4068

Glucosinolates and Derived Products in : Gas-Liquid Chromatographic Determination of the Aglucon Derivatives from

:MELVIN E. DAXENBICHLER and CECIL H. VANETTEN U.S. Department of Agriculture, Northern Regional Re8earch Center, Agricultural Research Service, Peoria, IL 61604

A procedure is described for identifying and 4' X 2 mm id column packed with l'/c EGSS-X quantitatively determining the on 100-120 mesh Gas-Chrom Q (5) (Applied Sci­ found in edible cabbage. The intact glucosino­ ence Laboratories, Inc., State College, PA). Col­ lates are extracted and their aglucons are reo umn preparation and methods of peak area inte­ leased into solvent after a special enzymatic gration have been described elsewhere (5). Helium hydrolysis step. The resulting carrier gas (40 psig) was supplied at rotameter and oxazolidinethiones are separated by a gas­ setting of 30-40 ml/min. Hydrogen (20 psig) was liquid chromatographic procedure that allows supplied from generator at 30-40 mllmin, and air the indh'idual aglucons to he identified and (30 psig) was introduced at 600 ml/min. Tem­ their amounts to he estimated b~' comparison peratures were set at 204 and 240·C at inlet and with an internal reference. detector, respective!)'; electrometer was usually operated at 3 X 10-10 ,amp. Many horticultural crops contain natural toxi­ cants which might be increased inadvertently in Reagentll the development of new varieties (1). Recently, (a) Methyl palmitate.-Nu-Chek-Prep. Inc., Box 172, Elysian, MN. we reported the grand total and the specific (b) .-Crystalline monohydrate (Ald­ amount of each present in 22 varie­ rich Chemical Co.). ties of edible cabbage (2). The glucosinolate (c) Allyl . - Eastman Organic products, which are related to conditions during Chemicals. enzymatic hydrolyses, may include organic nit riles as well as organic isothiocyanates, goitrin, Preparation of Samples and ion (3). From these results it were sampled, and extracts of unhy­ becomes evident that more detailed studies are drolyzed glucosinolates from them were prepared needed on the variability of glucosinolate com­ as described elsewhere (2). A portion of gluco­ position as related to environment and heredity sinolate extract is taken through ion exchange and and on the relative toxicities of the products enzymatic hydrolysis procedure that releases glu­ cose into aqueous phase and aglucons into methyl­ from them. This paper on gas-liquid chromato­ ene chloride phase (4). A portion of methylene graphic (GLC) analyses to measure the indi­ chloride is filtered through ca 2 g anhydrous vidual glucosinolates and the companion paper Na2S04' An aliquot, equivalent to 10-30 g fresh on the determination of total glucosinolate con­ cabbage, is transferred to 100 ml round-bottom tent (4) describe in detail the method we applied flask. Cautious concentration of methylene chlo­ to cabbage in our initial survey (2, 3). ride solution allows retention of desired compo­ nents. Compounds ma~' be concentrated by evapo­ METHOD rating soh'en! under stream of nitrogen while Apparatus container is immersed in water and ice bath. A Packard Co. Model 7401 gas chromatograph Rotary evaporator connected to water aspirator was equipped with Model 846 temperature pro­ may also be used, but no heat should be applied grammer and included other accessories as fol­ to sample flask. In either case. soh'ent e\'aporation lows: flame ionization detectors. Model 873 devi­ is stopped when ca 2 ml remains, sample is trans­ ation temperature controller, Model 824 dual flow ferred, with rinses, to vial, and methylene chlo- controllers. Model 843 dual electrometer. Model 834 dual bi-polar high voltage supply. and Model The mentIon of finn names or trade products does not 804 coiled column air O\·en. Two glass eolumns Imply that they are endorsed or recommended by the De­ X partment of Agriculture over other firms or similar products were used: 6' 2 mm id column packed with 3o/c not mentioned. Apiezon L on 80-100 mesh Gas-Chrom Q, and Received November 29. 1976. DA..XE:\BICHLER &: VA:>ETTE:\: GLC OF GLUCOSI:\OLATES 1:\ CABBAGE 951

Table 1. GLC of Isothlocyanates and goltrln from glucoslnolates in cabbage

Glucosinolate Detector Amt. ppmd precursor response Peak No.· Compound measuredb (trivial name) factor" Fig. 1 Fig. 2

1 allyl NCS sinigrin 2.0 17.5 5.9 2 3·butenyl NCS gluconapin 2.0 1.1 5.7 3 4·pentenyl NCS glucobrasslcanapin 2.0 0.4 4 3·methylthiopropyl NCS glucoibervirin 2.0 1.6 5 benzyl NCS 2.0 0.8 0.2 6 4·methylthiobutyl NCS glucoerucin 2.0 0.4 0.6 7 phenylethyl NCS 2.0 1.0 1.4 8 goitrin [(S)·5·vinyloxazolid ine·2·thione] 2.5 2.1 18.8 9 3·methylsulfinylpropyl NCS glucoiberin 2.5 n.8 23.3 10 4·methylsulfinylbutyl NCS 3.2 12.1 53.6 11 4·methylsulfonylbutyl NCS glucoerysolin 3.2 4.2 2.5 12 methyl palmitate 1.0

• Refers to Figs 1 and 2. b NCS = isothiocyanate. C Factor to normalize weight/area vs. methyl palmitate response. d Calculated as component measured in fresh weight of cabbages for the curves shown. ride solution containing known amount (0.5-0.6 tral data. However, some white cabbages and all mg) of methyl palmitate is added. Vial with the red cabbages we analyzed gave elution curves sample and internal standard is placed in small more like those in Fig. 2. They are higher in the beaker and surrounded by ice and water, and longer chain components, namely, the isothio­ sample again is concentrated under stream of cyanates and goitrin from the butenyl, 4-methyl­ nitrogen until solution volume is ca 0.2-0.4 ml. Sample should not go to dryness, because more sulfinylbutyl, and 2-hydroxy-3-butenyl glucosin­ volatile constituents are lost after removal of the olates (progoitrin). methylene chloride. The detector response factors used in the cal­ culations are also listed for each component in Gas.Liquid Chromatograph,. Table 1. The response factor normalizes the area A 2--6 1'1 aliquot of concentrate containing added that the component would show on a chromato­ methyl palmitate is injected onto each column. gram relative to the area shown by an equal Program is initiated when response is obtained weight of methyl palmitate. The actual detector from solvent. Oven temperature is initially 40°C response factors from weighed amounts of pure and programmed at 4·/min to 204°C. Upper limit compounds were determined for the major com­ is held ca 20 min and then column is cooled to ponents, i.e., allyl, 3-methylsulfinylpropyl, and 4­ 40·C for another injection. GLC separation re­ quires ca 1 hr. methylsulfinylbutyl isothiocyanates and goitrin. Each component, ppm = (peak area cabbage The response factors given for the other com­ component X response factor/peak area methyl ponents, usually present in much smaller palmitate) X (mg methyl palmitate/kg sample) amounts, are empirical values the same as those determined for components of similar retention Results and Discussion times. Table 1 lists the components identified from At the beginning of this investigation we relied cabbage and the numbers corresponding to the heavily on the literature (6-8) as to which GLe elution peaks shown in Figs 1 and 2. The aglucons to ex-pect in domestic cabbage. Other elution cun'es in Fig. 1 are typical of most white references (3, 9-11) give compilations of seeds cabbages we have examined in that the major rich in particular glucosinolates. From our seed isothiocyanates are from sinigrin and 3-methyl­ collection we selected those of known glucosino­ sulfinylpropyl glucosinolate. Figure 1 contains late compositions for GLC analysis and mass elution peaks of the organic isothiocyanates and spectral data for comparisons with cabbage com­ goitrin from all the glucosinolates we have found ponents. Sources of compounds prepared to de­ in cabbage. Except for pentenyl and 4-methyl­ termine .response factors were: goitrin from sulfonylbutyl isothiocyanate, the identity of each Crambe abyssinica, 3-methylsulfinylpropyl iso­ component was determined from GLC-mass spec- thiocyanate from Lesquerella gordonii, 3-methyl- JOURXAL OF THE AOAC (Yol. 60, No. of, 19iij

------.... , '-, I"~ ,..-"", _... --.....,----\ ; 8 " If V 6 /~ _ ..."'- .... I I v \ \,r--- .', ~, - a. " \ I , f 5 7 \12{ 9 \, , 4 \, ,/ \10/\, , \ , , I, 11 , \ , , I, I 2 \ , I, I , I, I I, I, 110I I, I I, I If 1) I I \ ,I

o 5 10 15 20 25 30 35 40 45 Time, min FIG. l--GLC elution curves representative of aglucon derivatives from white cabbage: -- -, curve from EGSS-X column: --, curve from Apiezon column. Analysis as described in text.

,,------,/--,4 5 6 I,',-'1 ,/"- ----,,,-'I,r..,"-- I"~ 8 ---- Ii ' 112 ~ , \9/", ,r. /~\~-\/ I 1 7 : / 1 , \ 10 11 I ,I III \ I, 'I I \ ,' I, \ \ II \1 I , 1 , \\ \ , 1 \1 I I \\ I \ 1\ \ i It 2 \ :: \ \ I \1 \I : \1 \: ~\i :\ \i\1 II I 7 \ ~ l1\\.fI f\ 3 4~: 5 ~08\.9~1O L; 12 'V"-J'-- '--_\ ----'100---'--,"..,/, I I I o 5 10 15 20 25 30 35 40 45 Time, min FIG. 2--GLC elution curves representative of aglucon derivatives from red cabbage: ---, curve from EGSS-X column: ---, curve from Apiezon column. Analysis as described In text. sulfonylpropyl isothiocyanate from Rapistrum paired compounds most likely to be analogous. rugusom, 4-methylsulfinylbutyl isothiocyanate We have not yet found a good source of the 4­ from Diplotaxis tenuijolia, and allyl isothio­ methylsulfonylbutyl isothiocyanate for further c~'anate from crystalline sinigrin. Of these, 3­ comparisons. methylsulfonylpropyl isothiocyanate was not The GLC separations and other information found in our cabbage preparations; however, its reported here have been e};tended to include retention on the 2 columns relative to the reten­ some of the organic nitriles formed instead of tion of 3-meth~'lsulfinylpropyl isothiocyanate the isothiocyanates and goitrin (3). The method supports our tentative assignment of the peak is also currently being applied to other horticul­ following that of 4-methylsulfinylbutyl isothio­ tural crops of the Cruciferae family that in cyanate to be 4-methylsulfonylbutyl isothio­ some cases contain glucosinolates not found in cyanate, since we e:..:pect the behavior of the cabbage. DA..XEXBICHLER &: VANETTEX: GLC OF GLUCOSI:\OLATES IX CABBAGE 953

Acknowledgment (5) Daxenbichler, M. E., Spencer, G. F., Klei­ We thank G. F. Spencer for the mass spectra. man, R., VanEtten, C. H., & Wolff,!. A. (1970) Anal. Biochem. 38, 374-382 (6) Josefsson, E. (1967) Phytochemistry 6, 1617­ REFERENCES 1627. (1) Senti, F. R., & Rizek, R. L. (1974) The Effect (7) Clapp,' R. C., Long, L., Jr, Dateo, G. P., of FDA Regulations (GRAS) on Plant Breed­ Bissett, F. H., & Hasselstrom, T. (1959) J. ing and Processing, C. H. Hanson (Ed.), Am. Chern. Soc. 81, 6278-Q281 Publication 5, Crop Science SOclety of (8) Bailey, S. D., Bazinet, M. L., Driscoll, J. L., America. Madison, 'VI, pp. 7-20 & McCarthy, A. 1. (1961) J. Food Sci. 26, (2) VanEtten, C. H., Daxenbichler, M. E., Wil­ 163-170 liams, P. R., & Kwolek, W. F. (1976) J. (9) Kjaer, A. (1960) Prog. Chem. Org. l'v'at. Prod. Agric. Food Chem. 24, 452-455 18. 122-176 (3) Daxenbichler, M. E., VanEtten, C. H., & (10) Ettlinger, M. G., & Kjaer, A. (1968) Recent Spencer, G. F. (1977) J. Agric. Food Chem. Adv. Phytochem. 1,59-144 25, 121-124 (11) Daxenbichler, M. E., VanEtten, C. H .. Brown, (4) VanEtten, C. H., & Daxenbichler, M. E. F. S., & Jones, Q. (1964) J. Agric. Food (1977) JAOAC 60, 946-949 Chem. 12, 127-130

Supplied by U.s. Dept. of Agriculture National Center for Agricultural . Utilization Research, Peoria, Dfinois

Reprinted from the Journal of the Association of Official Analytical Chemists, Vol. 60, July 19i7.