Article pubs.acs.org/JAFC
Multi-Mycotoxin Screening Reveals Separate Occurrence of Aflatoxins and Ochratoxin A in Asian Rice Chee Wei Lim,*,† Tomoya Yoshinari,‡ Jeff Layne,§ and Sheot Harn Chan† †
Food Safety Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, Singapore 169078 National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan § Phenomenex, 411 Madrid Avenue, Torrance, California 90501, United States ‡
ABSTRACT: The determination of important regulated mycotoxins in rice has been reported previously but not in the individual matrix of white, brown, red, and basmati rice with respect to the matrix effect, recovery, and stability. A total of 190 Asian rices were examined for regulated mycotoxin contamination by the LC-ESI-MS/MS method. Significant variation (p < 0.05) in the matrix effect was observed for fumonisins. Methanol improved the limits of detection (LOD) for HT-2 from 50 μg/ kg to 2.3 μg/kg by promoting ionization efficiency of the ammonium-adduct. LOD and limits of quantitation ranged from 0.1 to 18 μg/kg and 0.2−31 μg/kg, respectively. All analytes degraded by more than 50% on storage, except fumonisins. Acetic acid (1%) provided significant improvement (p < 0.05) in recovery for all analytes in selected white rice from Thailand and China. Mean recovery ranged from 70 to 120%. RSD values were lower than 15% for all analytes. Five AFB1 and single OTA positive samples were detected. No correlation between mycotoxin contamination and rice species (r = 0) exists. KEYWORDS: LC-ESI-MS/MS, regulated multimycotoxins, rice
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INTRODUCTION Mycotoxins are toxic secondary metabolites produced by molds that grow on grains and cereals in the field or during storage.1 Factors such as cultivation practices and inappropriate postharvest storage conditions are two reasons that can contribute toward creating the ideal conditions for mycotoxigenic mold growth, among others. The Food and Agricultural Organization (FAO) estimated that almost a quarter of the world’s grains were contaminated,2 and this poses a serious threat to human health because most of these mycotoxins are known to be carcinogenic to humans.3 In particular, aflatoxin B1 is hepatotoxic and was classified as a Class 1 human carcinogen by the International Agency for Research on Cancer, a body under the World Health Organization in 1993.4 Rice (Oryza savita L.) forms the major source of starch intake in an Asia Pacific diet.5 FAO predicts the world trade in rice to be 37.5 million tonnes in 2013.6 Despite the possible risk of mycotoxin contamination in rice, there are fewer reports on regional mycotoxin contamination profile in this staple food crop compared to others such as maize and wheat. In fact, the European Union Commission Regulation 1881/2006 laid down maximum limits of 5 μg/kg for aflatoxin B1 and 10 μg/kg for total aflatoxin content (aflatoxins B1, B2, G1, and G2) in rice.7 Maximum limits for other major mycotoxins have not been specified for rice. Several studies have shown that some rice can be naturally contaminated with various combinations of important mycotoxins such as aflatoxins, tricothecenes, ochratoxin A, fumonisins, zearalenone, citrinin, and sterigmatocystin.8,9 The current study was mainly devoted to verify whether major mycotoxins are present in rice of Asian origin and to understand their co-occurrence. In addition, their stability in individual rice extracts of white rice, brown rice, red rice, and basmati rice were investigated for the first time by means of the LC-ESI-MS/MS method. © 2015 American Chemical Society
MATERIALS AND METHODS Instrumentation. Analysis of aflatoxins, DON, fumonisins, T-2 and HT-2 toxins, OTA, ZEA, and their respective stable carbon isotopic standards was performed using MRM, on a hyphenated liquid chromatography (Agilent 1290 Infinity; Agilent Technologies, Palo Alto, CA, U.S.A.) tandem mass spectrometry (QTrap 5500; AB Sciex, Foster City, CA, U.S.A.) system (LC-MS/MS). The LC system consisted of four solvent reservoirs, a built-in degasser, two binary pumps, and a refrigerated autosampler. A 0.3 μm inline filter (Agilent Technologies, Palo Alto, CA, U.S.A.) was employed to minimize potential contamination to the LC column. Mycotoxins were separated using a Kinetex C18 column (2.6 μm, 100 × 3.00 mm; Phenomenex, Torrance, CA, U.S.A.) at a flow rate of 0.6 mL/min. Mobile phase selection was based on conditions reported previously.10 Briefly, mobile phases A and B were composed of 99% methanol (MeOH) and 1% methanol (in water) respectively, both containing 1 mM ammonium formate (NH4-formate) and 0.1% acetic acid. Gradient elution was applied as follows: 100% B hold 0.5 min, 100−10% B over 7.5 min and hold 1.5 min, 10−100% B over 0.1 min and hold 1.9 min. The separated analytes were then ionized using electrospray ionization and detected by tandem mass spectrometry. Concurrent polarity switching was applied. The application of concurrent polarity switching created a scan time penalty of 50 ms per analyte on the QTrap 5500 instrument. Positive polarity was applied for the analyses of aflatoxins (AFB1, AFB2, Received: Revised: Accepted: Published: 3104
September 7, 2014 February 26, 2015 February 27, 2015 February 27, 2015 DOI: 10.1021/acs.jafc.5b00471 J. Agric. Food Chem. 2015, 63, 3104−3113
Article
Journal of Agricultural and Food Chemistry
Table 1. MS/MS Parameters for the 11 Regulated Mycotoxins of AFB1, AFB2, AFG1, AFG2, OTA, FB1, FB2, HT-2, T2, DON, and ZEAa precursor ion
Q1 (m/z)
Q3 (m/z)
AFB1
[AFB1+H]+
313.1
AFB2
[AFB2+H]+
315.2
AFG1
[AFG1+H]+
329.1
AFG2
[AFG2+H]+
331.1
OTA
[OTA+H]+
404.0
FB1
[FB1+H]+
722.5
FB2
[FB2+H]+
706.5
HT-2
[HT-2+NH4]+
442.2
T-2
[T-2+NH4]+
484.3
DON
[DON+CH3COO]−
355.1
ZEA
[ZEA-H]−
317.0
C-AFB1 C-AFB2 13 C-AFG1 13 C-AFG2 13 C-OTA 13 C-FB1 13 C-FB2 13 C-HT-2 13 C-T-2 13 C-DON 13 C-ZEA
[13C- AFB1+H]+ [13C- AFB2+H]+ [13C- AFG1+H]+ [13C- AFG2+H]+ [13C- OTA+H]+ [13C- FB1 +H]+ [13C- FB2 +H]+ [13C- HT-2 +NH4]+ [13C- T-2 +NH4]+ 13 [ C- DON +CH3COO]− [13C- ZEA -H]−
330.2 332.0 346.1 348.0 424.0 756.5 740.5 464.2 508.3 370.2 335.0
241.0 285.0 287.2 259.0 243.1 115.1 313.0 115.1 239.0 102.0 334.4 704.4 336.3 688.4 263.1 215.0 215.2 185.1 295.2 265.0 273.0 175.1 300.9 273.2 257.0 330.1 250.1 374.4 358.3 278.4 322.1 310.2 290.0
13 13
DP (V)
EP (V)
55
10
60
10
54
10
60
10
30
10
91
10
96
10
50
10
60
10
−30
−10
−70
−10
120 82 92 60 100 70 90 50 60 −30 −70
10 10 10 10 10 10 10 10 10 −10 −10
CE (V)
CXP (V)
49 37 37 43 37 89 41 81 27 93 57 43 51 41 21 27 29 31 −15 −21 −25 −30 33 42 38 33 34 37 47 18.5 29 −12.7 −25
RT (min)
10
6.2
10
6.0
10
5.8
10
5.5
10
8.1
10
6.8
4 10 19 20 18 11 −17 −16 −10
7.7
24 18 20 10 12 10 13 13 18 −17 −10
6.7 7.2 4.0 8.0 6.2 6.0 5.8 5.5 8.1 6.8 7.7 6.7 7.2 4.0 8.0
a Q1, first quadrupole; Q3, third quadrupole; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential; RT, retention time.
Table 2. Purity (%) of Individual Mycotoxins and Their Respective Stable Carbon Isotopically Labelled Standards (ISTD) and Calibration Levels (μg/L) AFB1 AFB2 AFG1 AFG2 OTA FB1 FB2 HT-2 T-2 DON ZEA
purity of standard (%)
purity of 13C ISTD (%)
S1 (μg/L)
S2 (μg/L)
S3 (μg/L)
S4 (μg/L)
S5 (μg/L)
S6 (μg/L)
98.0 98.0 98.0 98.0 98.0 97.6 98.0 98.8 99.7 99.4 99.0
99.0 99.0 99.0 99.0 98.7 97.8 98.7 98.9 98.8 99.5 99.2
0.199 0.198 0.265 0.185 0.504 25 20.8 5 5 5 5
0.498 0.495 0.5 0.463 1.26 62.5 51.9 10 10 10 10
0.996 0.99 1.32 0.925 2.52 125 104 25 25 25 25
1.99 1.98 2.64 1.85 5.04 250 208 50 50 50 50
3.98 3.96 5.28 3.70 10.1 500 416 100 100 100 100
5.97 5.94 7.92 5.55 15.1 750 624 150 150 150 150
kV for positive ionization polarity and −4.5 kV for negative ionization polarity. The source temperature and nitrogen gas flows (GS1 and GS2) were set to 500 °C and 40 psi, respectively. A summary of precursor ions, their respective retention times, and MS conditions is listed in Table 1. Materials and Reagents. Acetonitrile (MeCN) and MeOH were of HPLC grade from Labscan (a brand from
AFG1, AFG2), ochratoxin (OTA), fumonisins (FB1, FB2) HT-2 and T-2, and their respective stable carbon-labeled isotopes (13C-AFB1, 13C-AFB2, 13C-AFG1, 13C-AFG2, 13C-OTA, 13CFB1, 13C-FB2, 13C-HT-2 and 13C-T-2). Negative polarity was applied for the analyses of deoxynivalenol (DON) and zearalenone (ZEA), and their stable carbon-labeled isotopes (13C-DON and 13C-ZEA). Electrospray voltages were set to 5.5 3105
DOI: 10.1021/acs.jafc.5b00471 J. Agric. Food Chem. 2015, 63, 3104−3113
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Journal of Agricultural and Food Chemistry
to a 1 g (w) sample in a 50 mL taper tube and shaken for 30 min. Then, the mixture was centrifuged at 8000 rpm (3226g) for 6 min at room temperature. Next, 1:1 dilution (v2) using mobile phase B was performed. Individual isotopic standards were added to the diluted supernatant and shaken vigorously by hand, and the samples were then purified using a 0.2 μm PTFE syringe filter before they were transferred into 1.5 mL HPLC amber vial with deactivated inserts. Finally, 10 μL of purified supernatant was injected into the LC-MS/MS for analysis. By applying this sample purification protocol for method validation and commercial sample screening, a solute to solvent ratio of 1:8 can be achieved. For commercial rice samples screening, a scaled-up extraction protocol was applied to a 25 g sample weight portion. Data Processing for LC-MS/MS. The data were analyzed using the quantitation wizard built into Analyst software 1.6.0 for windows (AB Sciex, Foster City, CA, U.S.A.). Transition ions representing individual analytes were summarized in Table 1. The most abundant transition ion of each isotope was used to perform quantitative analysis. Evaluation of Matrix Effect and Detection Limit. The intention of performing matrix effect assessment is to examine ion signal enhancement/suppression due to coextractants originating from the rice matrix over a practicable concentration range. A 1 g rice sample was first extracted, followed by spiking at six concentration levels. Isotopic dilution was not applied. The application of the post-extraction spiking strategy identified ion signal enhancement/suppression due to matrix interference and distinguished them from effects due to recovery. Spiking levels (A, in μg/kg) were determined as
Sigma-Aldrich). HPLC grade NH4-formate, acetic acid, and benzene were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Water was purified by passing through a Purelab Option-Q water purification system (a brand from Elga, U.K.). Analytical standards of aflatoxins, OTA, and fumonisins were purchased from Sigma-Aldrich, whereas HT-2, T-2, DON, and ZEA were purchased from Biopure (Tulln, Austria). Stable carbon isotopic standards of all 11 mycotoxins were purchased from Biopure (Tulln, Austria). Purity of the standards and their respective 13C-labeled isotopes is summarized in Table 2. Stock preparation of Aflatoxins and OTA was reproduced from previous work.10 Briefly, an individual stock solution of aflatoxins was prepared by dissolving in benzene (to extend the shelf life of aflatoxins). For OTA, methanol was selected as solvent of choice. Stock solutions of FB1 and FB2 were prepared in 50% MeOH and then diluted using mobile phase B. Next, individual stock solution of HT-2, T-2, DON, and ZEA was prepared in MeCN. From these individual stock solutions, mixed calibrants of all 11 analytes were freshly prepared, by dilution, using mobile phase B at six concentration levels as shown in Table 2. For aflatoxins, an additional drying step (under a gentle stream of nitrogen gas) was required to remove benzene prior to preparation of mixed calibrants. 13C-AFB1, 13 C-AFB2, 13C-AFG1, 13C-AFG2, 13C-OTA, 13C-FB1, 13C-FB2, 13 C-HT-2, 13C-T-2, 13C-DON, and 13C-ZEA were applied for spiking experiments and commercial sample screening. Concentrations ranged from 1 μg/L for 13C-AFG2 to 20 μg/ L for 13C-FB2. All chemicals were used without further purification. Sample Preparation and Extraction. Rice samples from 9 Asian countries were investigated in this work, as follows: P.R. China (6 samples, white rice), India (31 samples, parboiled rice and basmati rice,), Japan (26 samples, white rice and brown rice), Malaysia (6 samples, white rice and brown rice), Myanmar (2 samples, broken rice), Pakistan (15 samples, basmati rice), Taiwan (10 samples, white rice), Thailand (79 samples, white rice, brown rice and red rice) and Vietnam (15 samples, white rice and brown rice) were purchased from a local distributor in Singapore. An aggregate sample of 1 kg (minimum) was required for retail sampling (European Union Commission Regulation 401/2006). White rice, brown rice, red rice, and basmati rice were randomly selected from the pool of 190 rice varieties and tested for mycotoxins before the selected sample was used for the matrix effect assessment, recovery, and individual analyte stability study. Before performing extraction, an individual rice sample (approximately 1 kg) was milled using Ultra Centrifugal Mill ZM 200 from Retsch (a brand from Haan, Germany), which was fitted with a ring sieve with an aperture size of 0.5 mm. For a sample size of 1 kg, three batches of milling were required because the miller has a capacity limit of 400 g for rice samples. A household blender was then used to blend the milled samples. To ensure minimum carry over between rice samples’ preparation, potato starch powder was applied to both the miller and the household blender, and then discarded. Sample extraction was developed with the intention of ease of execution and low cost of operation for a routine laboratory. Rice extraction was performed by applying a modified sample extraction protocol reported by Sulyok et al.11 and Varga et al.12 for maize analysis. A 1 g sample weight was used to determine matrix effects and recovery. Single extraction using an equal volume of MeCN and purified water containing 1% acetic acid was applied. Briefly, 4 mL (v1) of extraction solvent was added
A = [(spiking concentration/w)] × v1 × 2
(eq A.1)
Six levels of spiking concentration were generated by applying eq A.1, as summarized in Table 2 (n = 3). Matrix effect (ME) was evaluated as13 ME = (peak area of matrix‐matched/peak area of matrix‐free) × 100
(eq A.2)
Linearity was assessed using a residual plot and statistical test for goodness-of-fit. Limits of detection (LOD) and limits of quantitation (LOQ) were determined as (2.33 × standard deviation)/gradient of slope and LOD + (1.64 × standard deviation)/gradient of slope, respectively.14 Recovery, Intraday, Interday Repeatability and Stability Studies. Fumonisins are known to suffer from poor recovery in maize based on reports published previously.11,12 Losses can occur during sample preparation and during storage. In this study, we examined the fate of all 11 analytes (including fumonisins) in rice through spiking experiments, and by using normal and deactivated glass inserts for storage. Briefly, 1 g aliquots (n = 6) of rice samples were spiked at three concentration levels (standard 2, 3, and 5) and extracted using protocols described in the section Sample Preparation and Extraction. Matrix-free calibration curves at six concentration levels (Table 2) were freshly prepared for recovery study. Isotopic dilution was applied. Recovery (extraction efficiency, R) was assessed as R = [(mean measured concentrationstandard2,3,5) /(spiked concentration standard2,3,5)] × 100
(eq A.3)
using the respective calibration curves of individual mycotoxin. For analyte stability study, two sets of matrix-free calibrants were required. The first set of calibrants was used for recovery 3106
DOI: 10.1021/acs.jafc.5b00471 J. Agric. Food Chem. 2015, 63, 3104−3113
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Journal of Agricultural and Food Chemistry Table 3. Summary Table of Mycotoxins’ Surveys Reported Recently country
matrix
sample size
mycotoxin
Austria
rice (basmati, whole grain, long grain short grain, puffed)
AFB1, B2, G1, G2
81
Brazil
rice and its processing fractions
total aflatoxin, OTA, ZEA, DON, citreoviridin
230
Canada
rice (white, brown, red, black, basmati, jasmine)
AFB1, AFB2, OTA, FB1, FB2, FB3
200
Egypt
rice, maize
40
India (Review) Iran
rice
AFB1, AFB2, AFG1, AFG2, total aflatoxin, FB1, FB2 aflatoxins
rice
T-2
140
Malaysia (Review) Nepal
cereals, nuts, spices
aflatoxins, OTA, tricothecenes, ZEA, fumonisins
-
rice
48
Nigeria
rice
21
Pakistan
rice (white, brown, sella)
fumonisin, beauvericin, gibberellic acid, moniliformin, tricothecenes AFB1, AFB2, AFG1, AFG2, OTA, ZEA, DON, FB1, FB2, FB3, patulin, T-2 AFB1, AFB2, AFG1, AFG2
Pakistan
basmati rice
AFB1, AFB2, AFG1, AFG2
2047
Sweden
rice (basmati, brown, jasmine)
AFB1, AFB2, AFG1, AFG2, OTA
99
Turkey
rice
total aflatoxin, AFB1, OTA
100
United Kingdoms Vietnam
rice and rice products (long grain, easy cook, basmati, specialty, brown, short grain, flaked, ground) rice
aflatoxins, fumonisins, OTA, sterigmatocystin, tricothecenes, ZEA AFB1, citrinin, OTA
100
-
519
100
ref (Reiter et al., 2010) (Almeida et al., 2012) (Bansal et al., 2011) (Madbouly et al., 2012) (Reddy et al., 2008) (Riazipour et al., 2009) (Afsah-Hejri et al., 2013) (Desjardins et al., 2000) (Makun et al., 2011) (Firdous et al., 2012) (Asghar et al., 2013) (Fredlund et al., 2009) (Aydin et al., 2011) (FSA, 2002) (Nguyen et al., 2007)
for results obtained described in the section Screening of Commercial Samples by assigning rice species (count) as (X) and mycotoxins positive samples as (Y).
experiments. The same set of sample vials used for recovery experiments were measured 1 month later using freshly prepared calibrants. Sample vials were stored at 4 °C when not in use. This way, information related to individual analyte stability in solution and in matrix can be obtained. In addition, effects of acetic acid on recovery for all analytes including fumonisins in selected white rice were investigated. This study was motivated by a report on degradation of fumonisins in various samples of Thai white rice.15 For this purpose, spiking experiments (at standard 2 concentration) were conducted in triplicates for each composition of acetic acid (extraction solvent containing 0.1% and 1% acetic acid, respectively) by applying the protocols described in the section Sample Preparation and Extraction. To distinguish losses due to degradation from losses due to extraction inefficacy, single concentration calibrant (standard 2) was prepared in supernatant. Measurements were performed 12 h later to simulate degradation in matrix reported previously.15 Isotopic dilution was then applied. Recovery for individual analytes was calculated using eq A.3. Screening of Commercial Samples. A total of 190 rice samples were screened for aflatoxins, OTA, fumonisins, HT-2 and T-2 toxins, DON, and ZEA. Duplicate testing was performed on suspect samples. An average of the two values was reported. Owing to a lack of reference materials containing all 11 mycotoxins in rice, compensation for recovery for individual analyte was made by spiking. Statistical Analysis. Two-tailed Student’s t-test was applied to identify significant differences between two sets of experimental data at 95% confidence level,16 where applicable. To determine the correlation between mycotoxins contamination and rice species, Pearson Correlation (r) was calculated
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RESULTS AND DISCUSSION
MS Method, Mobile Phase Selection, and Chromatography. Applying MS for multimyotoxins detection in rice is not new.17,18 Common to these publications, aflatoxins, fumonisins, deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 toxins in rice were examined using analyte-specific sample purification columns. In this study, we applied a simple diluteand-shoot workflow to determine aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), deoxynivalenol (DON), fumonisins (FB1 and FB2), T-2 and HT-2 toxins, ochratoxin A (OTA), and zearalenone (ZEA) in rice. Our choice of mycotoxins is based on mycotoxins’ occurrence reports published previously, as shown in Table 3.19−33 For MS method selection, we applied scheduled MRM because only targeted analytes at their respective retention time were monitored. The application of scheduled MRM provided the benefit of decreasing the number of concurrent MRMs monitored at the same time.34 This way, we were able to achieve reproducible peak symmetry and optimum data quality by adhering to the guideline of 10 to 15 data points per peak even when concurrent polarity switching was applied for the entirety of our experiment.35 In our work published previously, MS3 was applied as an accurate tool to identify and quantify aflatoxins and OTA in chilli matrices because it exhibited superior resilience to ion ratio distortion for AFG2 and OTA.10 In this study, however, the application of scheduled MRM was fit-for-purpose for quantification of multimycotoxins in rice because ion ratio distortion was absent. 3107
DOI: 10.1021/acs.jafc.5b00471 J. Agric. Food Chem. 2015, 63, 3104−3113
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Figure 1. LC-MS/MS separation and detection showing current polarity switching for concentration S1 (1a) total ion chromatograms of AFG2, AFG1, AFB2, AFB1, HT-2, FB1, T-2, FB2, and OTA. (1b) Total ion chromatograms of DON and ZEA prepared in mobile phase B and 25% MeCN. (1c) and (1d) show distorted ion chromatograms of DON and FB1 (as well as their respective isotopes) at 50% MeCN strength.
AFB1, AFB2, AFG1, and AFG2 to be made. In order to improve the ionization efficiency of NH4-adduct for HT-2 toxin under positive polarity,10 we chose methanol over acetonitrile as mobile phase A. Experimentally, LOD for HT-2 from 50 μg/kg to 2.3 μg/kg in white rice. Similarly, the addition of 0.1% acetic acid converted DON into its acetate adduct for MRM quantitation.36 The transitions monitored for DON in this study is different from those reported previously12 because the QTRAP 5500 did not generate useful fragmentation pattern for [DON + H]+. We attribute the difference in fragmentation reaction for [DON + H]+ to be collision-cell-specific. Selection of methanol and water as mobile phases of choice (containing 1
For mobile phases selection, we considered the ionization efficacy of aflatoxins and HT-2 toxin in acetonitrile and methanol individually. For aflatoxins, we reported previously that the addition of 1 mM ammonium formate to both the extraction solvent and mobile phase would yield a combined ion signal improvement of 3 orders of magnitude based on triplicate measurements10 using triple stage mass spectrometry (MS3). Under MRM mode, adding 1 mM ammonium formate to the extraction solvent provided insignificant ion signal enhancement for aflatoxins. The addition of ammonium formate to both mobile phases A and B alone enabled measurements for sub μg/L (∼0.01 μg/L) concentrations of 3108
DOI: 10.1021/acs.jafc.5b00471 J. Agric. Food Chem. 2015, 63, 3104−3113
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Journal of Agricultural and Food Chemistry
Table 4. Matrix Effect (%) Determined at Six Concentration Levels for the 11 Regulated Mycotoxins of AFB1, AFB2, AFG1, AFG2, OTA, FB1, FB2, HT-2, T2, DON, and ZEA in White Rice, Brown Rice, Red Rice, and Basmati Ricea average matrix effect (%) S1
S2
S3
S4
S5
S6
a
AFB1
AFB2
AFG1
AFG2
OTA
FB1
FB2
HT-2
T2
DON
ZEA
rice
77 97 77 83 45 40 65 65 51 45 84 66 62 51 97 77 50 47 80 52 62 64 98 49
63 98 72 71 44 56 63 74 48 46 74 67 62 53 86 69 52 46 79 55 63 68 97 43
81 68 90 73 41 66 71 75 51 60 89 81 70 67 95 88 51 57 83 59 64 77 94 45
91 50 73 69 49 58 60 79 57 48 76 82 66 60 85 94 54 50 76 65 64 69 92 55
123 268 172 113 58 147 110 89 88 123 147 101 107 156 179 135 92 129 160 106 107 176 186 75
197 250 259 214 157 180 219 160 140 191 198 155 167 225 219 170 139 170 182 127 150 190 197 100
1959 5730 2476 1831 1172 741 1770 1290 471 701 729 550 402 550 545 425 300 268 409 382 299 188 447 429
96 131 115 90 72 128 104 86 76 112 118 98 102 138 140 113 82 111 121 84 84 130 133 58
104 312 141 92 76 146 114 90 83 128 138 100 100 155 143 120 88 122 137 92 102 150 149 72
79 138 59 70 84 131 62 89 76 113 109 85 86 122 112 114 85 116 98 76 95 112 110 57
102 264 152 95 72 125 126 89 103 118 151 110 120 147 182 146 105 111 148 106 120 139 168 79
white brown red basmati white brown red basmati white brown red basmati white brown red basmati white brown red basmati white brown red basmati
Average matrix effect (%) = (peak area of matrix-matched/peak area of matrix-free) × 100.
ion signal enhancement effect in all rice matrices at all six concentration levels, ranging from 100 to 5730%. Ion signal suppression for aflatoxins ranged from 40% to 97%. For OTA, T2 and HT2 toxins, DON, and ZEA, effect of ion signal enhancement or suppression were observed. Mechanisms such as competition for available charges and access to droplet surface for gas phase emission,37 among others, could explain the wide ranging matrix effect results shown in Table 4. Although matrix effect in some rice was reported previously,38 there was no mention of significant variation in matrix effect for fumonisins in white rice, brown rice, red rice, and basmati rice. Application of dilute-and-shoot strategy alone was insufficient to compensate for matrix effect. Isotopic dilution was therefore applied. Evaluation of Recovery, Linearity, Intraday, Interday Repeatability and Stability. We investigated the recovery performance of all analytes in white rice, brown rice, red rice, and basmati rice. From Table 5A, we found there is no evidence to show mycotoxins can degrade in white rice extract from Thailand/China. However, we did observe recovery losses for all analytes when 0.1% acetic acid was applied to the initial extraction solvent (Table 5B) for some rice samples from Thailand/China. From Table 5C, the presence of 1% acetic acid in the extraction solvent provided significant recovery improvement (p < 0.05) for all analytes (including fumonisins) in white rice samples from China/Thailand. Although the use of 1% acetic acid for extraction of mycotoxins was reported previously,11,38 the data reveal the reason it is used. Our recovery study aimed to assess method accuracy, precision, and repeatability by performing spiking using rice blanks (n = 6) made at three concentration levels. Recoveries were evaluated using matrix-free calibration curves with internal
mM ammonium formate and 0.1% acetic acid) would provide the optimum chemical environment needed to examine individual analyte on our proposed LC column. There were challenges related to peak shape reproducibility when we performed rice extraction without applying dilution using mobile phase B. Briefly, we found the peak shapes for both DON and FB1, as well as their respective isotopes were severely distorted. Performing 1:1 dilution (25% MeCN) using mobile phase B preserved peak shape integrity for both DON and FB1. Figure 1a,b showed the total ion chromatogram representing individual mycotoxins when prepared in water dominant mobile phase B (respective isotopically labeled peaks not shown) and 25% MeCN. Figure 1c,d showed distorted peaks for DON and FB1 in extraction solvent (50% MeCN) without 1:1 dilution. Although peak shape integrity is easily dismissed by some researchers as cosmetic imperfection due to poor chromatographic skills, we observed significant variation for precision (RSD > 50%) at three spiking concentrations in rice for both DON and FB1. Clearly, adopting good chromatographic separation practices provided data confidence through repeatability assessment. Evaluation of Matrix Effect. Matrix effect or the occurrence of ion-suppression or enhancement in quantitative analysis using LC-ESI-MS/MS was reported previously.37 It has a significant impact on method integrity by giving inaccurate and unreliable results, thereby affecting reproducibility, linearity, and recovery values. Thus, matrix effect was evaluated at six concentration levels using both matrix-free and matrixmatched calibration curves to assess analyte performance without internal standards. A summary of matrix effect on LC-MS/MS using MRM is tabulated in Table 4. In particular, fumonisins showed severe 3109
DOI: 10.1021/acs.jafc.5b00471 J. Agric. Food Chem. 2015, 63, 3104−3113
Article
Journal of Agricultural and Food Chemistry
provide significant improvement for method accuracy for fumonisins, which represented the major contributing factor toward uncertainty calculation. Therefore, we can conclude that deactivated inserts improved measurement uncertainty by providing greater accuracy in the determination of fumonisins in rice. Hence, we recommend fresh preparation of mycotoxin calibrants and store in deactivated inserts, when prepared in water-enriched solvent system such as mobile phase B. Therefore, for the first time, information related to aflatoxins, OTA, fumonisins, T-2 and HT-2 toxins, DON and ZEA stability in rice matrix and in solution has been made available. Coefficient of determination values obtained were between 0.9991 and 0.9998 for all analytes examined. LOD and LOQ values for individual mycotoxins are summarized in Table 6. Measurement uncertainty (k = 2) values were calculated39 and reported for rice samples tested positive for mycotoxins. Screening Commercial Rice Samples for Multimycotoxins. Screening commercial rice samples for multimycotoxins would provide occurrence data in the supply chain to support prospective maximum regulatory limits revision. A survey involving 190 rice samples from nine countries was conducted of which six rice samples were tested positive for mycotoxins. This constituted a mere 3.2% compared to contamination figures published previously (Table 3). One possible reason for the difference in mycotoxin-contaminated rice values presented in this study and those reported previously in Table 3 could be due to sampling strategy. A summary of samples tested positive for mycotoxins is shown in Table 7. Information related to contamination levels, mycotoxins, source of production, and types of rice were provided. Table 7 provided no evidence for co-occurrence of mycotoxins. The results showed that AFB1 concentration levels ranged from 1.1 to 3.2 (±0.1 to 0.2) μg/kg, which was below the EU maximum limits of 5 μg/kg7. From a wide variety of rice species analyzed, basmati rice samples have the highest risk of contamination from AFB1 based on occurrence. Basmati rice produced by Pakistan represent four out of six rice samples tested positive for AFB1, as shown in Table 7. A sample of Indian parboiled rice for use by diabetic patients was positive for AFB1 at 1.7 ± 0.1 μg/kg. Only one sample of broken rice produced by Myanmar was positive for OTA at 46.6 ± 4.2 μg/ kg. All rice samples were negative for AFB2, AFG1, FB1, FB2, DON, T2, HT-2, and ZEA. Our rice survey identified Pakistan as a major aflatoxin-contaminated rice producer in the supply chain. This observation is consistent with data reported previously by RASFF in 2008, which identified Pakistan as the major aflatoxin-contaminated rice producer. There are parallels in rice contamination profiles between our report and those published previously.19,21,30 Common to these survey outcomes, AFB1 represented the single major contaminant in all basmati rice samples examined. Indeed, Reiter et al.21 showed that 22 out of 24 basmati varieties which were positive for AFB1 in Sweden were produced by Pakistan and India. In yet another series of similar rice profiling exercises conducted previously,30,32 basmati rice samples of localized Pakistan produce represented the host crops for AFB1 contamination. The prevalence of AFB1 in rice samples produced by localized Pakistan farmers10 explained the need for increased level of official controls on imports for basmati rice from Pakistan.40 Indeed, a study conducted by Asghar et al.29 over a period of 6 years revealed a persistent AFB1 fingerprint (1.2 μg/kg) in the supply chain. The level detected
Table 5. Recovery for AFB1, AFB2, AFG1, AFG2, OTA, FB1, FB2, HT-2, T2, DON, and ZEA in White Rice Extract (Thailand/China) with 0.1% Acetic Acid (A); Effect of Acetic Acid Composition (in Extraction Solvent) on Recovery for AFB1, AFB2, AFG1, AFG2, OTA, FB1, FB2, HT2, T2, DON, and ZEA in White Rice from Thailand/China (B, C)a mean recoveries (%, RSDn = 3) (A)
(B)
(C)
analytes
0.1% acetic acidb
0.1% acetic acidc
1.0% acetic acidc
AFB1 AFB2 AFG1 AFG2 OTA T2 HT2 FB1 FB2 DON ZEA
115 (2) 91 (9) 110 (8) 108 (3) 110 (9) 107 (7) 102 (3) 109 (6) 96 (2) 94 (7) 97 (9)
82 43 51 55 48 58 78 57 46 63 46
(4) (6) (9) (5) (5) (2) (9) (2) (9) (5) (9)
110* (9) 88* (3) 94* (4) 87* (10) 86* (8) 99* (5) 102* (8) 94* (1) 82* (4) 102* (8) 103* (10)
a
Experiments for recovery were performed at S2 spiking concentration in triplicates, with isotopic dilution. bMeasurements were performed 12 h later to simulate “degradation” in rice extract. cMeasurements were performed immediately after extraction. *Indicates significantly different from values obtained at 0.1% acetic acidb composition, (p < 0.05) at 95% confidence interval.
standards added. Mean recoveries, intraday and interday relative standard deviation (RSD) values were calculated and shown in Table 6. RSD values were less than 15% for all analytes. From Table 6, satisfactory recovery values ranged from 70% to 120% were achieved for all analytes in all rice matrices. The application of single extraction protocol containing 1% acetic acid provided recovery advantage for fumonisins in all rice matrices without the need to apply double extraction protocols to address fumonisins’ losses such as in maize.12 The sample extraction protocol presented in this study is also suitable for maize analysis (based on FAPAS 04223 Proficiency Test) because it produced a satisfactory z-score value of
