Research Advances in Detection Methods of Plant Growth Regulators for Agricultural Products

Shuyuan XU Lei WANG Haitao ZHAO Huihui CAO Shuai WANG Sining TANG Jianhua WANG

Abstract In recent years, plant growth regulators are widely used in agricultural products. As the toxicity of plant growth regulator residues has gained increasing concerns, trace analysis methods for plant growth regulators have been developed. In this paper, the major methods with advantages and disadvantages for the detection and pre-treatment of plant growth regulator residues in agricultural products were summarized, including gas chromatography (GC), high performance liquid chromatography (HPLC), chromatographic technique combined with mass spectrometry, enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE) and so on. Meanwhile, the development prospects were also discussed.

Key words Agricultural products; Plant growth regulator; Detection method

Received: October 12, 2020  Accepted: December 21, 2020

Supported by Tangshan Science and Technology Planning Project (20150210C); Hebei Provincial Phase II Modern Agricultural Industry Technology System Innovation Team Building Project (HBCT2018120207, HBCT2018160403).

Shuyuan XU (1966-), female, P. R. China, senior veterinarian, devoted to research about functional ingredients and food additives.

Lei WANG (1982-), male, P. R. China, associate researcher, devoted to research about functional ingredients and food additives.

#These authors contributed equally to this work.

*Corresponding author.

Phytohormones are organic substances synthesized in plants that have a significant effect on plant growth and development. They are also called natural plant hormones or plant endogenous hormones, which can be transported to other organs of plants from the produced parts or tissues to regulate the growth, development and differentiation of plants separately or in a coordinated manner in terms of cell division and elongation, tissue and organ differentiation, flowering and fruiting, maturation and senescence, dormant germination, and tissue culture in vitro［1］. In recent years, as the types of plant growth regulators continue to increase, their applications in agriculture have become increasingly widespread. At present, food safety problems caused by the abuse and improper use of plant growth regulators have gradually increased, and food safety accidents caused by this have also occurred frequently［2］. The residues of plant growth regulators in food have therefore become one of the main factors affecting food safety in China. From the perspective of food safety, it is necessary to study and establish a new method for the analysis of plant growth regulator residues in agricultural products. Therefore, strengthening the rapid and effective detection of plant growth regulator residues in agricultural products is of great practical significance for ensuring food safety and promoting human health and social and economic development.

Pretreatment Technologies for Detection of Plant Growth Regulators

The detection of plant growth regulators is an analysis of trace components in a complex mixture system, and in order to ensure the accuracy and reliability of the analysis results, the samples must be pre-treated. Due to the complex composition of various types of agricultural products and the large differences in the physical and chemical properties of different plant growth regulator varieties, the pretreatment of samples is particularly critical for the detection of plant growth regulators. The sample pretreatment mainly includes two steps of extraction and purification.

Extraction of plant growth regulators

The requirement of sample extraction is to extract the target compound as much as possible and extract as little interfering substances as possible. First, a suitable extraction solvent is selected based on the principle of "Like dissolves like ", which has a polarity similar to that of the plant growth regulator to be tested, and low toxicity, and does not react with the sample. For non-polar plant growth regulators, non-polar solvents can be used for extraction, or mixed solvents can be used for extraction; and for samples with higher water content, such as fruits and vegetables, polar solvents should be used. Commonly used extractants are methanol, acetone, ethanol, acetonitrile and so on.

Methanol is the most commonly used extraction solvent. Yu et al.［3］ used methanol and antioxidants to extract abscisic acid (ABA), zeatin (ZT), gibberellin (GA3) and indoleacetic acid (IAA) from cucumber fruit overnight. After HPLC analysis, the recoveries were 79.47%, 88.02%, 74.65%, and 88.18%, respectively. The extraction method added a small amount of antioxidants, which were beneficial to protecting the sample and effectively preventing the decomposition of plant hormones. Yang et al.［4］ used 80% cold methanol to soak the samples overnight in a refrigerator at 4 ℃, and extracted from the apricot flower buds such 8 endogenous hormones as adenine, ZT, GA3, IAA, 6-benzylaminopurine, colchicine, ABA, and indolebutyric acid (IBA), the recoveries reached 96.1%, 97.7%, 103.5%, 98.2%, 99.0%, 101.4%, 95.4%, and 102.3%, respectively. The method extracted many kinds of plant hormones, the recoveries of which were high.

Ethanol and acetone are also often used as extractants for plant growth regulators. Song et al.［5］ used 80% cold ethanol and 2% polyvinylpyrrolidone (PVP) to extract such 4 plant hormones as GA3, GA4, IAA and ABA from rice seedlings, and determined them by gas chromatography, obtaining the recoveries up to 85%, 89%, 81% and 91%, respectively. They also pointed out that if mature tissues are used as materials, methanol extraction should be the basis. Using acetone as the extractant, the GC-MS method was used to detect ethephon residues in fruits and vegetables in western Japan with satisfactory results.

Meanwhile, acetonitrile is also used for the extraction of plant hormones. Chen et al.［6］ extracted GA3 and IAA in cucumbers with 100% cold acetonitrile, and used high performance liquid chromatography for detection and analysis, and the average recoveries were 80.12% and 81.33%, respectively. Andrzej et al.［7］ believed that the use of acetonitrile to extract plant growth regulators in samples could greatly reduce the dissolution of acids and other impurities, but because acetonitrile itself is very toxic, it is harmful to the human body and the environment, so it is less used in the extraction of plant growth regulators.

Zhang et al.［8］ used microwave-assisted extraction technology and ultrasonic extraction technology to extract 6-BA from soybean sprouts, black bean sprouts, mung bean sprouts, cauliflower, and broccoli. The results showed that the extraction efficiency of the two extraction methods could well meet the requirements of analysis and detection. Hu et al.［9］ also used microwave-assisted extraction method to extract and detect IAA and IBA in beans and cereals, with satisfactory results. Sancho et al.［10］ used a high-speed stirring homogenizer to accelerate solvent extraction of paclobutrazol residues in pears. The extraction time of the method was 2 min, and the recovery reached 82% to 102%, so it is fast and efficient.

Sample purification

The purpose of purification is to remove as much interfering substances as possible in samples and improve the sensitivity of methods. Commonly used purification methods include liquid-liquid extraction, solid phase extraction, gel permeation chromatography (GPC), thin layer chromatography (TLC) and so on.

Liquid-liquid extraction

The liquid-liquid extraction method is simple to operate, and is often used in the pretreatment process of plant growth regulator detection in the early days. Ethyl acetate is an excellent extraction solvent, especially for samples with higher sugar content, and its feature of less extraction impurities is more prominent. Tang et al.［11］ used the method of 3 times liquid-liquid extraction and purification with ethyl acetate when establishing a method for the detection of plant growth regulators in white radish. The average recovery was between 77.56% and 105.08%, and the relative standard deviation was 3.48%-15.88%. Anna Sannino［12］ used acetone to establish a method for detecting 24 pesticide residues including paclobutrazol in lemon juice, in which  ethyl acetate-cyclohexane liquid-liquid extraction was adopted for purification, and the recovery of paclobutrazol was 87.0%-89.0%. Sharma et al.［13］ and Li［14］ extracted vegetable and fruit samples with acetonitrile, purified the extracts by liquid-liquid extraction with n-hexane and dichloromethane, and determined the residues of forchlorfenuron in grapes and kiwi fruit by high pressure liquid chromatography, and the results met the requirements for trace analysis.

Solid phase extraction

Compared with liquid-liquid extraction, solid-phase extraction (SPE) has a high degree of commercialization and a wide variety of products. Commercial solid phase extraction columns are uniformly packed and consume less solvent, thereby reducing pollution and improving the stability of the method. Therefore, solid-phase extraction is fast, simple, efficient, and has good reproducibility. It is especially suitable for extracting trace samples, and can basically meet the requirements in actual testing. It is the most commonly used purification method in the pretreatment of plant growth regulator testing. Maki Kobayashi et al.［15］ used an HLB column and PSA solid phase extraction column to analyze the acetone extract of forchlorfenuron from fruits, GeLiya et al.［16］ used an Oasis MAX solid phase extraction column to analyze the gibberellin GA1 and GA3, and Sharma D et al.［17］ used a Florisil column to purify the methanol extract of paclobutrazol from mangoes. They all achieved good purification effects. Xu et al.［18］ used acetonitrile to extract 205 pesticides including paclobutrazol in apple samples, and purified the extract by PSA and C18 solid phase extraction column, and the recoveries of paclobutrazol at the three different addition levels of 0.01, 0.02 and 0.04 mg/kg were 92.9%, 96.5% and 96.4%, respectively.

Gel permeation chromatography

Gel permeation chromatography (GPC) used in sample purification can better shorten the time and improve the purity. Chen et al.［19］ extracted endogenous hormones such as IAA, GA3, ZT, and ABA from chestnuts. After centrifuging the extract, the supernatant was purified by PVPP column and DEAE Sephadex A-25 column, and then used a Sep-Pak C18 cartridge to collect the endogenous plant hormones in the sample. After eluting with 50% methanol aqueous solution, the extract was analyzed finally by high performance liquid chromatography. The average recoveries were 98.80%, 97.81%, 89.60% and 101.54%, respectively. The method greatly simplified the pre-treatment operating procedures and improved the recovery efficiency.

QuEChERS solid phase extraction

The QuEChERS solid-phase extraction technique was first proposed by Anastassi-adas et al.［20］. It is a rapid pretreatment technique developed on the basis of dispersive solid-phase extraction, which has the characteristics of quick, easy, cheap, effective, rugged and safe, and has been widely used in the analysis of plant growth regulators［21-22］. Lee et al.［23］ compared three extraction methods: liquid-liquid extraction (LLE), pressurized liquid extraction (PLE), and QuEChERS. Among them, the QuEChERS method had higher recovery and precision than those of other two methods.

Thin layer chromatography

On the basis of solvent extraction, thin layer chromatography (TLC) is also a commonly used method for further separation and purification of samples. Choosing an appropriate developing agent is one of the key factors of the TLC method. Wu et al.［24］ used chloroform with Rf values of 0.83, 0.63, and 0.44 as the developing agent for the separation and purification of such as three plant growth regulators as 5-nitroguaiacol sodium salt, sodium salicylate, and sodium paraben, respectively, achieving very good separation effects, and the recovery reached 102.7%, 102.3% and 96.45%, respectively.

Shuyuan XU et al. Research Advances in Detection Methods of Plant Growth Regulators for Agricultural products

Analysis method of plant growth regulators

The analysis techniques currently applied to plant growth regulators mainly include gas chromatography, gas chromatography-mass spectrometry, high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry, and various spectral analysis methods.

Gas chromatography (GC) and gas-mass spectrometry (GC-MS)

The GC method is a common method for the analysis of pesticide residues. It is used for the determination of easily volatile pesticides, and mostly used for the determination of ethephon in the detection of plant growth regulators［25］. Tseng et al.［26］ used headspace gas chromatography to analyze ethephon residues in apples, tomatoes, grapes, kiwifruit and sugarcane based on the characteristic of ethephon decomposing to produce ethylene at high temperatures. Chu et al.［27］ also used headspace gas chromatography to determine the ethephon in concentrated pineapple juice. The pretreatment of the headspace sampling method is extremely simple, and the method is sensitive and stable, and very suitable for the detection of ethephon in food. The GC-MS method has stronger qualitative ability and lower detection limit. It also has many applications in the determination of plant growth regulators. Wu et al.［28］ determined the residues of 10 plant growth regulators in bean sprouts, and Claudia et al.［29］ determined indole-3-acetic acid, abscisic acid, jasmonic acid, and salicylic acid in tobacco roots.

High performance liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology

LC and LC-MS/MS techniques are the most common analysis methods for plant growth regulators. Compared with GC, the pretreatment of LC technique is more flexible, simple, and does not require derivatization reaction. When combined with a mass detector, it can achieve a lower detection limit and a more powerful qualitative ability. Zhang［30］ studied in detail the LC and LC-MS/MS determination methods of various plant growth promoter residues in fruits and vegetables. Hu et al.［31］ used HPLC-diode array detector (DAD) to establish a method for the determination of the bishydrazide plant growth regulator JS-118 in cabbage and soil samples. Lu et al.［32］ established an analysis method for 2,4-D, gibberellin, paclobutrazol and 6-BA in watermelon. Zhou et al.［33］ used the HPLC-DAD method to develop a method for common plant growth regulators in 7 kinds of fruits and vegetables. The 7 compounds were well separated and the detection limit was as low as 0.004 mg/kg.

Spectroscopy

Fluorescence analysis is an important method for analysis at trace, ultra-trace and even molecular level. It has the advantages of good selectivity, high sensitivity, less sampling, simple and fast, etc., and has been widely used in the analysis of plant growth regulators in recent years. Wan et al.［34］ found that the ethanol solution of ABA did not emit fluorescence, but the system would emit strong fluorescence (excitation wavelength 373 nm, emission wavelength 490 nm) after adding concentrated sulfuric acid and heating in boiling water. Base on this, they established a fluorescence analysis method. Huang et al.［35］ studied the effects of acidity, light time, temperature and coexistents on fluorescence intensity based on the nature of plant growth regulators naphthaleneacetic acid (NAA) and IAA that can emit fluorescence under ultraviolet light excitation, and established a new method for the simultaneous determination of NAA and IAA components through multivariate calibration without separation.

Other analytical methods

Enzyme-linked immunosorbent assay (ELISA) has the advantages of high specificity, high sensitivity, quickness and simplicity, and easy promotion, but its disadvantage lies in the high false positive rate. Watanabe et al.［36］ established an ELISA method for the determination of inabenfide in rice, which achieved a good correlation when compared and verified with HPLC and GC methods. Capillary electrophoresis technology is a combination of classic electrophoresis technology and microcolumn technology. It not only has high separation efficiency and short time, but also has the effect of online enrichment of samples, which is very beneficial to trace plant growth regulators and endogenous plant hormones. Cheng［37］ used dual-wavelength capillary electrophoresis to determine four growth promoters, and the detection limit could reach 1.2-6.0 μg/ml. Kubilius et al.［38］ used SPE pretreatment combined with capillary micellar electrokinetic chromatography to analyze maleic hydrazide in potatoes and onions, and the limit of quantification reached 2 mg/kg.

Conclusions

China is one of the countries with the largest number of regulators in the world. In order to avoid potential health hazards to humans caused by their residues, to break the trade technical barriers of developed countries and regions against the regulator residues in Chinas exported fruits and vegetables, in addition to increasing the guidance on their use, the improvement of analysis and detection technology is also an important and arduous task. Due to the wide varieties of regulators, different chemical structures and properties, complex components to be tested, and generally low residual levels, more effective pre-treatment techniques and more sensitive detection techniques are required.

In addition, under the premise of ensuring qualitative and quantitative accuracy, the analysis of modulators with pesticides, germicides, herbicides and other agrochemical residues is also a need for multi-residue testing, and adequate attention should also be given to the analysis and detection of the metabolites of modulators and their potential risks. It is foreseeable that with the advancement of science and technology and the continuous development of modern detection methods, the analysis methods of regulators in fruits and vegetables will become more mature, and their applications in agrochemical residue analysis is bound to become more and more extensive, and will gradually be used in daily agrochemical residue monitoring.

References

［1］ MENDES AFS, CIDADE C, OTONI WC, et al. Role of auxins, polyamines and ethylene in root formation and growth in sweet orange［J］. Biologia Plantarum, 2011, 55(2): 375-378.

［2］ XU QJ. Research on the application technology of plant growth regulators on greenhouse vegetables［J］. Modern Agricultural Science and Technology, 2010(2): 206-207. (in Chinese)

［3］ YU YM, LIU CX, ZHU YY. An improved HPLC method for detecting inner hormones in cucumber fruit［J］. Shandong Agricultural Sciences, 2008(7):97-99. (in Chinese)

［4］ YANG TX, WEI AZ, ZHENG Y. Simultaneous determination of 8 endogenous hormones in apricot floral bud by high performance liquid chromatography［J］. Chinese Journal of Analytical Chemistry, 2007, 35(9): 1359-1361. (in Chinese)

［5］ SONG P, CAO XZ, SHEN ZD. Determination of four kinds of plant hormones (IAA, ABA, GA3, GA4) in rice seedlings by gas liquid chromatography［J］. Journal of Jiangsu Agricultural College, 1997, 18(2): 11-13. (in Chinese)

［6］ CHEN XP, WANG XF, SUN XL. Determination of gibberellin abbeymycin and abscisic acid in cucumber by high-performance liquid chromatography［J］. Shandong Agricultural Sciences, 2005(1): 65-67. (in Chinese)

［7］ ANDRZEJ M, RAKOCZY A, HUTTERMANN A. Improvements in methods for determination of abscisic acid and indole-3-acetic acid by high-performance liquid chromatography［J］. Journal of Chromatography A, 1986(357): 399-408.

［8］ ZHANG J, WANG LL, LIU HY, et al. Determination of N-6-benzylaminopurine in vegetables by high performance liquid chromatography［J］. Analytical Chemistry, 2010, 38(1): 147-150. (in Chinese)

［9］ HU YL, LI YW, ZHANG Y, et al. Development of sample preparation method for auxin analysis in plants by vacuum microwave-assisted extraction combined with molecularly imprinted clean-up procedure［J］. Analytical and Bioanalytical Chemistry, 2011, 399(10): 3367-3374.

［10］ SANCHO JV, POZO OJ, ZAMORA T, et al. Direct determination of paclobutrazol residues in pear samples by liquid chromatography-electrospray tandem mass spectrometry［J］. Journal of Agricultural an Food Chemistry, 2003, 51(15): 4202-4206.

［11］ TANG LJ, TAN T, WAN YQ. Determination of plant growth regulators in white radish by ultrasonic extraction-high performance liquid chromatography-electrospray ionization mass spectrometry［J］. Food Science, 2012, 33(14): 136-141. (in Chinese)

［12］ SANNINO A, BOLZONI L, BANDINI M. Application of liquid chromatography with electro-spray tandem mass spectrometry to the determination of a new generation of pesticides in processed fruits and vegetables［J］. Chromatogr A, 2004, 1036(2): 161-169.

［13］ SHARMA D, AWASTHI MD. Behaviour of for chlorfenuron residues in grape, soil and water［J］. Chemosphere, 2003(50): 589-594.

［14］ LI RJ, YU JL, SONG GC, et al. Degradation dynamic and residue determination of forchlorfenuron in Chinese goosebeery fruit and soil［J］. Shandong Agricultural Sciences, 2009(2): 78-80. (in Chinese)

［15］ KOBAYASHI M, TAKANO I, TAMURA Y, et al. Clean-up method of forchlorfenuron in agricultural products for HPLC analysis［J］. Journal of the Food Hygienic Society of Japan, 2007, 48(5):  148-152.

［16］ LIYA GE, CLARE YUNN CHYN PEH, JEAN WAN HONG YONG, et al. Analyses of gibberellins by capillary electrophoresis-mass spectrometry combined with solid-phase extraction［J］. Chromatogr A, 2007(1159): 242-249.

［17］ SHARMA D, AWASTHI MD. Uptake of soil applied paclobu-trazol in mango (Mangifera indica L.) and its persistence in fruit and soil［J］. Chemosphere, 2005, 60(2): 164-169.

［18］ XU XL, LI L, ZHONG WK, et al. Multi-residue analysis of 205 crop pesticides using mini-solid phase extraction-large volume injection-GC-MS［J］. Chromatogr A, 2009, 70(1-2):  173-183.

［19］ CHEN JH, CAO Y, LI LZ. High-efficient liquid chromatographic determination of the internal hormones in Castanea mollissima Blume［J］. Journal of Central South Forestry University, 2004, 24(5): 39-41. (in Chinese)

［20］ ANASTASSIADES M, LEHOTAY SJ, STAJNBAHER D, et al. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce［J］. J JAOAC Internat, 2003, 86(2): 412-431.

［21］ WANG J, CHOW W, CHEUNG W. Application of a tandem mass spectrometer and core-shell particle column for the determination of 151 pesticides in grains［J］. J Agr Food Chem, 2011(59): 8589-8608.

［22］ HUANG HH, XU GM, ZHOU Y, et al. Determination of several natural plant growth regulators in fruits by high performance liquid chromatography-tandem mass spectrometry ［J］. Food Safety and Quality Detection Technology, 2014, 5(4): 1133-1141. (in Chinese)

［23］ LEE JM, PARK JW, JANG GC, et al. Comparative study of pesticide multi-residue extraction in tobacco for gas chromatography-triple quadrupole mass spectrometry［J］. J Chromatogr A, 2008, 1187(1): 25-33.

［24］ WU Y, BAO FR, ZOU JF. TLC Determination of sodium o-nitrophenate, sodium p-nitrophenate and sodium 5-nitroguaiacate in plant-growth-helping substance［J］. Chemical World, 2000(9): 490-491. (in Chinese)

［25］ CHEN WJ, ZHANG YH, LI YC, et al. Research progress on analysis methods of commonly used plant growth regulators in fruits and vegetables［J］. Food Science, 2012, 33(11): 283-289. (in Chinese)

［26］ TSENG SH, CHANG PC, CHOU SS, et al. A rapid and simple method for the determination of ethephon residue in agricultural products by GC with headspace sampling［J］. Journal of Food and Drug Analysis, 2000, 8(3): 213-217.

［27］ CHU XG, YONG W, CAO HX, et al. Rapid determination of ethephon residues in concentrated pineapple juice by head-space gas chromatography［J］. Chinese Journal of Chromatography, 2001, 19(3): 286-288. (in Chinese)

［28］ WU PG, TAN Y, ZHANG J, et al. Fractional purification combined with gas chromatography-mass spectrometry to determine 10 plant growth regulators in bean sprouts［J］. Analytical Chemistry, 2014, 42(6): 866-871. (in Chinese)

［29］ CLAUDIA B, ANIA K, JOACHIM K, et al. Comprehensive chemical derivatization for gas chromatography-mass spectrometry-based multi-targeted profiling of the major phytohormones［J］. Journal of Chromatography A, 2003, 993(1/2): 89-102.

［30］ ZHANG Y. HPLC and HPLC-MS/MS methods for detecting plant growth promoter residues in fruits and vegetables［D］. Urumchi: Xinjiang University, 2012: 25-46. (in Chinese)

［31］ HU JY, DENG ZB, QIN DM. Determination of diacylhydrazines: Type insect growth regulator JS-118 residues in cabbage and soil by high performance liquid chromatography with DAD detection［J］. Bulletin Environmental Contamination Toxicology, 2009, 83(6): 803-807.

［32］ LU YM, YI GB, CHEN CB, et al. Study on method of determination of four plant growth regulater residues in watermelons［J］. Journal of Instrumental Analysis, 2011, 30(2): 186-189. (in Chinese)

［33］ ZHOU YM, XIN X. Determination of plant hormone residues in vegetables and fruits by high performance liquid chromatography［J］. Food Science, 2010, 31(18): 301-304. (in Chinese)

［34］ WAN Y. A study on the fluorescence properties of abscisic acid［J］. Journal of Xuzhou Normal University: Natural Sciences Edition), 2002, 20(2): 39-41. (in Chinese)

［35］ HUANG CF, CHEN HL, LONG WQ. Application of multivariate calibration to simultaneous fluorescence determination of α-naphthylaceticacid and indole-3-aceticacid［J］. Journal of Jinggangshan University: Natural Science, 2008, 29(6): 52-53, 74. (in Chinese)

［36］ WATANABE E, TSUDA Y, WATANABE S, et al. Development of an enzyme immunoassay for the detection of plant growth regulator inabenfide in rice［J］. Analytica Chimica Acta, 2000, 424(2): 149-160.

［37］ CHEN Y. Simultaneous separation and determination of four auxins by capillary electrophoresis with dual wavelengths［C］//Proceedings of the 3rd Chromatography Academic Exchange Conference in Central and Western China. Xian: Chinese Chemical Society, Shaanxi Provincial Chemical Society, 2010: 217-218. (in Chinese)

［38］ KUBILIUS DT, BUSHWAY J. Determination of maleic hydrazide in potatoes and onions by capillary electrophoresis［J］. Journal of Agricultural and Food Chemistry, 1998, 46(10): 4224-4227.