Journal of Hazardous Materials 273 (2014) 287–292

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Rapid and sensitive suspension array for multiplex detection of organophosphorus pesticides and carbamate pesticides based on silica–hydrogel hybrid microbeads Xuan Wang a , Zhongde Mu b , Fengqi Shangguan b , Ran Liu a , Yuepu Pu a , Lihong Yin a,∗ a b

Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, Jiangsu, China State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu, China

h i g h l i g h t s • Silica–hydrogel hybrid microbeads were used to develop suspension array. • The results in detecting pesticides agree well with those from LC–MS/MS. • The method showed the good capability for multiplex analysis of pesticides residues.

a r t i c l e

i n f o

Article history: Received 1 October 2013 Received in revised form 2 February 2014 Accepted 4 March 2014 Available online 16 March 2014 Keywords: Photonic Suspension array Fluorescent immunoassay Pesticides

a b s t r a c t A technique for multiplex detection of organophosphorus pesticides and carbamate pesticides has been developed using a suspension array based on silica–hydrogel hybrid microbeads (SHHMs). The main advantage of SHHMs, which consist of both silica and hydrogel materials, is that they not only could be distinguished by their characteristic reflection peak originating from the stop-band of the photonic crystal but also have low non-specific adsorption of proteins. Using fluorescent immunoassay, the LODs for fenitrothion, chlorpyrifos-methyl, fenthion, carbaryl and metolcarb were measured to be 0.02 ng/mL, 0.012 ng/mL, 0.04 ng/mL, 0.05 ng/mL and 0.1 ng/mL, respectively, all of which are much lower than the maximum residue limits, as reported in the European Union pesticides database. All the determination coefficients for these five pesticides were greater than 0.99, demonstrating excellent correlations. The suspension array was specific and had no significant cross-reactivity with other chemicals. The results for the detection of pesticide residues collected from agricultural samples using this method agree well with those from liquid chromatography–tandem mass spectrometry. Our results showed that this simple method is suitable for simultaneous detection of these five pesticides residues in fruits and vegetables. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Over recent years, the application of anthropogenic pesticides to agricultural products around the world to protect against insects, fungi and other pests has become common practice, but overuse and incorrect use can pose risks to human health and the environment [1,2]. Since pesticides are widely used and are inadequately supervised, these serious problems of food safety have become a considerable cause of public concern all over the world. According to incomplete statistics, about 70% of the total quantity of pesticides used consists of organophosphorus (OP) pesticides and carbamate (CM) pesticides [3]. These are therefore the pesticides

∗ Corresponding author. Tel.: +86 25 83272583; fax: +86 25 83272583. E-mail address: [email protected] (L. Yin). http://dx.doi.org/10.1016/j.jhazmat.2014.03.006 0304-3894/© 2014 Elsevier B.V. All rights reserved.

most widely used in agriculture due to their relatively low persistence under natural conditions and high effectiveness for insect eradication. Regulations have been established by government agencies and international organizations to ensure that concentrations of pesticides in food and the environment are lower than the maximum residue limits (MRLs) [4]. As a consequence, the development of suitable analytical methods to support these MRLs is required. Therefore, a rapid, sensitive and reliable quantitative analysis method for pesticides is of tremendous importance. There has been great progress in the development of detection methods for OP pesticides and CM pesticides such as liquid/gas chromatography–mass spectrometry [5–7], high performance liquid chromatography [8] and enzyme-linked immunosorbent assays (ELISAs) [9–11]. However, chromatography based methods have significant drawbacks, such as the requirement for sophisticated equipment, skilled operators and time-consuming sample

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preparation steps. Although traditional ELISAs have recently gained prominence as alternative methods for the analysis of pesticides, due to their simplicity and cost-effectiveness, they need large sample volumes and numerous washing and preparation steps. In addition, these methods cannot detect several pesticides simultaneously using a small volume of sample and are thus unsuitable for screening large numbers of samples. Recently, some new techniques were developed for the simultaneous determination of small analytes. Most of these assays are based on planar microarrays, which are encoded by the coordinates of their positions, while the kinetics of molecular reaction on the carriers is diffusion limited [12]. The suspension array has evolved from the planar microarray. This uses self-encoded microcarriers as elements to produce a high-throughput and efficient screening technology. It offers distinct advantages in the forms of faster binding kinetics, greater flexibility, higher sensitivity and lower sample consumption [13–15]. Several encoding technologies have been developed for suspension arrays, such as the use of fluorescent dyes, quantum dots and photonic crystals. Although encoding with fluorescent dyes has been commercialized by Luminex and other companies [16], there still exist some problems. For instance, there are mutual interference between the fluorescent encoding signal and the fluorescent detection signal during the detection procedures; fluorescent dyes are easily quenched and bleached. Recently, photonic crystal encoding has been proposed for suspension arrays due to low fluorescent background and high mechanical stability after calcination [17]. Furthermore, the encoding uses the characteristic reflection peak originating from the stop-band of the photonic crystal, which does not interfere with the detection signal [18]. Polyethylene glycol diacrylates (PEG-DA) hydrogels have been widely used in biosensing applications for many years and have excellent biological characteristics, such as resistance to surface protein adsorption, good hydrophilicity and biocompatibility. Thus, silica and hydrogel are frequently used in fabrication of encoded microbeads [18,19]. However, the suspension array base on silica photonic crystal microbeads (SPCMs) has certain problems such as non-specific adsorption and reduced activity of surface-bound biomolecules while the encoding of hydrogel photonic beads is instable since the hydrogel is soft and easy to transform [18,20,21]. In order to solve the problems above, we fabricated silica–hydrogel hybrid microbeads (SHHMs) by combining complementary silica and hydrogel materials. There are two advantages of SHHMs as microcarriers for suspension array [20,22–27]: on the one hand, they have characteristic reflection peaks originating from the stop-band of the photonic crystal for encoding; on the other hand, blended hydrogel composed of PEG-DA and AA (acrylic acid) could supply the functional carboxyl groups for connecting with antigens or antibodies and reduce non-specific adsorption. As far as we know, there have been few reports on the application of such suspension array for the detection of pesticides based on the combination of silica and hydrogel. In this paper, we report the design of a new type of suspension array for multiplex detection of OP pesticides and CM pesticides using SHHMs as encoding elements. Fenitrothion (FNT), chlorpyrifos-methyl (CLT), fenthion (FT), carbaryl (CBL) and metolcarb (MTL) were selected as model analytes to explore the feasibility and capability of the suspension array for the determination of pesticide residues. The suspension array is based on an indirect competition immunoassay and a biotin–streptavidin signal amplification system for pesticides detecting. The selected pesticides are detected through the competition of the specific monoclonal antibodies between the multiple pesticides and the antigens (the pesticides conjugated with BSA), which are immobilized on different types of SHHMs in solution. If the sample contained the target pesticides, the antigens on the surface of SHHMs would compete with the target pesticides for binding with

Scheme 1. Schematic illustration of the suspension array. (A) The antigens of the pesticides were covalently immobilized on the SHHMs. (B) The antigens of the pesticides on the surface of beads and the free target pesticides were allowed to compete for their corresponding mAbs in solution. (C) SecAb-biotin solution was added. (D) SA-PE was added to the solutions and the fluorescence intensities of beads were measured.

mAb (monoclonal antibody) and secAb-biotin (goat anti-mouse IgG secondary antibody, labeled with biotin), which are subsequently recognized by SA-PE (streptavidin-R-phycoerythrin). Then, the fluorescein signals would decrease when more target pesticides were available. Scheme 1 shows a schematic illustration of the detection procedures. With the use of spiked agricultural samples, the accuracy of the developed suspension array was confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). 2. Materials and methods 2.1. Materials CLT standard, FNT standard and bovine serum albumin (BSA) were brought from Sigma Chemicals. FT standard, CBL standard and MTL standard were purchased from Aladdin Reagent (Shanghai, China). Monodispersed silica nanoparticles were synthesized using the Stöber method [28]. Monoclonal antibodies (mAbs) and antigens (the pesticides conjugated with BSA) were customized from Zoonbio Biotechnology Co., Ltd. (Nanjing, China). Goat anti-mouse IgG secondary antibody, labeled with biotin (secAb-biotin) was purchased from Boster bio-engineering Co., Ltd. (Wuhan, China). PEG-DA with weight-average molecular weights of 700 and 2hydroxy-2-methylpropiophenone (HOMPP) photoinitiator were purchased from Aldrich. Acrylic acid (AA) was obtained from Sinopharm Chemical Reagent Co., Ltd. N-Hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and streptavidin-R-phycoerythrin (SA-PE) were purchased from Pierce (Rockford, IL, USA). 2-Morpholinoethanesulfonic acid (MES) was purchased from Amresco LLC (Solon, USA). All other reagents were of the best grade available and used as received. All buffers were prepared with water purified in a Milli-Q system (Millipore, Bedford, MA). PBS–TBS was prepared from PBS buffer (0.01 M pH 7.4) by adding Tween-20 (0.05%, v/v), BSA (1%, v/v) and sodium azide (0.02%, v/v). The blocking buffer consisted of PBS containing 5% (v/v) BSA. 1 mg/mL CHLM, FNT, FT, CBL and MTL stock standard solutions were prepared in methanol and stored at −20 ◦ C. 2.2. Instrumentation A custom made capillary microfluidic device was used to generate SPCMs with five different kinds of characteristic reflection spectra [29]. Photographs of beads were taken with an

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Fig. 1. Photographs and SEM images of five SHHMs. Photographs of five SHHMs and their reflection spectra (A) and SEM images (B and C).

optical microscope (OLYMPUS BX51) equipped with a CCD camera (Media Cybernetics Evolution MP 5.0). The antigen–antibody reaction was carried out in a constant temperature shaker (Eppendorf Thermomixer comfort 5355). The microstructures of SHHMs were characterized using scanning electron microscopy (SEM, Hitachi, S-300N). Reflection spectra of the beads were recorded using a microscope equipped with a fiber optic spectrometer (Ocean Optics, USB2000). The fluorescence intensity of beads was recorded with an inverted fluorescence microscope (Olympus IX51) equipped with a fiber optic spectrometer (Ocean Optics, QE65000).

in solution competed with the antigens immobilized on SHHMs by competitive bonding with a fixed supply of free mAbs in solution. After incubation for 30 min in the constant temperature shaker at 37 ◦ C, the vessels were washed three times with PBS–TBS. SecAbbiotin solution (diluted in PBS–TBS) was then added for another incubation under the same conditions. Because the test vessel was flat-bottomed, SHHMs can roll slowly at the bottom, ensuring that antigens on the surface of SHHMs were in full contact with antibodies with free binding sites in solution. After washing, SA-PE was added and incubated for 25 min. Then, the encoding signal and the fluorescence signal were measured.

2.3. Fabrication of SHHMs and probe immobilization

2.5. Sampling and analysis of agricultural samples

As the precursor of SHHMs, five kinds of SPCMs, with different characteristic reflection spectra, were fabricated by a microfluidic device [18,30]. The SHHMs were prepared by allowing the pregel solution fill the voids between SPCMs. The pregel solution was composed of different proportions of PEG-DA, AA and HOMPP (1%, V/V). There are three steps to fabricate the SHHMs. These are hydrophilic treatment with piranha solution (30% hydrogen peroxide and 70% sulfuric acid) for 6 h, immersion in the pregel solution for 30 min and exposure to UV light for the polymerization of the pregel solution left in the voids of SPCMs for 2 min. Due to capillary forces, the gap between the SPCMs is filled with hydrogel. After the hydrogel skins were peeled off, five kinds of SHHMs with different characteristic reflection peaks were obtained (Fig. 1). Subsequently, antigens of the pesticides were covalently immobilized on SHHMs by an EDC/NHS-mediated reaction. The beads were immersed in the MES buffer containing 2 mM EDC, 5 mM NHS and antigens at room temperature overnight. The amino groups of the antigens then combined with the carboxyl groups on the surface of beads. After washing, a blocking buffer was used at room temperature to block the unbound active sites.

Apple, lettuce, cabbage, cucumber and tomato from a local farm were chosen for recovery studies to validate our suspension array for simultaneous detection of the selected pesticides. All the samples were separated into two groups to compare the suspension array technology with LC–MS/MS. As confirmed by LC–MS/MS, these samples did not contain residues of the selected pesticides. When samples were finely chopped, each 1.0 g of the samples was spiked with different levels of these five pesticide standards dissolved in methanol (1.0 mL) and then shaken for 2 h. After centrifugation for 5 min (5000 rpm), the supernatant was transferred and evaporated to near dryness. Subsequently, the residue was dissolved in 1.0 mL of 10% methanol–PBS and shaken for 5 min. The obtained samples were analyzed by the established suspension array. We also measured all the samples spiked with different concentrations of these five pesticides using LC–MS/MS according to the Chinese National Standard method (GB/T 20769-2008).

2.4. Multiplex detection of pesticides

To produce SHHMs, PEG-DA hydrogel was applied to perfuse SPCMs as the bioinert nature of hydrogels offers the advantage of reduced non-specific adsorption on the surface of beads, which benefits the improvement of sensitivity. Furthermore, it provided a protective environment for the immobilized proteins and inhibited degradation and fouling [31]. As shown in Fig. 1A, five types of SHHMs were produced and the characteristic reflection peaks of SHHMs were red shifted by 10–15 nm compared with SPCMs. However, the colors of beads did not change and the encodings of SHHMs were not affected. We examined the effects of different proportions

The multiplex detection method developed to determine the selected pesticides is considered to be a competitive binding test, based on the antigen coated format, and using CHLM-BSA, FNTBSA, FT-BSA, CBL-BSA and MTL-BSA as the coating conjugates. To simultaneously detect the selected pesticides, five types of SHHMs with probe immobilization, five kinds of mAb solutions and five kinds of standard pesticide solutions of different concentrations were added and mixed thoroughly in a vessel. The free pesticides

3. Results 3.1. Optimization of hydrogel composition

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Fig. 2. Optimization of experimental conditions. Effects of different amounts of antigens (A) and incubation time (B) on fluorescence intensities. Each point was obtained by detecting 5 SHHMs.

of PEG-DA and AA on the fluorescence intensities as shown in Fig. S1 (CHLM-BSA as a model). The fluorescence intensities reached a maximum value when the composition of blended hydrogel (group 3) is 89% PEG-DA (V/V) and 10% AA (V/V). Although AA is introduced to supply the carboxyl functionality for protein antigen conjugation, the lowest AA proportion obtains the highest fluorescence intensities. It could be explained in two cases. On the one hand, high proportion of PEG-DA will result in more hydrogel exposed on the surface of beads. On the other hand, hydrogel could swell when the proportion of PEG-DA is over 40% [27]. These will increase the probability of combination with more protein antigen as more functional carboxyl groups in hydrogel could be exposed on the surface. Meanwhile, high proportion of PEG-DA leads to small mesh size and adsorbed protein on the surface of beads is easily cleared out. Hence, this reduces non-specific adsorption (group 3). The prepolymer solution containing PEG-DA (89%, V/V), AA (10%, V/V) and HOMPP (1%, V/V) was used throughout this study. Fig. 1B and C shows the SEM images of SHHMs. 3.2. Optimization of experimental conditions In order to establish optimal experimental conditions for multiplex detection of these five pesticides, the experimental parameters were investigated systematically, including the amount of antigens, antibodies and incubation time (in this work, the measured fluorescence intensities are taken as representing the effect of all factors). First, we studied the effect of different amounts of antigens. In

order to discover the optimized addition ratio, the concentration of reacted antigens was varied when the other parameters were controlled. There were differences between the obtained fluorescence intensities and the different amounts of antigens added (Fig. 2A). With the increase of the quantity of antigens, the fluorescence intensities reached a maximum and then decreased. In addition, the fluorescence intensities increased slowly when approaching the maximum. Therefore, 40, 60, 60, 80 and 80 ng was chosen as the optimized additional amounts of CHLM-BSA, FNT-BSA, FT-BSA, MTL-BSA and CBL-BSA, respectively. With excess antigens, the fluorescence intensities apparently decreased, which might be because the excess antigens accumulated randomly on the surface of beads. Due to steric hindrance and inter-molecular repulsion, the reaction between antigens and antibodies might be affected. The reaction efficiency of the immunoassay depends on the concentration of antigen and antibody because only when the dose of antigen and antibody are in optimum proportion, can the macromolecule immune complexes be formed. Using optimal additions of antigens, the chess board titration was used to optimize the dose of mAbs and secAb-biotin (Table 1). When the concentration of secAbbiotin was constant, the fluorescence intensities of all the pesticides showed an increase when increasing the concentration of mAbs. At the same time, the fluorescence intensities reduced when the dose of secAb-biotin was increased. On the basis of the highest obtained fluorescence intensities, the optimal dose of mAb for CHLM, FNT, FT, MTL and CBL was selected as 0.4, 0.6, 0.8, 2 and 1 ng, respectively. In addition, 5 ng was chosen as the optimal dose of secAb-biotin.

Table 1 Fluorescence intensities obtained by chessboard titration for pesticides (X ± SD, n = 5). Pesticide

Dose of mAb (ng)

Intensity (a.u.) Dose of secAb-biotin (ng) 5

10

20

CHLM

0.1 0.2 0.4

808.34 ± 66.23 1192.33 ± 25.38 1540.23 ± 47.55

679.53 ± 18.89 1005.66 ± 72.67 1311.29 ± 54.44

515.7 ± 19.65 723.98 ± 17.92 989.53 ± 25.44

FNT

0.15 0.3 0.6

1076.38 ± 26.24 1875.35 ± 21.35 2322.78 ± 35.47

755.25 ± 38.45 1344.89 ± 28.34 2019.76 ± 37.45

538.53 ± 15.65 782.68 ± 38.93 1659.55 ± 51.54

FT

0.2 0.4 0.8

2025.95 ± 37.23 3215.45 ± 52.22 4645.23 ± 67.65

1091.89 ± 22.43 2263.25 ± 30.33 3654.79 ± 75.81

1158.85 ± 19.78 1735.75 ± 49.45 2576.37 ± 29.35

MTL

0.5 1 2

5148.65 ± 105.32 7628.92 ± 115.68 9905.37 ± 174.22

4276.95 ± 110.52 5749.25 ± 128.83 8268.75 ± 154.38

3892.78 ± 79.54 4738.29 ± 89.73 6736.55 ± 135.33

CBL

0.25 0.5 1

3735.66 ± 68.94 5385.42 ± 137.82 6472.33 ± 107.27

3345.58 ± 82.44 4775.48 ± 79.45 5748.78 ± 118.34

2643.56 ± 48.69 4053.68 ± 101.77 4286.39 ± 93.55

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Table 2 Standard curves for multiplex detection of pesticides by the proposed method. Pesticide

Standard curve

R2

FNT CHLM FT CBL MTL

y = −30.785 + 2411.886/[1 + (x/8.667)0.638 ] y = −33.930 + 1519.385/[1 + (x/11.275)0.701 ] y = 50.808 + 4362.412/[1 + (x/2.667)1.417 ] y = 20.769 + 6077.307/[1 + (x/9.257)1.121 ] y = 149.225 + 9782.329/[1 + (x/2.109)1.436 ]

0.994 0.999 0.993 0.999 0.996

Intensity (x ± SD, n = 5) Blank control

In the competitive immunoassays, the incubation time for the antigen–antibody interaction had significant influence on the analytical performance of the immunoassay. The incubation time was tested using different time intervals, from 10 to 50 min. As shown in Fig. 2B, with the increase of incubation time, all the fluorescence intensities increased and then tended to reach their maximum values after 30 min. Longer incubation times did not significantly change the fluorescence intensities. Considering the optimal analytical performance and further development of this method to high sample throughput, an incubation time of 30 min was selected for the following study. Compared with the 1–3 h at 37 ◦ C required for a traditional microwell plate ELISA, our suspension array required a shorter incubation (only 30 min). In general, the conditions optimized for suspension array were shown in Table S4. The optimized amount of antigens for the selected pesticides was 40, 60, 60, 80 and 80 ng. The optimal dose of mAb for these five pesticides was 0.4, 0.6, 0.8, 2 and 1 ng. The optimal dose of secAb-biotin was 5 ng. The incubation time was 30 min. 3.3. Standard curve plotting and multiplex detection for the five pesticides In order to provide homogeneous solutions, a buffer with a small amount of methanol (less than 10%) is often used to prepare standard or sample solutions. This does not influence the competitive immunoassays significantly. In this work, the standard concentrations of pesticides under analysis were prepared by diluting stock standard solutions with 10% methanol–PBS. For simultaneous detection, these five pesticide standard solutions were mixed before being used for the suspension assay. As shown in Fig. 3, standard curves were obtained for the multiplex detection of the selected pesticides in 10% methanol–PBS. The fluorescence intensities were plotted against the logarithm of the concentration of pesticides using a four-parameter logistic model as follows [32–35]: y=

A1 − A2 1 + ([Ag]/[Ag0 ])

p

+ A2

2287.74 1502.44 4511.86 6156.36 9832.18

± ± ± ± ±

91.36 48.33 95.47 177.78 223.45

Min DC 2231.29 1445.72 4450.25 6110.53 9780.40

± ± ± ± ±

82.65 59.33 81.24 155.88 183.65

where [Ag] is the concentration of the pesticide, [Ag0 ] is the pesticide concentration at inflection, A1 and A2 are the fluorescence intensities at zero analyte concentration and infinite analyte concentration and p is the slope factor. The equations for the standard curves are also shown in Table 2. There were negative logistic correlations between fluorescence intensities and the concentrations of pesticides. All the determination coefficients (R2 ) for these five pesticides were greater than 0.99, which demonstrates excellent correlations. The ranges of detection were 0.02–1562.5 ng/mL, 0.012–937.5 ng/mL, 0.04–1250 ng/mL, 0.05–819.2 ng/mL and 0.1–218.7 ng/mL for FNT, CHLM, FT, MTL and CBL, respectively. MRLs regulated by the European Union (EU) for fruits and vegetables are as follows: 10–50 ng/mL for FNT, 50–500 ng/mL for CHLM, 10 ng/mL for FT and 10 ng/mL for CBL. No data are available for MTL. Because there were no significant differences between the obtained fluorescence intensities of the blank control and the groups of the minimum detectable concentration (Min DC), the Min DC could be considered as the LOD. Thus, the LODs of FNT, CHLM, FT, MTL and CBL were 0.02, 0.012, 0.04, 0.05 and 0.1 ng/mL, respectively, which are much lower than the MRLs reported in the European Union pesticides database. Hence, the method described can meet current standards for the detection of these five pesticides in fruits and vegetables. The proposed method appears to be an excellent method for simultaneously determining these five pesticides. 3.4. Cross-reactivity Cross-reactivity (CR) is an important analytical parameter concerning specificity and reliability of multiplex immunoassays. Several OP pesticides and CM pesticides were tested for CR using the proposed suspension array. The chemical structures of the pesticides used in this study are presented in Fig. S2. The CR values of the antibodies were calculated using the formula: CR% =

C  0

C

× 100

where C0 is the concentration of the target pesticide at 50% inhibition and C is the concentration of the cross-reacting pesticide at 50% inhibition. As shown in Table S3, the CR values of all the different pesticides were very small, typically below 5%. There was no cross-reaction between the different antibodies and any of the other heterologous pesticides. Hence, these OP pesticides and CM pesticides did not interfere with the performance of the multiplex immunoassay. Clearly, the suspension array is very specific. 3.5. Analysis of spiked vegetable samples

Fig. 3. Standard curves of the multiplex detection for five pesticides.

The performance of the suspension array was evaluated by comparing the results obtained by the proposed method using spiked samples (pesticide concentrations 25, 50 and 100 ng/mL) with those obtained from the analysis of similar samples by LC–MS/MS. For each concentration level, three replicate experiments were performed. As demonstrated in Table S1, the recovery rates for CHLM, FNT, FT, MTL and CBL were in the range 88.9–106.2%,

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82.6–106.3%, 85.4–105.5%, 88.7–104.3% and 85.6–103.5%, respectively. These recovery rates showed good agreement with those of the LC–MS/MS method, which were in the range 85.9–104.3%, 88.4–105.6, 86.7–103.6%, 84.6–102.7 and 85.3–105.6% for CHLM, FNT, FT, MTL and CBL, respectively (Table S2). The relative standard deviations (RSDs) were