Application of a mixed-mode solid-phase extraction and cleanup procedure for LC/MS determination of thiabendazole and carbendazim in apple juice

Recently, a mixed-mode solid-phase extraction (SPE) procedure was developed for rapid extraction and cleanup for determination of the fungicides thiabendazole and carbendazim in various fruit juices. This paper reports the application of that sample preparation procedure to the liquid chromatographic/mass spectrometric determination of these fungicides in apple juice with detection by positive electrospray ionization mass spectrometry (ESI/MS). Response was linear for sample concentrations from 2 to 500 microg/L (ppb). Recoveries averaged 74% (9% RSD) for carbendazim and 93% (9% RSD) for thiabendazole. After SPE cleanup, no matrix supression was observed for the ESI+ response for either compound studied. The method was applied to the analysis of incurred residues in 4 store-bought apple juices; carbendazim levels ranged from 10 to 70 microg/L and thiabendazole levels ranged from less than 2 to 130 microg/L.     

固相萃取-HPLC法同时测定浓缩柑橘汁中噻菌灵、多菌灵的残留量

建立了一种可同时测定浓缩柑橘汁中噻菌灵和多菌灵残留量的反相HPLC分析法。浓缩柑橘汁样品用水适当稀释后,经过调节pH值、离心、过滤,用混合相固相萃取小柱(m ixed-mode SPE)进行提取、净化,用配有二级管阵列检测器(DAD)的液相色谱仪检测,外标法定量。使用噻菌灵和多菌灵对照品进行添加回收试验,噻菌灵、多菌灵的回收率分别为80.8%~87.1%、82.9%~87.7%,二者的测定下限均为0.020 mg/kg,测定结果的相对标准偏差均不大于7.38%(n=10)。     

超高效液相色谱-串联质谱法快速测定浓缩果汁中噻菌灵和多菌灵的残留量

建立了同时测定浓缩果汁中噻菌灵和多菌灵残留的超高效液相色谱-串联质谱快速检测法。样品用乙酸乙酯提取,以ACQUITY UPLC BEH C18色谱柱(50 mm×2.1 mm, 1.7 μm)进行超高效液相色谱分离,以电喷雾电离串联质谱正离子多反应监测(MRM)模式进行测定,以基质匹配标准溶液外标法定量。结果表明:在试验条件下,噻菌灵和多菌灵在0.5~10 μg/kg范围内线性关系良好,相关系数大于0.99,不同基质中的检出限(S/N=3)范围为0.12~0.23 μg/kg。在0.5、1.0和5.0 μg/kg 3个水平下噻菌灵和多菌灵的加标回收率为76.98%~108.7%,相对标准偏差(RSD)为2.95%~9.99%。同时,本研究对浓缩果汁中噻菌灵和多菌灵残留检测的基质效应进行了考察。本方法具有操作简便、快速、准确的特点,可用于浓缩果汁中噻菌灵和多菌灵残留量的日常检测。     

Determination of seventeen polar/thermolabile pesticides in apples and apricots by liquid chromatography/mass spectrometry

A simple liquid chromatography/mass spectrometry (LC/MS) approach for the determination of widely used representatives of polar/thermolabile pesticides in fruits was developed and validated. The group of pesticides comprised benzimidazoles and azoles (carbendazim, thiabendazole, imazalil, propiconazole, prochloraz, epoxiconazole, flusilazole, tebuconazole, bitertanol); N-methylcarbamates (carbaryl, carbofuran, methiocarb); and phenylureas and benzoylphenylureas (linuron, diflubenzuron, triflumuron, teflubenzuron, flufenoxuron). Matrixes (apple, apricot) were extracted with acetonitrile and crude extracts were cleaned up by solid-phase extraction (SPE) using either mixed cation exchange or hydrophilic lipophilic balance cartridges. LC separation of pesticides was performed on a reversed-phase column, Discovery C18. Electrospray ionization and ion trap MS/MS detection were applied. For most pesticides, overall recoveries ranged from 75 to 122%, and repeatability (as relative standard deviation) from 5 repetitive determinations of recovery ranged from 3 to 21%. Carbofuran was the only compound for which recovery was not satisfactory due to its loss in the SPE cleanup step. Limits of detection were 0.1-3 microg/kg for benzimidazole and azole fungicides and carbamate insecticides. For urea insecticides, detection limits were slightly higher (3-10 microg/kg).     

Determination of thiabendazole in fruit juices by a new monoclonal enzyme immunoassay

A competitive, indirect enzyme-linked immunosorbent assay (ELISA) for thiabendazole has been developed and applied to the analysis of fruit juices spiked with this fungicide. The immunoassay is based on a new monoclonal antibody derived from a hapten functionalized at the nitrogen atom in the 1-position of the thiabendazole structure. To our knowledge, such a structure has not been previously used to obtain antibodies to thiabendazole. The I50 value and the detection limit of the ELISA for standards were 0.2 and 0.05 ng/mL, respectively. Fruit juices were analyzed by diluting samples in assay buffer, without extraction or cleanup. Samples were not even centrifuged or filtered to remove fruit pulp. Under these conditions, the immunoassay was able to accurately determine thiabendazole down to 1 ng/mL in orange and grapefruit juices, down to 5 ng/mL in banana juice, and down to 20 ng/mL in apple and pear juices. Sensitivity differences of the ELISA were caused by the minimum dilution required by each juice to minimize matrix effects: 1/10 for orange and grapefruit juices, 1/50 for banana juice, and 1/100 for apple and pear juices. In an attempt to further increase the sensitivity of the immunoassay for matrixes showing the strongest interferences, apple and pear juices spiked with thiabendazole at low levels (1-20 ng/mL) were extracted with ethyl acetate before analysis. This simple procedure entailed a significant reduction of matrix effects, which in fact allowed us to determine accurately as low as 5 ng/mL thiabendazole in apple and pear juices. Irrespective of whether samples were analyzed by the direct dilution method or after extraction, the simplicity, sensitivity, and sample throughput of this monoclonal immunoassay makes it a very convenient method for the routine monitoring of thiabendazole residues in fruit juices.     

Analyses of pesticide residues in fruit-based baby food by liquid chromatography/electrospray ionization time-of-flight mass spectrometry

A liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) method has been developed for the determination of 12 pesticides (namely, carbendazim, thiabendazole, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil, iprodione, diphenylamine and procymidone) in fruit-based baby food (multi-fruit jars and juices intended for infant consumption). The developed method consists of a sample treatment step based on liquid-liquid extraction using acetonitrile, followed by a clean-up step based on dispersive solid-phase extraction (SPE) with a primary-secondary amine (PSA). Multi-fruit and apple juices were processed by a SPE procedure using Oasis HLB cartridges. Subsequent identification and quantitation was accomplished by LC/ESI-TOFMS analysis: the confirmation of the target pesticides was based on accurate mass measurements of selected ions (protonated molecules ([M+H]+) and fragment ions). Confirmation studies were accomplished at low concentration levels (10 microg kg-1) and accuracy errors lower than 2 ppm were obtained in most cases. Baby food extracts spiked at 10 microg kg-1 fortification level yielded average recoveries in the range 78-105% with relative standard deviations less than 10% for most of the analytes. Limits of detection (LODs) were between 0.1 and 4 microg kg-1 depending on the pesticide studied. Finally, the proposed method was applied to a total of 33 baby food samples from Spain and the United Kingdom. Although imazalil, thiabendazole and carbendazim were detected in a high number--over 60%- of baby food samples, none of the samples tested were found to be above the 0.01 mg kg-1 EU standard.     

Determination of carbendazim,thiabendazole, and<Emphasis Type="Italic"> o-</Emphasis>phenylphenol residues in lemons by HPLC following sample clean-up by ion-pairing

An efficient analytical method is presented involving effective sample clean-up with solid-phase extraction and HPLC-UV analysis for the simultaneous determination of carbendazim, thiabendazole, and o-phenylphenol residues in lemons. Sample preparation involves extraction with acetonitrile acidified with trifluoroacetic acid and an ethyl acetate/petroleum ether mixture. Purification of the crude extract was carried out with liquid–liquid partitioning after addition of an aqueous ammonia solution. Final clean-up was performed on polymeric reversed-phase cartridges pretreated with sodium dodecyl sulfate. Chromatographic analysis was performed on a reversed-phase HPLC column isocratically eluted with an acetonitrile/water/ammonia mixture and UV detection at 254 nm. The chromatographic method is repeatable, reproducible, and sensitive. Fungicide recoveries from lemon samples fortified at levels of 5 and 1 mg kg^–1 were 81–85% for carbendazim, 96–98% for thiabendazole, and 81–106% for o-phenylphenol with coefficients of variation of 2.5–7.4%. Detection limits for carbendazim, thiabendazole, and o-phenylphenol in lemons were 0.21, 0.27, and 0.51 mg kg^–1, respectively.     

Trace determination of carbendazim, fuberidazole and thiabendazole in water by application of multivariate calibration to cross-sections of three-dimensional excitation-emission matrix fluorescence

The simultaneous determination of carbendazim, fuberidazole and thiabendazole was accomplished by cross-section (CS) fluorimetry in combination with multivariate calibration algorithms. The total luminescence information of the compounds was used to optimise the linear trajectories of the CS. A comparison between principal component regression (PCR) and two partial least squares (PLS) algorithms, PLS-1 and PLS-2, with different pre-processing methodologies was made. The final model, which applied the PLS-1 method, built using pesticide standard and emission spectra, was successfully used for the determination of these compounds in synthetic mixtures. However, a different PLS-1 multivariate calibration model, based on CS through the total luminescence spectroscopic data, was necessary for determining the cited pesticides in water samples. Mean centring was the best pre-processing technique in both PLS-1 models. This later calibration model was built from ultra-pure water samples spiked with known carbendazim, fuberidazole and thiabendazole concentrations, after solid-phase extraction (SPE). The method, which had a precision better than 5%, was shown to be suitable for carbendazim, fuberidazole and thiabendazole monitoring in water samples at trace levels.     

Liquid chromatographic-mass spectrometric determination of post-harvest fungicides in citrus fruits

Liquid chromatography (LC)-atmospheric pressure ionisation (API)-mass spectrometry (MS) has been used to determine residues of five fungicides in oranges with a minimum sample cleanup. Atmospheric pressure chemical ionisation (APCI) and electrospray (ES) were compared and both gave similar results in terms of sensitivity and structural information. The main ions were [M+H]+ for carbendazim, imazalil, thiophanate methyl and thiabendazole, and [M+H-C4H9NHCO]+ for benomyl. Samples were extracted with sodium sulphate and ethyl acetate. Although benomyl and thiophanate methyl were transformed through the extraction procedure to carbendazim, the method showed good precision (<13%) and recovery (>70%), except for thiophanate methyl (50%), whilst also yielding limits of detection (<0.03 mg kg(-1)) that are adequate for the determination of the studied fungicides in oranges.     

Quantitative determination of incarvillateine in Incarvillea sinensis by solid phase extraction and high performance liquid chromatography

Method for rapid quantitative analysis of incarvillateine in Incarvillea sinensis by high-performance liquid chromatography (HPLC) has been developed. The sample preparation involves solid phase extraction (SPE) with a mixed-mode reversed-phase and cation-exchange cartridge. The linear calibration range for incarvillateine was 0.002-0.5 mg/ml. The limit of detection was 0.35 microg/ml (S/N=3). Intra- and interday precisions were less than 0.36% (n=6) and 1.61% (n=18), respectively. The recovery of incarvillateine was 97.61-102.44% with the relative standard deviation (RSD) ranging from 0.63 to 1.93% (n=3). This method was proposed as a simple, rapid and accurate method for quantitative determination of incarvillateine content in various samples of Incarvillea sinensis collected from different areas of China.     

Mixed-mode chromatography for fractionation of peptides, phosphopeptides, and sialylated glycopeptides

A mixed-mode chromatographic (MMC) sorbent was prepared by functionalizing the silica sorbent with a pentafluorophenyl (PFP) ligand. The resulting stationary phase provided a reversed-phase (RP) retention mode along with a relatively mild strong cation-exchange (SCX) retention interaction. While the mechanism of interaction is not entirely clear, it is believed that the silanols in the vicinity of the perfluorinated ligand act as strongly acidic sites. The 2.1 mm x 150 mm column packed with such sorbent was applied to the separation of peptides. Linear RP gradients in combination with salt steps were used for pseudo two-dimensional (2D) separation and fractionation of tryptic peptides. An alternative approach of using linear cation-exchange gradients combined with RP step gradients was also investigated. Besides the attractive forces, the ionic repulsion contributed to the retention mechanism. The analytes with strong negatively charged sites (phosphorylated peptides, sialylated glycopeptides) eluted in significantly different patterns than generic tryptic peptides. This retention mechanism was employed for the isolation of phosphopeptides or sialylated glycopeptides from non-functionalized peptide mixtures. The mixed-mode column was utilized in conjunction with a phosphopeptide enrichment solid phase extraction (SPE) device packed with metal oxide affinity chromatography (MOAC) sorbent. The combination of MOAC and mixed-mode chromatography (MMC) provided for an enhanced extraction selectivity of phosphopeptides and sialylated glycopeptides peptides from complex samples, such as yeast and human serum tryptic digests.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

Analysis of selected pharmaceuticals in fish and the fresh water bodies directly affected by reclaimed water using liquid chromatography-tandem mass spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple commonly used therapeutic classes was developed for biological tissues (fish), reclaimed water, and the surface water directly affected by irrigation with reclaimed water. One gram of fish tissue homogenate was extracted by accelerated solvent extraction with methylene chloride followed by mixed-mode cation exchange solid phase extraction (SPE) cleanup and analyzed by liquid chromatography-tandem mass spectrometry. Compared to previously reported methods, the protocol produces cleaner extracts resulting in lower method detection limits. Similarly, an SPE method based on Oasis HLB cartridges was used to concentrate and cleanup reclaimed and surface water samples. Among the 11 target compounds analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin, and fluoxetine were consistently detected in reclaimed water. Caffeine, diphenhydramine, and carbamazepine were consistently detected in fish and surface water samples. Bioaccumulation factors for caffeine, diphenhydramine, and carbamazepine in mosquito fish (Gambusia holbrooki) were calculated at 29?±?26, 821?±?422, and 108?±?144, respectively. This is the first report of potential accumulation of caffeine in fish from a water body directly influenced by reclaimed water.

Validation of a method for the determination of multiclass pesticide residues in fruit juices by liquid chromatography/tandem mass spectrometry after extraction by matrix solid-phase dispersion

A multiresidue method was developed and validated for the determination of pesticide residues (omethoate, dimethoate, carbendazim, propoxur, thiabendazole, carbaryl, pirimicarb, azinphos-methyl, methidathion, and iprodione) in fruit juices. The samples were extracted by matrix solid-phase dispersion with diatomaceous earth and analyzed by liquid chromatography/tandem mass spectrometry. The method detection limits were <0.2 ppb for all pesticides; the relative standard deviations for analyses of samples fortified over the range of 2-50 ng/g were <9%, and the recoveries for each pesticide were all between 77 and 102%. The proposed method was used to analyze 21 commercial fruit juices; pesticide residues were found in 71% of the samples.     

Selective clean-up applicable to aqueous acetone extracts for the determination of carbendazim and thiabendazole in fruits and vegetables by high-performance liquid chromatography with UV detection

Fungicide residues in vegetables (benomyl, carbendazim, thiabendazole) are analyzed through a clean-up procedure that uses a portion of the aqueous acetone extract prepared for multiresidue methodology. A portion of the aqueous acetone extract (equivalent to 5 g of vegetables) is loaded onto an Extrelut-20 cartridge (the cartridge is filled with a coarse, large-pore diatomaceous material). Then, acetone is partially removed by an upward stream of nitrogen at 2l/min for 30 min. Benzimidazolic fungicides are recovered by percolating the cartridge with 100 ml of 0.1 M phosphoric acid solution, which also serves to convert benomyl to carbendazim. The percolating acid solution is drained on-line through a strong cation-exchange (SCX) solid-phase extraction cartridge with the aid of a slight vacuum. Benzimidazolic fungicides are retained on the SCX cartridge. The phosphoric acid solution is discarded together with the washings of the SCX cartridge, i.e., water followed by methanol-water (75:25), that remove unwanted coextractives. Finally, benzimidazolic fungicides are recovered by eluting the SCX cartridge with methanol-ammonium formate buffer (75:25). The final extract is then analyzed by reversed-phase HPLC with UV detection. Recoveries from crops such as apples, lettuce, strawberries and citrus fruits are generally greater than 80% and no interferences were observed. The clean-up is simple and straightforward, requires only disposable items, water solutions and a few milliliters of solvent and a minimum number of manipulations, and does not require concentration steps or electrical equipment.     

1,3,5‐Triethynylbenzene and melamine as monomers to synthesize three‐dimensional network porous aromatic frameworks based silica/florisil for determination of carbendazim and thiabendazole in spinach

In this study, the new and efficient three‐dimensional network porous aromatic frameworks materials called Silica‐PAFs‐a, Florisil‐PAFs‐a, Silica‐PAFs‐b, and Florisil‐PAFs‐b were first synthesized. The properties of materials were analyzed by five characterization methods. The materials were used as adsorbents in pipette‐tip solid‐phase extraction for the effective determination of carbendazim and thiabendazole in spinach sample. Meanwhile, the obtained materials were tested by static adsorption and dynamic adsorption. The result showed that the specific surface area of materials greatly increased after introducing three‐dimensional network porous aromatic frameworks. Microstructural modification exposed a large number of amino reactive groups that made them have a better adsorption amount for the two targets. The calibration graphs of carbendazim and thiabendazole in methanol were linear over 0.10–300.0 µg/mL, and the limits of detection and quantification were 0.00546 and 0.0182 µg/mL, and 0.00741 and 0.0247µg/mL respectively. A reliable analytical method was developed for recognition targets in spinach sample by Silica‐PAFs‐b with satisfactory extraction recoveries (96.25 and 100.51%). The proposed method using the material was applied for trace analysis of the carbendazim and thiabendazole residue.     

Determination of carbendazim, thiabendazole and fuberidazole using a net analyte signal-based method

The net analyte signal (NAS)-based method HLA/GO, modification of the original hybrid linear analysis (HLA) method, has been used to determine carbendazim, fuberidazole and thiabendazole in water samples. This approach was used after a solid-phase extraction (SPE) step, using the native fluorescence emission spectra of real samples, previously standardized by piecewise direct standardization (PDS). The results obtained show that the modification of HLA performs in a similar way that partial least-squares method (PLS-1). The NAS concept was also used to calculate multivariate analytical figures of merit such as limit of detection, selectivity, sensitivity and analytical sensitivity (γ⁻¹). With this purpose, blanks of methanol and ternary mixtures, with the target analyte at low concentration and the other two ranging according to the calibration matrix, were used, with different results. Detection limits calculated in the last way are more realistic and show the influence of the other components in the sample. Selectivity for carbendazim is higher than the corresponding values for fuberidazole and thiabendazole, whereas sensitivity, as well as the values obtained for their detection limits, are lower for carbendazim, followed by thiabendazole and fuberidazole. Results obtained by modification of HLA vary in the same way that the ones obtained by PLS-1.     

Determination of imidacloprid and benzimidazole residues in fruits and vegetables by liquid chromatography-mass spectrometry after ethyl acetate multiresidue extraction

A simple and sensitive method based on liquid chromatography-atmospheric pressure ionization-mass spectrometry is described for the determination of 4 benzimidazole pesticides (carbendazim, thiabendazole, benomyl, and thiophanate-methyl) and imidacloprid in vegetables and fruits. Food samples were typically extracted with ethyl acetate to draw the analytes into the organic phase. No cleanup step was necessary before injection into the liquid chromatographic (LC) system with electrospray mass spectrometric detection. The analytes were separated on a reversed-phase C8LC column. Limits of detection for the compounds were in the microg/L range. Results are reported for validation studies with fortified pear and tomato samples and for residues of the target compounds found in the pesticide residue monitoring program during 1998.     

Analysis of carbendazim and thiabendazole in lemons by CE-DAD

Summary A capillary electrophoretic method for the determination of two benzimidazole fungicides, carbendazim and thiabendazole, in lemons has been developed. The two fungicides were separated in a law-pH phosphate buffer containing acetonitrile. Reproducibility tests measuring both migration times and peak areas gave low relative standard-deviation values. Calibration graphs were linear and the detection limits, defined as a signal-to-noise ratio of 3:1, were 2.3 and 2.0 μg mL⁻¹ for thiabendazole and carbendazim, respectively. Analysis of carbendazim and thiabendazole residues in lemons was performed after lemon homogenization, extraction with ethyl acetate and purification through a series of separatory-funnel, acid-base partitions. This lemon clean-up procedure ensured no interference in the capillary electrophoretic analysis from lemon endogenous ingredients.     

苹果中多菌灵、噻菌灵和甲基托布津的高效液相色谱法分析

建立了同时检测苹果中多菌灵、噻菌灵和甲基托布津残留量的高效液相色谱(HPLC)分析方法.样品经乙酸乙酯提取,旋转蒸发仪浓缩,氮气吹干甲醇定容后,采用配有二极管阵列检测器(DAD)的HPLC测定,外标法定量.在添加不同浓度的标准品时,多菌灵、噻菌灵和甲基托布津的添加回收率分别为87.7%～118.7%、72.8%～80.3%、64.0%～66.8%.方法对多菌灵、噻菌灵和甲基托布津3种农药的检出限较低,分别为0.134、0.230和0.250mg/L,可以满足苹果汁中多菌灵、噻菌灵和甲基托布津的残留限量检测要求.检测果皮样品中的农药残留量,多菌灵的残留量为7.24×10-2mg/kg,噻菌灵和甲基托布津未检出,低于国标中规定的残留限量标准.