Determination of Glucosinolates in Mustard by ... - ACS Publications

Chapter 11

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: January 29, 1997 | doi: 10.1021/bk-1997-0660.ch011

Determination of Glucosinolates in Mustard by High-Performance Liquid Chromatography—Electrospray Mass Spectrometry 1

Carol L. Zrybko and Robert T. Rosen

2

1

Nabisco, Inc., 200 DeForest Avenue, East Hanover, NJ 07936 Center for Advanced Food Technology, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903-0231 2

A method was developed to determine glucosinolates in mustard seeds by reverse phase HPLC using volatile ion-pairing reagents followed by U V detection at 235 nm. Two external standards, phenethylglucosinolate and sinigrin were used to quantify results. The identities of individual mustard glucosinolates were confirmed by negative ion electrospray mass spectrometry as was L C peak purity. This L C / M S method may be used to identify species which are not commercially available as pure standards since mass spectrometry can be used to check for all known glucosinolate anions. Three mustard types were chosen from the Brassica species: yellow (Brassicahirta),brown, and oriental (Brassica juncea). Eleven mustard samples representing harvest areas of southwestern Canada were analyzed in triplicate for glucosinolate content. Percent coefficient of variation between triplicate samples of the same batch was often less than 10%. In mustard and other members of the Brassica species of the Cruciferea family of vegetables, the important nonvolatile precursors are the hydrophilic glucosinolates. The structure of glucosinolates, as seen in Figure 1, imparts strong acidic properties to the compound due to the sulfate group. Other important structural moieties include the glucose and cyano groups. Glucosinolates vary due to differences in the R group. The side group can be alkyl, branched alkyl, indole, aromatic, or unsaturated. The differences in the side chain impart different flavors to food. A l l glucosinolates are formed in plants from L-amino acids and sugars through a common biosynthetic pathway.

© 1997 American Chemical Society In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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R CH2OH

C NOSO3-


Figure 1- The structure of glucosinolates. Glucosinolates breakdown due to the action of enzymes and heat to give many flavor compounds. Myrosinases (thioglucoside glucohydrolases) are found compartmentalized within the plant, and are released when the cell walls break through bruising, sample grinding, or during normal growth as a result of damage and death of cells. Glucosinolates are easily degraded by the myrosinase enzyme at both acidic and basic pH to give such products as isothiocyanates, nitriles, thiocyanates, oxazolidine-2-thiones, hydroxynitriles, and epithionitriles, all of which impart flavor to mustards (7). Kale, kohlrabi, broccoli, cabbage, mustard, cauliflower, rapeseed, turnip, and rutabaga, of the Brassica group of cruciferous vegetables, are best known for their pungent flavor. In ancient times, these crops were thought to have medicinal power over headaches, gout, diarrhea, deafness, and stomach disorders (7). Glucosinolate breakdown products, specifically 3-indole carbinol, 3-indole acetonitrile, and 3,3'diindolylmethane, produced by myrosinase degradation of glucobrassicin, are considered Phase II anticarcinogens (2). These compounds have also been proven to increase enzymatic activity of glutathione-S-transferase (GSH), ethoxycoumarin O-deethylase (ECD), aryl hydrocarbon hydroxylase (AHH), and epoxide hydratase (EH), all of which in turn increase the polarity of lipophilic carcinogens so that they are more readily excreted in the urine. Other benefits from the glucosinolates in Brassica plants include decreased growth of microorganisms, fungi, and the ability to repel most insects (3). To date, most research into glucosinolate identification has been done by isolating glucosinolate breakdown products and then determining the original precursor (4). This method of back tracking was favored because it was inexpensive and fast. Analysis of glucosinolates has also relied heavily on GC/MS techniques on either glucosinolate degradation products such as the isothiocyanates or on the desulfated and trimethylsilyated (TMS) derivitized glucosinolates (5). Most often, in the case of MS, the isothiocyanates and other volatile components are identified rather than the glucosinolate itself (6). In this study, negative ion electrospray mass spectrometry is used because it detects molecular anions and allows for direct identification of glucosinolates, most of which are unavailable as pure standards. We apply a novel method of reversed phase HPLC utilizing volatile ion-pairing reagents followed by negative ion electrospray mass spectrometry to semi-quantify and identify the glucosinolates in mustard seeds. Formic acid and triethylamine (TEA) were used to produce the ionpairing agent, triethylammonium formate which interacted with the glucosinolates to increase retention and prevent their elution in the void volume.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Glucosinolates in Mustard

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Materials & Method Analytically pure sinigrin (98%) was purchased from Aldrich (St. Louis, MO), as were the ion-pairing reagents, formic acid and triethylamine. Gluconasturtiin (phenethylglucosinolate) was obtained from L K T Laboratories (St. Paul., Minn). Sinapic acid (98%) was supplied by Aldrich (St. Louis, MO). HPLC-grade methanol was purchased from Fisher Scientific (Pittsburgh, PA). HPLC-grade water was generated in the laboratory through the use of a Milli-Q U V Plus Ultrapure water system by Millipore (Milford, MA). The external standard, phenethylglucosinolate, was carefully weighed (10.27 mg) and dissolved in 10 ml HPLC-grade water to obtain a solution with a concentration of 1.027 mg/ml. Sinigrin monohydrate (10.27 mg) was also dissolved in HPLCgrade water to obtain the same concentration as the phenethylglucosinolate. Sinapine bisulfate (98%) was purchased through Spectrum Chemical (Gardenia, CA) and was used as an external standard to quantify sinapine found in all three mustard types. All standards were filtered using a 0.45 micron Acrodisc purchased from Gelman Scientific (Philadelphia, PA). Standards were stored in amber autosampler vials under refrigeration until needed. Brown, oriental, and yellow mustard seeds were obtained from various suppliers, Continental Grain (Alberta, Canada), North Dakota Mustard and Spice, and McCormick & Company (Hunt Valley, MD). Samples were prepared by grinding -25 grams of mustard seed in a 250 ml polypropylene centrifuge tube, Thomas Scientific (Swedesboro, NJ), with 100 ml methanol. A Beckman Polytron was used for three minutes to insure a homogeneous grind. The samples were then centrifuged at 2700 rpm for 5 minutes. The supernatant was poured off into a 250 ml round bottom flask and then evaporated using a Rotovap set at 55°C and an rpm of 55. Samples were evaporated to a volume of approximately 2 ml methanol. The samples were then dissolved in 100 ml HPLC-grade water. All samples were filtered using a 0.45 micron nylon Acrodisc as used during standard preparation. Analyses of mustard samples were performed by injecting a 20 ul aliquot of the sample into a Waters (Milford, M A ) high-performance liquid chromatograph linked to a Waters 490 ultra-violet detector set at 235 nm. A Waters 600E System Controller and a 712 WISP Autoinjector were also used. The entire HPLC system was controlled using the Waters Millenium data acquisition software program version 2.1. The column used was a Phenomenex (Torrence, CA) 5 μπι ODS(20) column measuring 250 mm X 4.6 mm with a 55 mm X 4.6 mm Waters C-18 guard column attached. The two mobile phase solvents, methanol and water, were prepared with 0.15% triethylamine and 0.18% formic acid added as ion-pairing reagents. Both solutions were filtered using a 0.45 μπι filter, sonicated for 30 minutes, and degassed with helium throughout the chromatographic procedure at a sparge rate of 50 ml/minute. The initial mobile phase was 100% HPLC-grade water. At ten minutes, the mobile phase was switched to a linear gradient of 100% water to 100%



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methanol over 60 minutes. After each run the initial mobile phase conditions were set and the system allowed to equilibrate. The flow rate was kept constant at 1 ml/minute. The column temperature was held at 40°C. Confirmation of peak identity was accomplished through the use of a Fisons (Danvers, M A ) V G Platform Π mass spectrometer connected to a V G Mass Lynx data system. The system was operated in the negative ion electrospray mode. The ion source temperature was set at 150°C while the cone voltage was -20V. The HPLC conditions were identical to those obtained off-line except that a Varian 9012 HPLC (Fernando, CA) was used along with a Varian 9050 UV-Vis detector. Quantification of the nonvolatile flavor precursors, sinigrin, progoitrin, sinalbin, and sinapine was determined using external standards. A solution containing 50% phenethylglucosinolate and 50% sinigrin monohydrate was analyzed to determine the response factors of both aromatic and alkenyl glucosinolates, respectively. The ratio of response factor was 1.19, indicating that the aromatic glucosinolate was more sensitive. The concentration of the aromatic compounds, sinalbin and sinapine, were calculated using this response factor since pure standards were commercially unavailable. Results and Discussion Glucosinolate Quantification. Yellow, brown, and oriental mustards were analyzed for their glucosinolate content. The major compounds identified by mass spectrometry can be seen in Table I. A chromatogram of yellow mustard can be seen in Figure 2, whereas Figure 3 shows a chromatogram of brown mustard. Many of the minor peaks seen on the HPLC chromatograms were not visible by the mass spectrometry method utilized. This indicates that these compounds are uncharged and therefore are not glucosinolates. Results for the quantification of compounds in yellow mustard {Brassica hirta) identified by mass spec, can be seen in Table II.

RT

Table I. Nonvolatiles Found in Yellow, Brown, and Oriental Mustards Peak m/z Identification # (observed)

10.5 minutes 1 11 2

358 388

21 33

3 4

424 354

35

5

586

Allylglucosinolate (Sinigrin) 2-hydroxy-3butenylglucosinolate(Progoitrin) p-hydroxybenzylglucosinolate (Sinalbin) 3,5-dimethoxy-4-hydroxycinnamoyl choline (Sinapine) Sinalbin + glucose


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Determination of Glucosinolates in Mustard by ... - ACS Publications

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