Microdetermination of esculetin in Nicotiana tabacum

ANALYTICAL

BIOCHEMISTRY

Microdetermination

56, 626629

of

(1973)

Esculetin

in Nicotiana

tabacuml

Quantitative analysis of certain furanocoumarins extracted from roots and leaves of two varieties of Heracleum lanatum Michx. was performed by glc with flame ionization detection (FID) (1). Esculetin, a related aromatic dihydroxy coumarin, was identified by paper chromat.ography (PC) in tobacco leaf and smoke (2), and tobacco flower (3), but it was not measured quantitatively because it occurred in such small amounts. Trimethylsilylated esculetin (TMS-esculetin) was separated from a standard mixture of TMS-plant phenolics by glc, using FID, and its molecular weight was determined by mass spectroscopy (4). Dieterman et al. (2) had difficulty separating esculetin from scopoletin by pc in some solvent systems and from caffeic acid in others. The preponderance of these two phenolics compared to esculetin in tobacco tissue required a complete physical separation before analysis, since a relatively large sample of tissue was needed for extraction of a measurable quantity of esculetin or its glucosides (esculin or cichoriin) (5). The objective of this work was to develop a rapid and sensitive quantitative analysis for esculetin in tobacco leaf tissue. EXPERIMENTAL

Reagents. All chemicals were reagent grade unless otherwise specified. The sources or handling of acetonitrile, n-propanol, HCI gas, bis(trimethylsilyl) trifluoroacetamide (BSTFA) , and caffeic acid were as previously described (6). Esculin and esculetin were commercial products; caffeic acid n-butyl ester was synthesized as previously describ’ed (7). Analytical method and preliminary pc separation. The final procedure, somewhat similar t.o that described for the analysis of scopoletin moieties in tobacco (8), was as follows: Weigh 1 g freeze-dried finely-ground tobacco into a flask, add 20 ml n-propanol, and reflux for 45 min. Filter through Whatman No. 1 filter paper and collect filtrate and 3 n-propanol washings. Pass dry HCl gas through the solution until saturated, then reflux for 1 hr. Take to dryness on a rotary evaporator. Add caffeic acid n-butyl ester as internal standard in sufficient amount to provide a satis1 The investigation reported in this project of the U. S. Dept. of Agriculture Station. Copyright All rights

paper (Ky No. and the Kentucky

626 @ 1973 by Academic Press, Inc. of reproduction in anp form reserved.

73-620) is a cooperative Agricultural Experiment

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TABLE 1 Retention (T) of Trimethylsilylated Esculetin and Caffeic n-Butyl Ester on 10% OV-101 80/90-Mesh Anakrom ASa Component Caffeic acid Esculetin

n-butyl

Acid

r 1.00 0.49

ester

0 Glc operating conditions: Electron-capture 190°C; inlet, detector, and outlet temperatures, argon carrier-gas flow rate, 100 cm3/min.

detector, 75 V; column 220, 215, and 22O”C,

temperature, respectively;

factory peak height. Add 1.80 ml acetonitrile and 0.20 ml BSTFA. Cover flask and mix until mixture is completely dissolved. Transfer most of the contents rapidly into an acylation tube, and then seal the tube. Heat at 150°C for 1 hr. Cool to room temperature, open the tube, and inject 1.0-4.0 ~1 into the glc. A Packard Model 7821 gas chromatograph’ was used with a Packard Model 810 tritium-foil electron-capture detector. A 6-ft glass column, 4 mm i.d., was used. The glc operating conditions and column packing are described in Table 1. One gram of tobacco from a University of Kentucky IRl reference cigarette, with specifications as briefly described (9)) was extracted by refluxing 1 hr in n-propanol. The sample was hydrolyzed with dry HCl gas, and taken to dryness on a rotary evaporat’or. The residue was dissolved in 95% ethanol, and concentrated with a stream of dry nitrogen gas. The resultant. total volume was streaked on a sheet of Whatman No. 1 paper, 46 X 57 cm, and developed in two separate, descending solvent systems. In the first, system, which consisted of n-butanol/acetic acid/water, 10: 1:3 (v/v/v), the sample paper was developed 16 hr, together with a separate paper containing standard esculetin. The esculetin zone of the tobacco sample at Rf equivalent to that of the standard (fluorescent under uv light) was eluted wit.h 95% ethanol, concentrated as before, and spot,ted on a second sheet of paper. In the second system, which consisted of isopropanol/formic acid/water, 5: 0.1: 95 iv/v/v), the paper was chromatographed 6 hr, along with the standard esculetin. The csculetin spot, was eluted as described above, taken to dryness, and silylated for glc analysis as in the final procedure above. RESULTS

AND

DISCUSSION

Esculin refluxed 1 hr in n-propanol saturated with dry HCl gas was hydrolyzed and yielded an equivalent amount of esruletin. Trimethylsil‘Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.

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ylated esculetin was separated completely from TMS-scopoletin and TMS-caffeic acid n-propyl ester on a 10% OV-101 column at a column temperature of 190°C. The large quantity of tobacco sample used (injection into glc about equivalent to extract from 1-2 mg tobacco) produced a profusion of overlapping peaks with FID. However, use of the sensitive and more selective tritium-foil electron-capture detector avoided this difficulty, and the resolution of individual peaks was good. A calibration curve was plotted using l-g samples of low-phenol Burley 21 tobacco, spiked with O-2 mg standard esculetin, from which aliquots of extracts, equivalent to 250 pg sample containing up to 0.50 pg esculetin, were injected into the glc. A linear relationship between esculetin concentrations and corresponding peak areas was observed. Relative retention times (T) of the TMS-esculetin and the internal standard TMS-caffeic acid n-butyl ester are shown in Table 1. Assays were performed on (1) a low-phenolic tobacco (Burley 21 variety) spiked with synthetic esculetin, (2) the same tobacco nonspiked, (3) a reference cigarette, and (4) horse-chestnut stems. The latter material is known as a rich source of esculin. All these samples showed measurable esculetin peaks except the nonspiked Burley 21 tobacco sample. The horse-chestnut stems contained 1.74% esculetin, which was 300 times the amount of esculetin measured in the reference cigarette tobacco. The lowest detection limit of esculetin at the given glc parameters was approximately 15 ng. An extract of IRl reference cigarette tobacco was separated by pc as described in Experimental, the esculetin spot was eluted, and a glc analysis was performed. The result was compared to that of another IRl reference cigarette tobacco assay carried through the analytical method without pc and subjected to glc analysis (see final procedure, Experimental). These results were 56 and 55 ,u.g/g, respectively. Thus, identification of the esculetin peak was further verified. Esculetin reportedly formed as an artifact from caffeic acid during pc, and storage in certain solvents (5) was not observed in t’he present procedure. Pre-

Esculetin

Content

TABLE in Various

2 Tobacco-Leaf

(mean

Sample Burley 21 (air-cured, FC-402 (air-cured, Reference cigarette tobaccos)

field-grown) field-grown) (standard blend

Samples rg/g Esculetin 31 average deviation) None

of flue-

and air-cured

detected 14 f 2 55 zk 4

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sumably, our solvent extractions of samples and the anhydrous hydrolytic-transesterification reaction mixtures prevented cyclization of caffeic acid. To verify this, horse-chestnut stem and reference cigarette-tobacco samples spiked with caffeic acid were carried through the analytical method without hydrolysis, and no esculetin peaks appeared. For determination of the analytical precision of the method, four individual analyses for esculetin were performed on each of three tobacco-leaf samples designated as low, intermediate, and high in phenolic content. Results are in Table 2. The peak areas were measured by planimetry at the retention time established with TMS-esculetin standard samples. SUMMARY

A glc method has been described for the quantitative analysis of tobacco (and other plant tissue) for esculetin (as free esculetin and the glucosides esculin or cichoriin) which used tritium-foil electron-capture detection of as little as 15 ng esculetin. Polyphenols were directly extracted from plant material, using n-propanol, and the esculetin glucoside was hydrolyzed with n-propanol saturated with HCl gas to produce free esculetin. After silylation, the TMS-esculetin was separated on 107% OV101, with caffeic acid n-butyl ester as the internal standard. REFERENCES 1. FURUYA, T., AND KOJIMA, H. (1967) J. Chromatog. 29, 382. 2. DIETERMAN, L. J., YANG, C. H., NAKAGAWA, Y., AND WENDER, S. H. (1959) J. Org. Chem. 24, 1134. 3. WATANABE, R., AND WENDER, S. H. (1965) Arch. Biochem. Biophgs. 112, 111. 4. PELIZZARI, E. D., CHUANG, CHIEN-MEI, Ku?, J., AND WILLIAMS, E. B. (1969) 3. Chromatog. 40, 285. 5, RUNECKLES, V. C. (1962) Chem. Ind. 893. 6. ANDERSEN, R. A., AND VAUGHN, T. H. (1970) J. Chromatog. 52, 386. 7. ANDERSEN, R. A., AND MOEGLING, G. (1969) Anal. Biochem. 27, 3Qg. 8. ANDERSEN, R. A., AND VAUGHN, T. H. (1972) Phytochemistry 11, 2504. 9. GRUNWALD. C., DAVIS, D. L., AND BUSH, L. P. (1971) Agr. Food Chem. 19, 1%.

T. H. R. A. Agricultural Research Service United States Department oj Agriculture and Department of Agronomy

University Lexington, Received

of Kentucky Kentucky 40606 August

1, 1%9’s; accepted

August

69, 19Y.9

VAUGHN

ANDERSEN