Article pubs.acs.org/JAFC
Simultaneous Quantification of Sinigrin, Sinalbin, and Anionic Glucosinolate Hydrolysis Products in Brassica juncea and Sinapis alba Seed Extracts Using Ion Chromatography Inna E. Popova* and Matthew J. Morra Department of Plant, Soil, and Entomological Sciences, University of Idaho, 875 Perimeter Drive, MS 2339, Moscow, Idaho 83844-2339, United States ABSTRACT: Although mustards such as Sinapis alba and Brassica juncea contain glucosinolates (sinalbin and sinigrin, respectively) that hydrolyze to form biopesticidal products, routine quality control methods to measure active ingredients in seed and seed meals are lacking. We present a simple and fast ion chromatography method for the simultaneous quantification of sinigrin, sinalbin, and anionic hydrolysis products in mustard seed to assess biological potency. Optimum conditions include isocratic elution with 100 mM NaOH at a flow rate of 0.9 mL/min on a 4 × 210 mm hydroxide-selective anion-exchange column. All anion analytes including sinigrin, sinalbin, SO42−, and SCN− yielded recoveries ranging from 83 to 102% and limits of detection ≤0.04 mM, with samples displaying little interference from plant matrix components. Sample preparation is minimized and analysis times are shortened to 110 g/m2 of the meal can result in onion damage.5 Biopesticidal potential of mustard seed and seed meals is commonly assessed by measuring glucosinolates using the ISO 9167-1 method and predicting the concentration of biologically active compounds assuming complete hydrolysis and theoretical reaction stoichiometry.17 However, such an approach can result in inaccurate estimates of biological activity, ultimately leading to inefficient pest control. For instance, biopesticidal activity would be overestimated when glucosinolate hydrolysis is incomplete due to low myrosinase activity or when glucosinolates are converted to other compounds during seed processing.18,19 Underestimation of biopesticidal activity occurs when a portion of sinalbin is converted to SCN− during sample processing as may happen at high temperature or at high relative humidity.13 Simultaneous analysis of both glucosinolates and their respective hydrolysis products in mustard meal prior to its application provides a more accurate representation of potential biological activity and also yields information on the efficiency of meal-processing methods. When mustard glucosinolates are hydrolyzed, equimolar amounts of glucose, SO42−, and biologically active compounds are released (Figure 1). For S. alba, measuring both sinalbin and SCN− allows an estimate of meal phytotoxicity by including contributions from glucosinolate hydrolysis as well as endogenous SCN−. Similarly, SO42−
INTRODUCTION Over 430 weed species have developed herbicide resistance and more than 500 species of insects are documented to have resistance to at least one insecticide.1−3 In addition to the demand for new pesticides in conventional agriculture, there is an intense need for synthetic pesticide substitutes in the rapidly increasing organic production sector.4 As a result, plants containing biologically active compounds potentially useful for pest control have gained increasing interest. For example, seed meal of yellow mustard (Sinapis alba) was shown to be efficient in controlling weeds (pigweed, wild oat, and ryegrass), and Oriental mustard (Brassica juncea L.) seed meal has been used as a broad-spectrum pesticide to control nematodes, insects, and fungi.5−7 The development and use of mustard-derived biopesticides is especially appealing because mustard crops are highly drought tolerant, agronomically important species that exhibit rotational and environmental benefits, making them excellent choices for increasing agricultural sustainability.8,9 Bioactive properties of mustard are caused by the presence of high concentrations of glucosinolates (up to 10% by weight).10 Whereas glucosinolates themselves are not pesticidal, in the presence of water they are converted by the endogenous enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147) to biologically active compounds and an equimolar amount of SO42−.5,6 The major glucosinolate in S. alba, sinalbin (4-hydroxybenzyl glucosinolate), is hydrolyzed to an unstable isothiocyanate that is nonenzymatically converted to SCN−, a phytotoxic compound (Figure 1).11,12 Sinigrin (2-propenyl glucosinolate), a major glucosinolate in B. juncea, is hydrolyzed to produce volatile and bioactive 2-propenyl isothiocyanate.13 The concentrations of biologically active compounds in mustard meal depend on endogenous glucosinolate concentration and the extent of glucosinolate hydrolysis, which can vary significantly with the plant variety, environmental growth conditions, myrosinase activity, and seed-processing technolo© 2014 American Chemical Society
Received: Revised: Accepted: Published: 10687
August 4, 2014 October 13, 2014 October 14, 2014 October 14, 2014 dx.doi.org/10.1021/jf503755m | J. Agric. Food Chem. 2014, 62, 10687−10693
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Figure 1. Myrosinase-catalyzed hydrolysis of mustard glucosinolates sinalbin and sinigrin. sulfatase (EC 3.1.6) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glucolimnanthin (3-methoxybenzyl glucosinolate) and sinalbin were isolated from meadowfoam (Limnanthes alba) and S. alba, respectively, in our laboratory. Acetonitrile, water, methanol, and other solvents were of HPLC or LC/MS grade. Solvents and all other chemicals (at least of analytical grade) were purchased from SigmaAldrich or ThermoFisher (Pittsburgh, PA, USA). Extraction of Intact Glucosinolates. Mustard seed and meal samples were ground and analyzed as schematically depicted in Figure 2. Prior to analysis, mustard samples were homogenized and milled
quantification provides a measure of glucosinolate hydrolysis based on reaction stoichiometry. Thus, for B. juncea, sinigrin and SO42− can be quantified prior to seed meal application to predict biopesticidal potential. Intact glucosinolates are ionic compounds that can be quantified by ion chromatography (IC) along with SCN− and SO42−; however, there is no reported method for simultaneous analysis of mustard glucosinolates and their anionic hydrolysis products. Our objective was to develop and evaluate an IC method for the simultaneous quantification of sinigrin, sinalbin, SO42−, and SCN− in mustard seed. The advantages of using IC for analysis include simplicity, selectivity, and relatively low cost. Ion chromatography is a commonly used technique in agricultural and environmental laboratories and, as such, a method that can be easily implemented in routine quality control analyses. The availability of a simple and inexpensive method of analysis is critical in encouraging the development of plant-derived biopesticides to improve agricultural sustainability.
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MATERIALS AND METHODS
Materials. Mustard seeds (S. alba and B. juncea) were obtained locally (Latah County, ID, USA). Two varieties of B. juncea (Pacific Gold and Kodiak) and one variety of S. alba (IdaGold) were used (Table 1). Oil contents of seeds and meals were analyzed gravimetrically after extraction with hexane. A sinigrin standard and
Table 1. Mustard Seeds and Seed Meals Used in IC Analysis
a
sample
species
variety
form
oil contenta (%)
Sa_1 Sa_2 Sa_3 Sa_4 Sa_5 Bj_1 Bj_2 Bj_3 Bj_4 Bj_5
S. alba S. alba S. alba S. alba S. alba B. juncea B. juncea B. juncea B. juncea B. juncea
IdaGold IdaGold IdaGold IdaGold IdaGold Pacific Gold Kodiak Pacific Gold Pacific Gold Kodiak
seed seed meal meal meal meal meal meal seed seed
32 41 22 29 18 33 26 29 46 40
Figure 2. Scheme for optimization of the IC method for analysis of sinalbin and sinigrin in mustard seed meals. with a coffee grinder to a fine powder. Seed meal (0.1 g) was extracted with 5.5 mL of 73% (v/v) methanol using an end-to-end shaker at room temperature for 1 h. To account for extraction recoveries, 1 mL of glucolimnanthin was added as an internal standard. Seed debris was separated by centrifugation at 2400 rpm for 20 min. An aliquot (1000 μL) of supernatant was (1) evaporated to dryness under a gentle stream of N2 in a heating block at 60 °C, reconstituted in water, diluted 10 times with deionized water, and analyzed by IC; (2) diluted 20 times with deionized water and analyzed directly by HPLC/TOF MS; or (3) desulfated and analyzed by HPLC/UV.
Oil content was calculated on a weight basis. 10688
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Desulfation of Glucosinolates for HPLC/UV Analysis. Desulfation of intact glucosinolates was carried out according to the ISO 9167-1 procedure.17 A 1-mL aliquot of the filtered methanol extract was transferred onto a column containing 500 mg of DEAESephadex A-25 anion exchanger and allowed to drain freely. The column was washed twice with 1 mL of deionized water and finally with 1 mL of 0.1 M ammonium acetate buffer (pH 4.0). One hundred microliters of a 1 mg/L sulfatase enzyme (Sigma-Aldrich) solution was added to the column, the column was covered to prevent evaporation, and hydrolysis was allowed to continue for 12 h. Desulfated glucosinolates were eluted directly into 2-mL HPLC autosampler vials with two consecutive 1-mL volumes of deionized water. HPLC/DAD/TOF MS Analysis. HPLC analysis was performed using an Agilent 1200 series HPLC with a diode array detection (DAD) system coupled to an Agilent G1969A TOF-MS equipped with an ESI source (Agilent, Santa Clara, CA, USA). Chromatographic separation of desulfated glucosinolates was conducted using a Phenomenex 4u Hydro-RP, 250 mm × 2 mm, 4 μm, column (Phenomenex, Torrance, CA, USA) maintained at 30 °C. The injection volume was 5 μL. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B). The gradient elution program started with 2% B and followed a linear gradient to 30% B during a 30min period, after which solvent B was increased to 70% in 1 min and held for 2 min; solvent B was then reduced to 2% at 32 min and held for 13 min. The flow rate was 0.25 mL/min. The UV signal was recorded at 229 nm with the reference set to 360 nm. The chromatographic separation of intact glucosinolates was performed using a Zorbax XDB-C18, 50 mm × 4.6 mm, 1.8 μm, column (Agilent) maintained at 30 °C. The injection volume was 5 μL. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). The gradient program started with isocratic elution using 5% B for 3 min, followed by a linear gradient to 70% B from 3 to 5 min, and then held at 70% B for 5 min; solvent B was then reduced to 5% at 10 min and held for 5 min. For the first 1.5 min of the analysis, the flow was diverted from the MS to prevent MS contamination and ion suppression with salts and other polar species. The flow rate was 0.4 mL/min, and spectra were recorded from 190 to 400 nm. Electrospray ionization was operated in the negative mode, and electrospray ionization potential was set at −3500 V. The collision-induced dissociation potential was set at 200 V. Gas temperature was 350 °C, drying gas (N2) flow rate was 12 L/min, and nebulizer pressure was 2.4 × 105 Pa. Analyses were conducted in a profile mode with an m/z range from 90 to 1000 amu. Quantification was performed using deprotonated molecules ([M − H]−) with m/z of 424.0378, 358.0272, and 438.0534 to quantify sinalbin, sinigrin, and glucolimnanthin, respectively. Ion Chromatographic Analysis of Intact Glucosinolates. Ion chromatography was performed using Dionex Ion Analyzer (Dionex, Sunnyvale, CA, USA) equipped with a GP40 gradient pump, an ED40 electrochemical detector, and an AS40 autosampler. Three Dionex, 4 × 210 mm anion-exchange columns were tested: Ion-Pac AS11, IonPac AS16, and Ion-Pac AS18. Sodium hydroxide (100 mM) was used as the mobile phase. Flow rate was varied from 0.5 to 0.9 mL/min. The detector stabilizer temperature was set at 30 °C with temperature compensation of 1.7% per °C. Anion suppressor current was set to 300 mA. The injection volume was 20 μL. The solution used for optimization included 1 mM sinigrin, 1.5 mM sinalbin, 1 mM glucolimnanthin, 0.1 mM SO42−, and 0.25 mM SCN− in deionized water. Calibration Curves, Accuracy, and Precision. Eight calibration standards (0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mM) containing equimolar concentrations of sinigrin, sinalbin, SO42−, and SCN− were included in calibration curves for glucosinolates and their hydrolysis products. Limits of detection (LOD) and limits of quantification (LOQ) for glucosinolates were determined under the optimized conditions for IC analysis based on a least-squares regression analysis as 3.3sy/b and 10sy/b, respectively, where b is the slope of the calibration curve and sy is the residual standard deviation of the regression line. The precision of the developed IC method was evaluated on the basis of intra- and interday reproducibility of analyses.
The accuracy of the IC method for glucosinolate analysis was evaluated on the basis of statistical differences in IC concentrations compared to concentrations detected by the ISO 9167-1 HPLC/UV and HPLC/TOF MS methods. Matrix Effects, Analyte Recoveries, and Sample Stabilities. The chemical compositions of the two mustard species are similar with the exception of the major glucosinolates. B. juncea contains sinigrin, but not sinalbin, and S. alba contains sinalbin, but not sinigrin. For evaluation of matrix effects, recovery, and sample stability, methanol extracts of S. alba (Sa_4) and B. juncea (Bj_5) were spiked with sinigrin and sinalbin, respectively, to yield a final glucosinolate concentration of 5 mM. Thus, for the evaluation of matrix effects on sinigrin, methanolic extracts of S. alba were used to prepare calibration standards. Similarly, methanolic extracts of B. juncea were used to evaluate matrix effects on sinalbin. Recoveries of analytes evaluated at three representative concentrations were expressed as percent of spiked concentrations in the extracts relative to concentrations of the same analytes prepared in water. Stabilities of the analytes were evaluated in B. juncea and S. alba extracts spiked with known amounts of sulfate, thiocyanate, and sinalbin or sinigrin, respectively. Analyte concentrations were quantified at 3, 6, 12, and 24 h after sample storage at room temperature. Oil contents of mustard meals and seeds analyzed in the present study ranged from 18 to 46% (Table 1), thus potentially introducing additional matrix effects. To test for oil-introduced matrix effects, sinigrin in whole and defatted (DF) seed of Bj_5 and sinalbin in whole and DF seed of Sa_4 were quantified and respective samples of the same species compared. A final concern was that the evaporation/reconstitution step included in the proposed IC method may cause glucosinolate loss (Figure 2). Sinigrin concentrations in three B. juncea samples (Bj_2, Bj_4, and Bj_5) were analyzed by HPLC/TOF MS to address this possibility. One set of extracts was evaporated and reconstituted in water as described previously under Extraction of Intact Glucosinolates (Figure 2), and another set of extracts were not evaporated, but diluted 20 times. Both sets of samples were analyzed using HPLC/TOF MS, and sinigrin concentrations in each respective sample were compared. Glucosinolate Hydrolysis in Solutions of Varied Methanol Concentrations. For evaluation of sinalbin and sinigrin hydrolysis in methanol solutions, 0.1 g of seed (Sa_2 and Bj_4) was hydrolyzed for 1 h in 5.5 mL of solution containing 10, 40, or 70% methanol (v/v). Extracts were prepared and analyzed by IC as described under Ion Chromatographic Analysis of Intact Glucosinolates. Statistical Analysis. All experiments were performed at least in triplicate and are presented as means ± one standard deviation. Calibrations were conducted both before and after the analysis of each sequence of samples. The accuracy of the proposed method was evaluated on the basis of statistical differences determined using oneway analysis of variance (ANOVA) with a p < 0.05 level of significance. Precision was evaluated using the relative standard deviations of standards at three representative concentrations as well as biological replicates. For evaluation of sample extract stability, mean values of peak areas for each analyte in freshly prepared samples were compared to corresponding peak areas at 3, 6, 12, and 24 h. Differences were assessed on the basis of Student’s t-test. All analyses were performed using JMP software (version 10, SAS Institute Inc., Cary, NC, USA).
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RESULTS AND DISSCUSION Optimization of the IC Method. IC conditions including anion-exchange column, mobile phase compositions, and the elution program were optimized to obtain baseline separation of mustard glucosinolates (sinigrin and sinalbin), their hydrolysis products (SO42− and SCN−), and the internal standard (glucolimnanthin). All three Dionex anion-exchange columns evaluated for analyte separation were hydroxideselective, cross-linked alkanol, quaternary ammonium-based columns. The Ion-Pac AS11 (45 μequiv) column has a low capacity, whereas Ion-Pac AS16 (170 μequiv) and Ion-Pac 10689
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elution with 100 mM NaOH at a flow rate of 0.9 mL/min on a Dionex Ion-Pac AS16 column (Figure 3).
AS18 (185 μequiv) columns have higher capacities. On the basis of preliminary results, conditions were initiated with 100 mM NaOH at a 0.5 mL/min flow rate. The Ion-Pac AS11 column provided a good separation for inorganic anions; however, glucosinolates were not baseline separated under initial isocratic conditions (Table 2). When Table 2. Performance Characteristics of Three Tested Anion-Exchange Columns for the Separation of Glucosinolates (Sinigrin, Sinalbin, and Glucolimnanthin) and Anionic Hydrolysis Products (SO42− and SCN−)a analyte
tr (min)
Tf
sulfate sinigrin glucolimnanthin sinalbin thiocyanate
2.6 2.9 3.6 3.7 4.5
1.0 1.2 0.8 1.5 1.1
sulfate sinigrin glucolimnanthin sinalbin thiocyanate
5.1 6.9 9.0 11.2 13.0
1.6 1.1 1.5 1.4 1.2
sulfate sinigrin glucolimnanthin sinalbin thiocyanate
5.1 9.4 19.8 22.0 35.0
1.2 1.4 2.0 2.0 1.6
k IonPac AS11 1.6 1.9 2.6 2.7 3.5 IonPac AS16 4.1 5.9 8.0 10.2 12.0 IonPac AS18 4.1 8.4 18.8 21.0 34.0
N
Rs
1146 1284 7056 5040 2374
nab 0.8 2.9 0.8 2.7
4879 6540 8100 5535 7792
na 5.7 5.7 4.3 3.1
149 704 2518 1912 2040
na 2.8 7.0 1.2 5.1
Figure 3. IC chromatogram for glucosinolates, SCN−, and SO42− obtained using a 0.9 mL/min flow rate.
Linearity and Detection and Quantification Limits of the IC Method. Calibration curves for all analytes were linear for the entire calibration range (Table 3). Regression analysis of triplicates for each concentration level yielded correlation coefficients (r) of 0.9998 and 0.9986 for sinigrin and sinalbin, respectively. The LOD for sinigrin and sinalbin were 0.03 mM (Table 3). These LOD are comparable with those of the HPLC/UV method (0.06 mM) and up to 5 times higher than those of the HPLC/TOF MS method (0.01 mM).20 The LODs for SO42− and SCN− were 0.02 and 0.04 mM, respectively. The LOQ for sinigrin, sinalbin, sulfate and thiocyanate were 0.09, 0.10, 0.07, and 0.09 mM, respectively. When concentrations of sinigrin and sinalbin in the final solution are at the LOQ (0.09−0.10 mM), the corresponding concentrations in mustard meal would be 5.37 and 5.70 μmol/g meal (Table 3). The typical concentrations of glucosinolates in mustard meal are significantly higher and range from 60 to 220 μmol/g meal. Similarly, the concentrations of SO42− and SCN− in mustard hydrolysates are >200 times higher than the corresponding LOQ. Thus, the proposed IC method is sufficiently sensitive for the quantification of sinigrin and sinalbin concentrations in mustard meal. When concentrations of glucosinolates in mustard are expected to be low, 10-fold dilution of methanol extracts (Figure 2) can be omitted and more concentrated extracts can be analyzed. Precision. Precision was evaluated by intraday and interday reproducibility of analysis. When standards were analyzed by the proposed method, intraday and interday relative standard deviations were 1.0−2.6 and 1.3−2.6%, respectively (Table 4 and Figure 4). The ranges of detected concentrations were 64− 189 and 126−201 μmol/g in DF meal for sinigrin and sinalbin, respectively. The relative standard deviations for sinigrin were 2−9% and 2−6% for sinalbin. The precision of the IC method for both glucosinolates was comparable with those of the reference HPLC/UV method (1−7%) and the HPLC/TOF MS method developed in our laboratory (2−6%)20 (Figure 4). Accuracy. The accuracy of the proposed method was compared to that of the ISO HPLC/UV and HPLC/TOF MS methods for concentrations of sinigrin and sinalbin in the same respective samples (Figure 4). No differences in the concentrations of sinigrin and sinalbin detected by IC, HPLC/UV, and HPLC/TOF MS methods were observed. Matrix Effects and Analyte Recoveries. When mustard meal is extracted, other compounds may be coextracted along
a
Separation was carried out in an isocratic mode with 100 mM NaOH at room temperature. Tf, tailing factor; k, retention factor; N, number of plates; Rs, resolution; tr, retention time. bna, not applicable.
gradient elution (10−100 mM NaOH in 15 min) was used, the critical SO42−/sinigrin pair could be valley separated, but not the glucolimnanthin/sinalbin pair. Glucolimnanthin and sinalbin have similar structures with comparable polarizabilities of 35.86 ± 0.5 × 10−24 and 37.83 ± 0.5 × 10−24 cm3, respectively (calculated using ACDLabs 12, Advanced Chemistry Development Inc., Canada). Decreasing the gradient steepness from 6 to 4 mM NaOH/min resulted in unacceptable SCN− peak broadening and long run times. The use of Ion-Pac AS16 and Ion-Pac AS18 columns provided baseline separation of the five analytes under isocratic conditions (Table 2). Lateeluting sinalbin and SCN− peaks exhibited significant broadening on the Ion-Pac AS18 column (peak widths were 2.0 and 3.1 min, respectively), and their retention factors exceeded 20. The Ion-Pac AS16 column provided a reasonable run time as all five analytes were baseline separated in 13 min with retention factors ranging from 4.1 to 12.0 and resolutions ranging from 3.1 to 5.7. The Ion-Pac AS16 column was used for all subsequent analyses. To increase throughput of the analysis, a series of runs with flow rates ranging from 0.5 to 0.9 mL/min were evaluated. An increase in flow rate of 0.1 mL/min resulted in a 10−12% decrease in retention time and a 6−14% decrease in peak width. Using a 0.9 mL/min flow rate, elution of the five analytes was achieved in 7.5 min with resolution and retention factors ranging from 2.4 to 5.4 and from 1.8 to 6.2, respectively. Thus, optimum conditions of the IC method were as follows: isocratic 10690
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Table 3. Calibration Curve Parameters, Limits of Detection (LOD), and Limits of Quantification (LOQ) for Two Intact Glucosinolates (Sinigrin and Sinalbin) and Anionic Hydrolysis Products (SO42− and SCN−) As Determined by the Proposed IC Method analyte
sinigrin
sinalbin
sulfate
thiocyanate
concentration range (mM) slope intercept correlation coefficient linearity (mM) range (mM) LOD (mM) LOQ (mM) LOD (μmol/g meal) LOQ (μmol/g meal)
0.016−2.0 1.12 0.00 0.9998 0.03−2.0 0.08−2.0 0.03 0.09 1.6 5.0
0.016−2.0 1.21 −0.01 0.9991 0.01−2.0 0.09−2.0 0.03 0.10 1.9 5.8
0.016−2.0 4.13 0.01 0.9995 0.03−2.0 0.08−2.0 0.02 0.07 1.2 3.7
0.016−2.0 1.61 −0.02 0.9997 0.01−2.0 0.08−2.0 0.04 0.09 1.6 5.0
Table 4. Reproducibility and Recovery of Two Intact Glucosinolates (Sinigrin and Sinalbin) and Anionic Hydrolysis Products (SO42− and SCN−) As Determined by the Proposed IC Method concentration (mM)
0.1 0.5 1.0
0.1 0.5 1.0 0.1 0.5 1.0
sinigrin
sinalbin
sulfate
thiocyanate
Intraday Reproducibility (Relative Standard Deviation) 1.1 1.3 2.1 2.1 1.5 1.2 2.2 2.6 1.2 1.0 1.9 2.2 Interday Reproducibility (Relative Standard Deviation) 1.3 1.5 2.6 2.4 2.1 1.9 2.3 2.5 2.4 1.4 2.2 2.0 Recovery (%) 85 ± 1 98 ± 8 101 ± 3 87 ± 9 100 ± 1 95 ± 1 98 ± 3 83 ± 1 102 ± 4 95 ± 4 101 ± 2 95 ± 1
with glucosinolates including proteins, phenolics, and carbohydrates.21,22 Whereas only anions are detected by IC, positively charged and neutral compounds may affect detected concentrations of target analytes. For example, if glucosinolates are strongly associated with other matrix components, they may not be quantified by IC.23 First, the effect of coextracted matrix components was evaluated (Table 4). Recoveries for all four analytes at three representative concentrations ranged from 83 to 102% as determined by comparing conductivity peak areas for each calibration standard spiked with mustard extracts containing the corresponding standard prepared in deionized water (Table 4). The effect of oil content in mustard seed on method performance was then evaluated. Concentrations of sinigrin in whole mustard seed (sample Bj_5) with 40% oil content did not differ significantly from sinigrin concentrations in the same DF seed meal (155 ± 4 vs 152 ± 2 μmol/g DF meal, respectively). Concentrations of sinalbin in whole mustard seed (sample Sa_4) with 29% oil content did not differ significantly from sinigrin concentrations in the same DF seed meal (165 ± 3 vs 165 ± 2 μmol/g DF meal, respectively) (data not shown). These results demonstrate that the proposed method can be used for the analysis of mustard meals with a wide range of oil contents. The possibility of analysis with no required oil removal shortens overall analysis time. Finally, the effect of methanol evaporation on glucosinolate concentrations was evaluated. The two most commonly used
Figure 4. Concentrations of sinalbin in S. alba Sa_1−Sa_5 samples (A) and sinigrin in B. juncea Bj_1− Bj_5 samples (B) in mustard meal determined by the developed IC method as compared with ISO 91671 HPLC/UV and HPLC/TOF MS methods. Concentration is expressed as a mean value of three replicates with a verticle line representing the associated standard deviation for that mean.
procedures for the extraction of intact glucosinolates from mustard meals are extraction with boiling water and extraction with 73% aqueous methanol. High temperature and methanol prevent enzymatic hydrolysis of intact glucosinolates by myrosinase naturally present in mustard. When glucosinolates are extracted by boiling water, the filtered supernatant can be analyzed by IC with no additional sample preparation. However, during boiling extraction mustard meal can swell up to 400% by weight and a colloidal suspension may be formed. Also, a significant amount of protein (constituting up to 45% of mustard meal by weight) coextracted by boiling water may interfere with analysis and typically requires precipitation with lead acetate or barium acetate.24 The use of methanol reduces the formation of colloids and minimizes coextraction of proteins. However, when aqueous methanol is used, methanol should be removed, and extracts require 10691
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concentration range of detected glucosinolates in mustard meal extracts were analyzed immediately after preparation and again at 3, 6, 12, and 24 h. On the basis of the results of Student’s t-test, peak areas of all four analytes at the three representative concentrations were not significantly different from the corresponding peak areas of freshly prepared samples (Figure 6). Glucosinolate Hydrolysis in Solutions of Varied Methanol Concentrations. One application for the proposed method is monitoring glucosinolate hydrolysis during extraction of biopesticidal compounds from mustard meal. To prepare extracts with the highest concentration of biologically active compounds, glucosinolate hydrolysis and recoveries of hydrolysis products are monitored. Intact glucosinolates are typically extracted with 70% methanol, whereas lower methanol concentrations result in partial glucosinolate hydrolysis and the associated production of multiple hydrolysis products. Thus, to demonstrate the applicability of the proposed IC method, we assessed the extent of glucosinolate extraction and hydrolysis in aqueous solutions containing 10, 40, and 70% methanol by quantifying sinigrin, sinalbin, SCN−, and SO42− by the proposed IC method (Figure 7). On the basis of stoichiometric conversion of glucosinolates to SO42− (Figure 1), the mass balance was within 90% of that expected for both mustards (Figure 7). For S. alba seed, the use of SCN− or SO42− resulted in similar total moles of products. These results demonstrate the use of ion chromatography for simultaneous analysis of intact mustard glucosinolates and their anionic hydrolysis products, providing a technique with high utility for studies involving the hydrolysis of glucosinolates contained in mustard seed and seed meals. The proposed method can be used for the fast analysis of intact glucosinolates in mustard seeds and meals for screening and quality control. The method may also be useful for
reconstitution in water prior to IC injection. On the basis of the HPLC/TOF MS analysis of methanolic B. juncea extracts and corresponding evaporated and reconstituted in water extracts, no significant differences were found and, consequently, no loss of glucosinolates by evaporation was observed (Figure 5). The relative standard deviation for both sets of extracts did not exceed 8%.
Figure 5. Effect of evaporation on sinigrin concentrations in B. juncea seed meal (Bj_2, Bj_4, and Bj_6 meal samples) as determined by the HPLC/TOF MS method. Concentration is expressed as a mean value of three replicates with a verticle line representing the associated standard deviation for that mean.
Stability of Sample Extracts. The stability of the extracts was evaluated during a 24 h time period, which is a reasonable time interval for sample preparation and analysis that includes a sample preparation time of
