An HPLC Method for Simultaneous Quantitation of Individual Isothiocyanates and Oxazolidinethione in Myrosinase Digests of Rapeseed Meal P.N. MAHESHWARI, D.W. STANLEY, J.l. GRAY, 1 and F.R. VAN DE VOORT, Department of Food Science, University of Guelph, Guelph, Ontario N 1G 2Wl ABSTRACT A simple, rapid and precise method for simulta- neous quantitation of individual isothiocyanates and oxazolidinethione in myrosinase digests of rapeseed meal has been developed. The method consists of inactivation of native myrosinase activity present in the seedmeal, followed by digestion with mustard myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) to hydrolyze rapeseed glucosinolates quanti- tatively to isothiocyanates and oxazolidinethione. These hydrolytic products are extracted in methylene chloride as soon as they are formed and finally re- solved by a reverse phase high pressure liquid chro- matography (HPLC) technique on a /a Bondapak CI 8 column using aqueous acetonitrile as solvent and an ultraviolet (UV) absorbance detector set at 254 nm. The lower limits of quantitation by this method in a single aliquot applied to the column were 0.2 gg for the isothiocyanates and 0.01 pg for the oxazoli- dinethione. Recoveries of allyl isothiocyanate, oxazo- lidinethione and sinigrin added to B. ]uncea, prior to digestion, were quantitative and averaged at 94.5, 93.0 and 91.2 percent with standard deviations of 1.5, 3.3 and 2.8 percent, respectively. The butenyl and pentenyl isothiocyanates and oxazolidinethione in Tower (B. napus) and Candle (B. campestris) rape- seeds, and aUyl isothiocyanate in B. ]uncea were the major hydrolytic products of glucosinolates. The identity of peaks corresponding to these compounds on a HPLC chromatogram was confirmed by mass spectroscopy. INTRODUCTION Rapeseed is the major oilseed crop of Canada (1), an important oilseed crop of India, and presently ranks fifth in world production of oilseeds (2). This crop is primarily used as a source of vegetable oil since the oil content reaches ca. 45% (dry, dehulled basis). The residual meal contains ca. 40% protein (d.b., N x 5.53) and is a good source of quality protein since rapeseed proteins possess a well balanced essential amino acid composition and are, for plant proteins, high in lysine and methionine (3,4). How- ever, the utilization of rapesed meal as a protein source in nonruminant rations and human diets is severely limited, mainly due to the presence of glucosinolates. Glucosi- nolates in rapeseed are a source of goitrogens. As such, the glucosinolates are relatively nontoxic, but their hydrolytic products, following the action of thioglucoside glucohydro- lase (EC 3.2.3.1), are isothiocyanates, thiocyanates, nitriles, and oxazolidinethiones which are goitrogenic (5). In addition, these hydrolytic products in nonruminants produce varying manifestations of toxicity ranging from depressed growth and weight gain, loss of reproductive potential, and enlarged thyroids and kidneys to death in rats and other species of experimental animals (6,7). Previously, several technological attempts have been Ipresent address: Department of Food Science and Human Nutrition, Michigan State University, E. Lansing, MI 48824. made to overcome the problems of toxicity associated with rapeseed. These include heat inactivation of the enzyme and the removal or destruction of glucosinolates and their hydrolytic products. An alternative approach considered genetic improvement and the removal of glucosinolates from rapeseed through plant breeding. In either case, several methods have been develoPed for monitoring the removal of glucosinolates including: (i) gas liquid chroma- tographic (GLC) analysis of trimethylsilyl derivatives of glucosinolates (8-12); (ii) enzymatic hydrolysis of the glucosinolates followed by quantitation of the released aglucons by spectrophotometry or gas chromatography (GC) (13-16); (iii) hydrolysis of glucosinolates by thioglu- coside glucohydrolase (EC 3.2.3.1) and measurement of the released glucose by enzymatic (17-20) and GLC assays (21), and the bisulfate ion by gravimetric (22) and volumetric techniques (23,24). However, many of these methods lack precision and reproducibility and often require additional clean-up steps before final quantitation. In addition, the aglucon assay procedures are generally suspect since en- zymatic hydrolysis may not go to completion or may proceed by other routes (25). Recently, high pressure liquid chromatography (HPLC) has been used for the detection of oxazolidinethione in milk (26) and separation of a mixture of standard organic isothiocyanates (27). In this paper, we describe a simple, rapid and precise HPLC method for the detection and simultaneous quantitation of individual isothiocyanates and oxazolidinethione in myrosinase digests of rapeseed meal. MATERIALS AND METHODS Materials Canadian grown rapeseed seeds of B. campestris variety Candle, and B. juncea (oriental mustard) were obtained from the Saskatoon Research Station of Agriculture Canada. These seeds were dehulled Using the Palyi pneu- matic small-seed dehulling unit (28). Dehulled rapeseed, B. napus variety Tower, was obtained locally. Laboratory seed meals were prepared by grinding dehulled rapeseeds in a Wiley mill and extracting them in n-hexane. Crystalline sinigrin monohydrate, No. $330-5, was purchased from Aldrich Chemical Co. Inc., Milwaukee, WI. Allyl isothiocyanate was purchased from Eastman Kodak Co., Rochester, New York. An authentic sample of goitrin [ (S)-5-vinyloxazolidine~2-thioneJ was obtained from another laboratory. Certified HPLC grade acetonitrile, certified ACS grade methylene chloride, n-hexane, citric acid, and sodium phosphate (dibasic) were purchased from Fisher Scientific Co. Glass-distilled deionized water was used throughout the course of this investigation. Phosphate-citrate buffer (pH 7.0) was prepared according to the method of Wetter and Youngs (14) by mixing 17.5 ml of 0.1 M citric acid solution with 82.5 ml of 0.2 M dibasic sodium phosphate solution and adjusting the pH to 7.0. The myrosinase preparation used in these experiments was obtained from B. /uncea (oriental mustard), according to the procedure of Schwimmer (29). 837