Jie ZHANG，Gang ZHANG，Zhanbin SHEN，Lumei DUAN，Wei ZHANG，Junxia LUO*，Jianbo ZHAO*

1. Zhengzhou Agricultural Product Quality Testing and Distribution Center，Zhengzhou 450006，China; 2. Zhengzhou Agricultural Technology Extension Centre，Zhengzhou 450006，China; 3 Henan Hengsheng Testing Technology Co.，Ltd.，Xinzheng 451100，China

Abstract [Objectives] The paper was to understand the detection effect of commercial enzyme inhibition colorimetric kit. [Methods] Six brands of kits were used to detect pesticide residues in vegetables. The detection results were compared with those of 50 kinds of organophosphorus pesticides and 10 kinds of carbamate pesticides detected by chromatography and mass spectrometry. According to different thresholds，the test results of different kits were evaluated，and the false positive rate，false negative rate and coincidence rate of each kit were obtained. The test results of commercial enzyme inhibition colorimetric kits were compared and analyzed. [Results] The detection effect of kit D was the best among the 6 brands of kits，and the coincidence rate was the highest under the 7 thresholds. There was a certain relationship between the detection effect of commercial enzyme inhibition colorimetric kit and the determination threshold of positive samples. The false positive rate decreased with the increase of determination threshold，and the false negative rate increased with the increase of determination threshold，but the coincidence rate with chromatography and mass spectrometry can not reach 100%. When the threshold was set to 20%，the effect was the best. The coincidence rate of 3 brands of kits with the results of chromatography and mass spectrometry was the highest，and none of the 6 kits involved in the comparison had the lowest coincidence rate under this threshold. [Conclusions] It is suggested to modify the threshold values in national standard and trade standard.

Key words Kit，Pesticide，F(xiàn)alse positive，F(xiàn)alse negative，Accuracy，Determination threshold

1 Introduction

Since 2001 when the Ministry of Agriculture started the pollution-free agricultural products action plan in 37 large and medium-sized cities in China with vegetable as the breakthrough point，the safety of agricultural products has received widespread attention. Agricultural products detection and supervision has become an important work of agricultural departments at all levels，and the detection and supervision of plant agricultural products mainly focuses on the supervision of pesticide residues.TheLawofthePeople’sRepublicofChinaonQualityandSafetyofAgriculturalProductsandtheFoodQualityandSafetyLawofthePeople’sRepublicofChinarevised in 2015 grant the legal status of rapid testing as a means of detection. At present，rapid detection technologies that are widely studied include enzyme inhibition spectrophotometry[1]，molecular imprinting technology[2]，immunoassay technology，etc.[3]. Detection of organophosphorus and carbamate pesticides by cholinesterase spectrophotometry is featured by high efficiency，simplicity and low cost[1]. Cholinesterase spectrophotometry is the mainstream technology for rapid detection of pesticide residues at home and abroad[4-5]，and has been playing an important role in the quality and safety supervision of agricultural products in China. The most widely used enzyme inhibition colorimetric pesticide residue test kit，which contains enzyme，substrate，chromogenic agent and buffer solution，is a non-quantitative and non-qualitative kit[6]. The enzyme in the kit is an important factor determining the quality of the kit，because enzymes from different biological sources have certain differences in structure，and show different sensitivity to organophosphorus and carbamate pesticides[1,7]. Different forms of cholinesterase exist in the same organism[8]，and these cholinesterases have different specific substrates[8-9]. Meanwhile，cholinesterase is not sensitive to thiophosphate pesticides in organophosphorus pesticides，resulting in low detection rate of organophosphorus pesticides[10]. Therefore，commercial kits show different sensitivities to different pesticides[11-13]. Meanwhile，reaction time，reaction temperature and pH value of buffer solution all affect the detection results of enzyme inhibition colorimetric method[14-15]. Many plants，such as lavender，fennel and wild ferns，contain natural substances that inhibit the activity of cholinesterase[16-20]. These factors will directly or indirectly cause false positive or false negative test results，making the test results of the kit deviate from the real situation of the sample. In order to understand the detection effects of commercial enzyme inhibition colorimetric kits，6 brands of commercially available enzyme inhibition colorimetric kits were selected to detect 53 vegetable samples from 15 kinds of fruits and vegetables，such as leek，Shanghai green and Chinese yam，from Zhengzhou market，respectively. The detection results were compared with those of chromatography and mass spectrometry under different determination thresholds to obtain the false positive rate，false negative rate and coincidence rate of each brand of kit，which was combined with the sensitivity of the kit to pesticides[11-13]to select high quality commercial enzyme inhibition colorimetric pesticide residue kit，in order to provide the theoretical basis for agricultural product quality and safety supervision and practical detection work.

2 Materials and methods

2.1 Materials

2.1.1Instruments. CL-BIII 16-channel pesticide residue rapid detector (Shanghai Bona New Technology Institute，Shanghai Fubo Agricultural Science and Technology Co.，Ltd.); WH-861 vortex mixer (Shanghai Kanghua Instrument Factory); HH.W21-Cr 420 electric-heated thermostatic water bath (Beijing 328 Scientific Instrument Co.，Ltd.); SHA-C dual constant temperature water bath oscillator (Jieruier Electric Appliance Co.，Ltd.); 5 mL，200 μL pipette (Thermo，USA); 2010 gas chromatograph (Shimadzu Corporation，equipped with AOC-20i automatic sampler，flame photometric detector and DB-1701 column，30 mm×0.53 mm，0.25 μm); 1290-649 ultra performance liquid chromatography-triple quadrupole mass spectrometer (Agilent Technologies Inc.，USA); Hettic homogenizer (Heidolph SilentCrusher M，Germany); N-EVAPTM-112 nitrogen blower (Organomation Associates，USA); Milli-Q Advantage A10 ultrapure water machine (Millipore Corporation，USA).

2.1.2Materials. (i) The rapid detection kits used in the test included A (Shenzhen)，B (Guangzhou)，C (Dongguan)，D (Xiamen)，E (Hubei)，and F (Shenzhen).

(ii) The pesticide standards included acephate，mevinphos，dichlorvos，naled，trichlorfon，quintiofos，fenitrothion，paraoxon，methamidophos，phosphamidon，phoxim，pirimiphos-methyl，famphur，chlorpyrifos-methyl，phosmet，azinphos-methyl，parathion-methyl，dichlofenthion，chlorpyrifos，pyrimithate，fenthion，terbufos，moncrotophos，trichloronat，bromophos-ethyl，sulfotep，ethion，propetamphos，pyrazophos，profenofos，coumaphos，fenchlorphos，parathion，phorate，omethoate，phosfolan，malathion，triazophos，phosalone，plondrel，azinphos ethyl，isocarbophos，diazinon，isofenphos-methyl，tetrachlorvinphos，EPN，dimethoate，fonofos，disulfoton，dicrotophos，isoprocarb，metolcarb，aldicarb sulfoxide，aldoxycarb，aldicarb，fenobucarb，carbaryl，methomyl，3-hydroxycarbofuran，carbofuran (mass concentration 1 000 mg/L，Agricultural Environmental Protection Scientific Research and Testing Institute).

(iii) Vegetable samples were derived from Zhengzhou market. Market inspectors used kits A or E to test vegetable samples with inhibition rates greater than 50%.

2.2 Preparation of reagents

2.2.1Preparation of reagents in kits. Each brand of pesticide residue detection kit contained buffer salt，enzyme，chromogenic agent and substrate. The buffer salt was dissolved in 500 mL distilled water immediately before use，and enzyme，chromogenic agent and substrate were prepared according to the instructions of each kit (Table 1).

Table 1 Preparation methods and use scales for reagents in several brand kits

2.2.2Preparation of standard products. The standard substances listed in Section2.1.2were diluted to 80 μg/mL reserve solution. Organophosphorus pesticides were grouped and diluted to 0.04 μg/mL mixed standard solutions，and the samples were detected by chromatography and mass spectrometry.

2.3 Detection methods

2.3.1Rapid detection method. The samples were cut into slices or sections of 1 cm size. After mixing，2 g of samples were weighed respectively，and tested with 6 brands of kits in accordance with the requirements of the national standard (GB/T 5009.199-2003RapidDetectionofOrganophorusandCarbamatePesticideResiduesinVegetables，hereinafter referred to as the national standard). Each sample was tested with each kit to get 3 parallel samples. After detection，the inhibition rate was read out and its average value was taken.

2.3.2Detection by chromatography and mass spectrometry. After the samples were mixed and homogenized，25 g of samples were weighed for pre-treatment according to the trade standard (NY/T 761-2008DeterminationofOrganophosphorus，Organochlorine，PrethroidsandCarbamatePesticideResiduesinVegetablesandFruits，hereinafter referred to as the trade standard). Afterwards，gas chromatographic flame photometric detector and liquid chromatography-mass spectrometry (LC-MS) were used for the determination，and each sample was detected once.

2.4 Data processingThe results of rapid detection in Section2.3.1were compared with the results of chromatography and mass spectrometry，and the false positive rate and false negative rate were calculated according to formulae (1) and (2).

False positive rate (%)=Number of samples with inhibition rate greater than the set value/ Number of undetected samples of 60 pesticides by chromatography and mass spectrometry×100%

(1)

False negative rate (%)=Number of samples with inhibition rate less than the set value/number of detected samples of 60 pesticides by chromatography and mass spectrometry×100%

(2)

The set values in the formula were certain inhibition rates，which were 10%，20%，30%，40%，50%，60%，and 70% in Table 2.

Table 2 Test effect of each brand of kit

3 Results and analysis

3.1 Detection effect of each brand of kitAs shown in Table 2，there were false positive and false negative results in the test results of all brands of kits compared with those of chromatography and mass spectrometry. According to the 50% determination threshold of national standard[14]，the detection results with false positive were D>E>C>A>B=F，and the detection results with false negative were A>B=F>C>E>D. The consistency with the detection results of chromatography and mass spectrometry was D>B=C=F>E>A，the maximum coincidence rate was 68% and the minimum coincidence rate was 50.9%. According to the 70% determination threshold of the trade standard (NY/T 448RapidDetectionMethodforOrganophosphorusandCarbamidePesticideResiduesinVegetables)，the number of false positive samples detected by kits A，C and E was larger than that by kits D and F，and larger than that by kit B. In terms of the generation of false negative samples，A>F>B>C=E>D. The consistency with the detection results of chromatography and mass spectrometry was B=C=D>E>F>A，the maximum coincidence rate was 60% and the minimum coincidence rate was 34%. Therefore，the detection effect of commercial enzyme inhibition colorimetric kits and their coincidence rates with detection results of chromatography and mass spectrometry was not only related to the source of enzyme in the kit，but also related to the size of threshold value.

3.2 Relationship between detection effect of each brand of kit and determination threshold

3.2.1Relationship between false positive rate and determination threshold. As shown in Fig.1，the false positive rate of samples detected by each kit decreased with the increase of determination threshold. When the determination threshold was set as 10% inhibition rate，the false positive rate detected by kit F was the highest (100%)，and that detected by kit A was the lowest (66.7%). When the determination threshold was set as 20% inhibition rate，the false positive rate detected by kit B was the highest (81.8%)，and that detected by kits C and D was the lowest (63.6%). When the determination threshold was set as 30% inhibition rate，the false positive rate detected by kits B and F was the highest (63.6%)，and that detected by kit A was the lowest (42.9%). When the determination threshold was set as 40% inhibition rate，the false positive rate detected by kit E was the highest (71.4%)，and that detected by kit B was the lowest (18.2%). When the determination threshold was set as 50% inhibition rate，the false positive rate detected by kit D was the highest (54.5%)，and that detected by kits B and F was the lowest (27.3%). When the determination threshold was set as 60% inhibition rate，the false positive rate detected by kit D was the highest (54.5%)，and that detected by kit B was the lowest (9.1%). When the determination threshold was set as 70% inhibition rate，the false positive rate detected by kits A，C and E was the highest (27.3%)，and that detected by kit B was the lowest (9.1%).

3.2.2Relationship between false negative rate and determination threshold. As shown in Fig.2，the false negative rate of samples detected by each kit increased with the increase of determination threshold. When the determination thresholds were set as 10%，20%，30%，40%，50%，60% and 70% inhibition rates，the false negative rates detected by kit A were the highest，which were 25%，34.4%，50%，53.1%，62.5%，83.9%，and 87.1%，respectively. The false negative rates detected by kit D were the lowest，which were 0，0，0，7.1%，14.3%，28.6% and 42.9%，respectively. When the determination threshold was set as 10% inhibition rate，the false negative rate detected by kit B was equal to that detected by kit D (0). When the determination threshold was set as 30% inhibition rate，the false negative rate detected by kit B was equal to that detected by kit A (50%).

3.2.3Relationship between coincidence rate and determination threshold. As shown in Fig.3，the coincidence rate of the samples detected by each kit did not reach 100%. However，no matter which determination threshold was selected，kit D had the highest coincidence rate and the detection effect was the best among several kits. The coincidence rate of other kits fluctuated slightly with the change of determination threshold. For kit A，when its coincidence rate reached the highest (58.5%)，the determination threshold was set as 10% inhibition rate; when the coincidence rate was the lowest (34%)，the determination threshold was set as 70% inhibition rate. For kit B，when its coincidence rate reached the highest (64%)，the determination threshold was set as 40% inhibition rate; when the coincidence rate was the lowest (44%),the determination threshold was set as 30% inhibition rate. For kit C，when its coincidence rate reached the highest (68%)，the determination threshold was set as 20% or 30% inhibition rate; when the coincidence rate was the lowest (56%)，the determination threshold was set as 40% inhibition rate. For kit D，when its coincidence rate reached the highest (76%)，the determination threshold was set as 30% inhibition rate; when the coincidence rate was the lowest (64%)，the determination threshold was set as 10% inhibition rate. For kit E，when its coincidence rate reached the highest (61.5%)，the determination threshold was set as 10% or 20% inhibition rate; when the coincidence rate was the lowest (46.2%)，the determination threshold was set as 40% inhibition rate. For kit F，when its coincidence rate reached the highest (60%)，the determination threshold was set as 20% or 50% inhibition rate; when the coincidence rate was the lowest (44%)，the determination threshold was set as 40% or 70% inhibition rate.

Fig.1 Broken line chart of false positive rate and determination threshold of each brand of kit

Fig.2 Broken line chart of false negative rate and determination threshold of each brand of kit

Fig.3 Broken line chart of coincidence rate and determination threshold of each brand of kit

4 Conclusions and discussion

In general，kit D had the best detection effect among 6 brands of kits involved in comparison，that is to say，under the 7 determination thresholds，the detection results of kit D had the highest coincidence rate with those of chromatography and mass spectrometry. Commercial enzyme inhibition colorimetric kit is widely used in routine supervision. However，since this kit can neither be quantified nor qualitative[6]，what it measures is the total content of one or two kinds of substances，and false positives and false negatives are common in the detection work，resulting in different coincidence rates with detection results of chromatography and mass spectrometry. There was a certain relationship between coincidence rate and determination threshold. With the increase of the determination threshold，the false positive rate decreased and the false negative rate increased，but the coincidence rate with the detection results of chromatography and mass spectrometry did not continue to increase. The coincidence rate of each kit with the detection results of chromatography and mass spectrometry reached the highest under a certain determination threshold，but the determination threshold that led to the highest coincidence rate varied among 6 kits. In this study，the best effect was achieved when the threshold value was set as 20%. Under this threshold value，the detection effect of 3 brands of kits had the highest coincidence rate with the detection results of chromatography and mass spectrometry. In addition，none of the 6 kits involved in comparison had the lowest coincidence rate under this threshold. Therefore，it is suggested that the determination threshold of positive samples in national standard (GB/T 5009.199-2003RapidDetectionofOrganophosphorusandCarbamatePesticideResiduesinVegetables) and trade standard (NY/T 448-2001RapidDetectionofOrganophosphorusandCarbamatePesticideResiduesinVegetables) should be modified to 20%.