Article pubs.acs.org/JAFC
Accurate Analysis and Evaluation of Acidic Plant Growth Regulators in Transgenic and Nontransgenic Edible Oils with Facile MicrowaveAssisted Extraction−Derivatization Mengge Liu,†,‡,∥ Guang Chen,*,†,‡,§,∥ Hailong Guo,†,‡ Baolei Fan,# Jianjun Liu,†,‡ Qiang Fu,⊥ Xiu Li,†,‡ Xiaomin Lu,†,‡ Xianen Zhao,†,‡ Guoliang Li,†,‡ Zhiwei Sun,†,‡ Lian Xia,†,‡ Shuyun Zhu,†,‡ Daoshan Yang,†,‡ Ziping Cao,†,‡ Hua Wang,†,‡ Yourui Suo,§ and Jinmao You*,†,‡,§ †
The Key Laboratory of Life-Organic Analysis, Qufu Normal University, Qufu 273165, Shandong, China Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Qufu Normal University, Qufu 273165, Shandong, China § Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China # Hubei University of Science and Technology, Xianning, 437100 China ⊥ Qinghai Normal University, Xining, 810008 China ‡
S Supporting Information *
ABSTRACT: Determination of plant growth regulators (PGRs) in a signal transduction system (STS) is significant for transgenic food safety, but may be challenged by poor accuracy and analyte instability. In this work, a microwave-assisted extraction−derivatization (MAED) method is developed for six acidic PGRs in oil samples, allowing an efficient (0.05), showing that the differences between experimental and predicted values were statistically insignificant (at the 95% confidence level).46 Together, these results demonstrated that values correlating well with experimental data can be predicted by regression of the multivariate model. Accordingly, the optimal variables combination (X1, 34.87; X2, 130.05; X3, 4.69; X4, 1.07; and X5, 6.24) was achieved, which was confirmed by being applied to three independent replicates of MAED operations, giving a peak area of 809.51, an experimental value quite approximate to the predicted 803.45. Thus, the optimal conditions for MAED are established: to a 50 mL vessel were added successively 3 g of edible oil, 6 mL of methanol, 5 mL of DMF, 40 mg of K2CO3, and 1 mL of DDCETS (1.04 × 10−2 mol L−1); then the vessel was placed in the microwave apparatus at 130 °C with shaking for 35 min. Method Validation. It can be seen from Table 2 that linear equations with excellent correlation coefficients of ≥0.9996 were developed over the wide ranges. As expected, quite low LODs (0.37−1.36 ng mL−1) and LOQs (1.11−4.22 ng mL−1) were obtained with higher analytical sensitivity than most of those reported,17,19−22,26−28,35,36 which should be attributed to the strong fluorescence responses and low interferences of this method. The satisfactory repeatability for retention times (0.07−0.26%) and peak areas (1.11−1.51%) demonstrated that FLD quantification was reliable, whereas no obvious analyte loss was observed. Benefiting from the high efficiency, the sensitivity and accuracy (RE%, intraday, −2.96 to 2.60%; interday −4.30 to 3.68%) and precision (RSD%, intraday, 8063
DOI: 10.1021/acs.jafc.5b02489 J. Agric. Food Chem. 2015, 63, 8058−8067
Article
Journal of Agricultural and Food Chemistry
Figure 3. Typical chromatograms for PGR standards (A), nontransgenic soybean oil (B), transgenic soybean oil (C), nontransgenic sunflower oil (D), transgenic sunflower oil (E), nontransgenic rapeseed oil (F), and transgenic rapeseed oil (G). Peaks: 1, gibberellin A3 (GA3); 2, indole-3-acetic acid (IAA); 3, indole-3-propionic acid (IPA); 4, indole-3-butyric acid (IBA); 5, 1-naphthaleneacetic acid (1-NAA); 6, 2-naphthaleneacetic acid (2NAA); 7, excessive DDCETS.
contents. In the signal transduction system, release of component depends on the PGRs in biosynthesis. GA3 promoted production of α-amylase, thereby regulating the content of sugar; thus, its higher level in nontransgenic samples (rapeseed oil, 8.86 μg g−1; sunflower oil, 7.66 μg g−1) should be consistent with the fact that nontransgenic foods contained higher levels of sugar than transgenic foods.47,48 Serine was adjusted by IAA; thus, its contents in transgenic rapeseed oil, nontransgenic soybean oil, and transgenic sunflower oil would be higher.11 NAA was much higher in nontransgenic rapeseed oil than in the other samples, probably indicating the higher content of fatty acid in the nontransgenic rapeseed.49 Transgenic food has been criticized and unrecognized, but
of this method. Thus, the proposed MAED-HPLC-FLD-MS method proved to be more powerful for the accurate, rapid, and convenient determination of PGRs. Sample Analysis. The developed method was applied to the simultaneous determination of six PGRs in edible oil samples including transgenic and nontransgenic soybean oil, sunflower oil, and rapeseed oil. Chromatography is shown in Figure 3, and PGRs contents are summarized in Table 3. Overall, remarkable differences were observed between transgenic and nontransgenic samples: 1-NAA was detected only in nontransgenic samples; IPA was detected only in transgenic rapeseed oil and nontransgenic soybean oil samples; GA3, IAA, IBA, and 2-NAA were found in all of the samples at different 8064
DOI: 10.1021/acs.jafc.5b02489 J. Agric. Food Chem. 2015, 63, 8058−8067
a
1 3 5
1 3 5
1 3 5
IBA
1-NAA
8065
2-NAA
10.850
8.290
3.790
3.8 2.5 2.3
1.6 1.9 1.0
3.4 2.1 1.8
_ _ _
2.4 2.8 1.4
0.03 0.10 0.19
_ _ _
2.79 8.68 14.57
0.03 0.09 0.15
4.48 15.01 25.10
5.61 18.21 30.01
0.034
_
2.870
0.030
4.830
5.900
1.5 1.7 1.2
_ _ _
2.1 3.0 1.3
0.7 0.9 1.5
1.5 1.7 2.1
1.1 1.8 2.9
RSDc (%)
0.04 0.12 0.19
0.03 0.13 0.26
1.78 5.99 9.76
0.02 0.06 0.09
3.03 9.77 15.82
3.23 9.85 16.99
mean (μg)
0.039
0.042
1.910
0.018
3.150
3.300
avb (μg/g)
nontransgenic
3.1 2.7 2.6
1.1 1.4 1.8
0.8 1.2 0.7
1.3 1.8 2.6
0.6 0.7 1.1
1.8 2.5 3.1
RSDc (%)
0.02 0.06 0.08
_ _ _
2.05 6.67 11.01
_ _ _
1.31 4.13 6.99
3.24 10.05 17.10
mean (μg)
soybean oil
0.017
_
2.160
_
1.360
3.340
avb (μg/g)
transgenic
1.8 0.7 1.3
_ _ _
1.8 1.2 1.2
_ _ _
2.3 2.7 2.6
2.0 1.4 1.9
RSDc (%)
0.04 0.12 0.21
0.03 0.11 0.19
2.79 8.59 14.99
_ _ _
2.05 6.71 11.01
7.56 23.16 38.51
mean (μg)
0.040
0.037
2.880
_
2.160
7.660
avb (μg/g)
nontransgenic
1.9 2.0 1.5
0.9 1.6 1.2
0.9 0.7 1.5
_ _ _
0.9 1.3 1.8
2.1 3.6 2.4
RSDc (%)
0.03 0.08 0.14
_ _ _
2.10 7.00 11.90
_ _ _
2.93 9.22 15.93
3.35 10.97 17.99
mean (μg)
sunflower oil
0.026
_
2.270
_
3.060
3.530
avb (μg/g)
transgenic
Weighed three amount levels of samples. bThe average contents of GA3, IAA, IPA, IBA, 1-NAA, and 2-NAA in oil samples. cThe relative standard deviation (n = 6). d−, not found.
10.67 32.73 54.79
8.15 24.88 42.10
3.67 11.65 19.12
_
_d _ _
1 3 5
IPA
2.930
1 3 5
IAA
1.9 0.9 2.5
avb (μg/g)
8.860
transgenic mean (μg)
avb (μg/g)
RSDc (%)
nontransgenic
2.87 8.98 14.68
8.71 26.98 44.46
1 3 5
GA3
PGRs
mean (μg)
weighed contenta g
rapeseed oil
3.2 1.3 1.9
_ _ _
1.6 0.6 2.4
_ _ _
1.1 1.9 3.1
0.7 1.3 1.9
RSDc (%)
Table 3. Contents of Gibberellin A3 (GA3), Indole-3-acetic Acid (IAA), Indole-3-propionic Acid (IPA), Indole-3-butyric acid (IBA), 1-Naphthaleneacetic Acid (1-NAA), and 2Naphthaleneacetic Acid (2-NAA) in Six Oil Samples (n = 6)
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02489 J. Agric. Food Chem. 2015, 63, 8058−8067
Article
Journal of Agricultural and Food Chemistry
and the Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine Shandong Province.
this study reflects the advantages of nontransgenic food as well as transgenic foods in terms of some nutrients. Consequently, PGR-induced nutrient variation is promising to be an important index for the objective evaluation of transgenic foods. On the other hand, for transgenic foods, what should be explored is which and how many genes are directly or indirectly modified. As the ending point of the signal transduction system, variations of genetic trait and composition are reflected by PGRs.3−5 GA3 was biosynthesized with the aid of two essential genes (copalyl pyrophosphate synthase and ent-kaurene synthase); thus, its higher levels in nontransgenic samples (rapeseed oil, 8.86 μg g−1; sunflower oil, 7.66 μg g−1) indicated these two genes would decrease after the plant was genemodified.10 Similarly, tryptophan monooxygenase and indoleacetamide hydrolase were higher in transgenic rapeseed oil.12,50 IPA was found in transgenic rapeseed oil (0.03 μg g−1) and nontransgenic soybean oil (0.018 μg g−1), implying that the tryptophan aminotransferase genes in transgenic rapeseed and nontransgenic soybean were relatively higher.14 The higher IBA level in nontransgenic rapeseed oil (3.79 μg g−1), transgenic soybean oils (2.16 μg g−1), and nontransgenic sunflower oils (2.88 μg g−1) might indicate the higher content of glutamate-γsemialdehyde participating in the biosynthesis of IBA via the tryptophan pathway.13 Therefore, the results might reflect the PGR-induced genetic variation in addition to artificial gene modification, which is an added benefit of PGRs determination.
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Notes
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02489. Figures of single-variable experiments and mass spectrum and tables of recovery, matrix effect, stability, method comparison, and content (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(G.C.) Mail: Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Qufu Normal University, Qufu, China. Phone: +86 537 4456305. Fax: +86 537 4456305. E-mail:
[email protected]. *(J.Y.) E-mail:
[email protected]. Author Contributions ∥
REFERENCES
M.L. and G.C. contributed equally to this work.
Funding
This work was supported by the Natural Science Foundation of Shandong Province, China (ZR2013BQ019) and the National Natural Science Foundation of China (General Program) (21475074). This research work was supported by the Open Funds of the Shandong Province Key Laboratory of Detection Technology for Tumor Markers (KLDTTM2015-6), the Undergraduate Technology Innovation Project (201410446021), the National Natural Science Foundation of China (General Program) (21475075), the 100 Talents Program of The Chinese Academy of Sciences (328), the National Natural Science Foundation of China (General Program) (21275089), the National Natural Science Foundation of China (21405093, 21405094, 81303179, 31301595, 21305076, 21302110, 21402106), the Natural Science Foundation of Shandong Province, China (ZR2014BM029), the Key Laboratory of Bioorganic Analysis Shandong Province, 8066
DOI: 10.1021/acs.jafc.5b02489 J. Agric. Food Chem. 2015, 63, 8058−8067
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DOI: 10.1021/acs.jafc.5b02489 J. Agric. Food Chem. 2015, 63, 8058−8067
