayre,-1925) whereas H u l t i n and T o r s e l l (1964) detected 0.003% alkaloid in E. offici17alts and 0.01% in E. frigida. MaterialThe material investigated was a commercial sample containing the dried, coarsely powdered aerial part of three microspecies of Euphrasia officinalis L., namely, E. tremorosa (Per s .) W a 1 1 r., E . confusa, P u g s I . and E. psetrdokerneri P u g s 1. 1 Part VII. H a r k i s s , K. J.: Planta medica 23, 182 (1973).

Commercial samples of Tolu Balsam have been analysed by gas-liquid chromatography and the aromatic acid and ester content compared with appropriate BPC standards. Modern samples show an increase in benzoic acid content, a decreased cinnamic acid content and the absence of benzyl cinnamate. Some samples were found to contain additional unreported constituents, namely, cinnamaldehyde and benzyl alcohol.