Automated Gel Permeation Chromatography Research Articles

Gas chromatography-isotope dilution mass spectrometry method validation for target pesticides in soybeans

ABSTRACTThe production of certified reference materials requires the application of highly accurate methods for characterisation. A gas chromatography-isotope dilution mass spectrometry method, setting ambitious performance criteria, was developed for eight selected pesticides in soybeans. Pressurised liquid extraction was followed by automated gel-permeation chromatography and solid-phase extraction clean-up. Pesticides identification respected a Commission Decision and guidelines of the Directorate General for Health and Food Safety (DG SANTE). Reliable quantification involved stable isotopically labelled analogues as internal standards. Validation, according to ISO/IEC 17,025 and DG SANTE guidelines, assessed linearity, LOD/LOQ, trueness, selectivity, precision, stability and robustness. Mean recoveries ranges (83–109%, relative standard deviations < 3%), repeatability (2.2–4.8%), day-to-day variation (0.6–4.2%) and combined uncertainty (1.2–4.2%) were fit for purpose. The method is highly accurate and suitable for certification of the selected pesticides in soybean matrix reference material.Chemical compounds studied in this article: Diazinon (PubChem CID: 3017); malathion (PubChem CID: 4004); chlorpyrifos (PubChem CID: 2730); captan (PubChem CID: 8606); endosulfan (PubChem CID: 3224); tebuconazole (PubChem CID: 86,102); iprodione (PubChem CID: 37,517); cypermethrin (PubChem CID: 2912).

Simultaneous determination of substituted diphenylamine antioxidants and benzotriazole ultra violet stabilizers in blood plasma and fish homogenates by ultra high performance liquid chromatography–electrospray tandem mass spectrometry

Analytical methods were developed for the determination of eight substituted diphenylamines (SDPAs) and six benzotriazole UV stabilizers (BZT-UVs) in blood plasma and fish homogenate matrices. Liquid-liquid extraction by methyl tert-butyl ether and denaturation by KOH following silica gel packed column clean-up was employed for blood plasma preparation. For the fish homogenate samples, ultrasonic assisted solvent extraction combined with automated gel permeation chromatography and silica gel packed column clean-up was used. The target compounds were determined by optimized ultra performance liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. The method limits of quantification (MLOQs) of the 14 analytes ranged from 0.002 to 1.5ngg−1 and 0.001 to 2.3ngg−1 (wet weight, w.w.) for blood plasma and fish homogenate, respectively. The total recoveries of the target compounds varied from 61% to 100% (mean 77±9%). Eleven targets including monobutyl- (C4), dibutyl- (C4C4), monooctyl- (C8), monobutyl monooctyl- (C4C8), dioctyl-(C8C8), monononyl- (C9), dinonly-(C9C9) and 4,4′-bis(α,α-dimethylbenzyl)-(diAMS) DPAs, as well as 2-(2H-Benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol (UV234), 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl) phenol (UV327) and 2-(2H-Benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV328) were identified in the environmental biota samples, with concentrations in the range of <MLOQ–934pgg−1, <MLOQ–4.2×103pgg−1 and <MLOQ–3.9×103pgg−1w.w. for bottle nose dolphin (Tursiops truncatus) plasma, northern pike (Esox lucius) plasma and white sucker (Catostomus commersonii) homogenate, respectively. This is the first report of an analytical method development for SDPAs in biotic matrices and BZT-UVs in blood plasma samples.

An improved method for simultaneous analysis of steroid and phenolic endocrine disrupting chemicals in biological samples

An improved method was developed for the simultaneous determination of eight steroid and phenolic endocrine disrupting chemicals, such as oestrone (E1), 17β-oestradiol (E2), oestriol (E3), 17α-ethynylestradoil (EE2), 4-nonylphenol (4-NP), bisphenol A (BPA), 4-tert-octylphenol (4-t-OP) and 4-cumylphenol (4-CP), in biological samples. The optimal extraction and clean-up procedures were investigated using microwave-assisted extraction (MAE), automated gel permeation chromatography (GPC) and solid phase extraction (SPE). As a consequence, the most efficient extraction was achieved by using MAE with methanol as solvent at an extraction temperature of 110°C for 20 min. The clean-up of extracts was carried out by GPC on a Biobeads S-X3 column with cyclohexane/ethyl acetate (1:1, v/v) as mobile phase. Target compounds were eluted in the fraction from 7–14 min retention time. Moreover, the cleanest extracts were obtained by solid phase extraction with C-18 cartridges after the elution with 15 mL ethyl acetate. The final sample extracts were derivatised using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (1% trimethylchlorosilane, TMCS) as derivatisation reagent with pyridine as the solvent. Quantification was performed by gas chromatography-mass spectrometry (GC-MS) with electron ionisation (EI) and selected ion monitoring (SIM) mode. The method was validated by spiked samples which showed good recovery and reproducibility. The overall recoveries ranged between 55.1% and 100.6%, with relative standard deviations (RSD) of 2.3–12.7% for the entire procedure. Method detection limits (MDL) ranged from 0.3 to 0.7 ng g−1 dry weight (dw). Performance of the method was demonstrated by its application on tissues from fish exposed to high concentration of EDCs in the laboratory. The developed method is a promising approach for the analysis of steroid and phenolic endocrine disrupting chemicals in various biological samples.

GC–MS Determination of PAHs in Fish Samples Following Salting-out-Assisted Solvent Extraction-Gel Permeation Chromatography

A fast and effective sample cleanup procedure for the quantification of polycyclic aromatic hydrocarbons (PAHs) in fish samples is presented. The procedure involved extraction of fish samples using acetonitrile and cleanup by an automated gel-permeation chromatography (GPC) following liquid–liquid partition into n-hexane. The extracted samples were analyzed by gas chromatography-mass spectrometry (GC–MS). Electron ionization was employed in a single analysis for the determination of PAHs in the selected ion monitoring mode. Three different solvents were studied for the extraction step: acetonitrile/n-hexane, methanol/n-hexane and acetone/n-hexane. The best solvent was found to be acetonitrile/n-hexane. The cleanup technique resulted in a good separation of analytes from co-extractive matrix compounds.

Analysis of multi-pesticide residues in the foods of animal origin by GC–MS coupled with accelerated solvent extraction and gel permeation chromatography cleanup

A new analytical method was developed to simultaneously determine residues of 109 pesticides (including isomers) in the foods of animal origin. Acetonitrile was selected for accelerated solvent extraction (ASE) for effectively extracting the pesticides from the fatty samples. The cleanup was performed with an automated gel permeation chromatography (GPC) cleanup system. The prepared samples were analysed with GC–MS in the selected ion monitoring mode (SIM) using one target and two qualitative ions for each analyte. Chlorpyrifos-d10 was used as an internal standard. The lowest limit of detection was 0.3μgkg−1 for some pesticides. The recoveries and relative standard deviations (RSDs) were checked by spiking untreated samples with pesticides at 0.05, 0.1 and 0.2mgkg−1. The average recoveries of most pesticides were from 62.6% to 107.8%. The precision values expressed as RSD were all ⩽20.5% (n=6). Good linearity (r⩾0.99) was observed between 0.05 and 1.5μgmL−1.

Pesticide multiresidue analysis of peanuts using automated gel permeation chromatography clean-up/gas chromatography-mass spectrometry

A method based on automated gel permeation chromatography (GPC) and on-line quantitative concentration was developed for the determination of residues of 38 typically used pesticides in high-oil peanuts. Pesticides were extracted using acetonitrile into an oil fraction containing little of the peanut matrix. After changing the solvent to the mobile phase (1 : 1 cyclohexane : ethyl acetate), clean-up was carried out using GPC, the final collected solution being automatically concentrated to a fixed volume and transferred into vials for gas chromatography (GC) injection. The pesticides were analysed by gas chromatography-mass spectrometry (GC/MS) in the selected ion monitoring (SIM) mode. Average recoveries (spiked levels of 0.02, 0.05, and 0.1 µg g−1) were between 70 and 117%. The relative standard deviation of the method was 3.5–21% (n = 6). This approach gave comparable results with a previously published method.

Fast isolation of hydrophobic organic environmental contaminants from exposed semipermeable membrane devices (SPMDs) prior to GC analysis

Semipermeable membrane devices (SPMD) represent a passive sampling technology that is becoming widely used for monitoring of surface waters pollution. While “classic” procedures employ dialysis to recover target compounds from exposed SPMDs, in the present study analytes were isolated from cut membrane together with sequestering medium (triolein) using hexane as an extraction solvent. This approach allowed us to reduce the time needed for accomplishment of isolation step from 48 h to only 1 h. Automated gel permeation chromatography (GPC) clean-up is employed in the following step to separate triolein from analytes fraction. Musk compounds (MCs), polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs) and several other persistent organochlorine compounds (OCs) were determined in the respective fraction by GC method employing selective detectors (MSD, ECD). As shown in a series of analyses of SPMDs deployed in various aquatic ecosystems, high recoveries and good repeatability of results together with a possibility to obtain the information on the pollution of sampling site at the day of sample arrival to laboratory make this newly implemented procedure an interesting alternative to time consuming dialysis.

Development of a accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis

A new analytical method has been developed for the quantification of 59 different persistent organohalogen compounds, such as polybrominated diphenyl ethers (PBDEs), polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), PCB metabolites, organochlorine pesticides (OCPs) in biological organ tissues. The optimum extraction and cleanup procedures were examined using accelerated solvent extraction (ASE), automated gel permeation chromatography (GPC) on Biobeads S-X3 and automated solid phase extraction (SPE) on silica-gel. The target compounds were divided into two fractions, non-polar compounds and more polar compounds, which in the latter fraction was subsequently methylated using diazomethane. Detection can be achieved by GC/MS in negative chemical ionization (NCI) mode. The average recoveries of the compounds spiked in swine liver, heart, kidney, and cattle adipose tissues were considered satisfactory, and it was confirmed that the method could be used in routine analysis.

GC/MS analysis of polybrominated diphenyl ethers in fish collected from the Inland Sea of Seto, Japan

Development of an analytical method for polybrominated diphenyl ethers (PBDEs) in fish and their concentration in Japanese marine fish were investigated. Fish homogenate was extracted with diethyl ether/hexane (1+3). The extract was cleaned up by automated gel permeation chromatography (GPC) and then by mini-column chromatography, which consisted of three layers of silica gel and sulfuric acid-impregnated silica gel. The PBDE fraction was concentrated and injected into a GC/MS with negative chemical ionization (NCI). Recoveries of the 15 individual PBDEs (BDE-15, 28, 37, 47, 66, 71, 75, 77, 85, 99, 100, 119, 153, 154, and 209) each at a fortification level of 4 ng/g lipid were in the range of 88–128% and the relative standard deviations (RSD) were 0.43–7.6% ( n= 4). Seven species of marine fish (conger eel, flounder, gray mullet, horse mackerel, red sea bream, sea bass, and yellowtail) were collected from the Inland Sea of Seto, and were analyzed with the developed method. Seven PBDEs (BDE-28, 47, 66, 99, 100, 153, and 154) were detected in all the samples. The most abundant PBDE congener was BDE-47 found in all the samples. Relatively high levels of PBDEs were found in the gray mullets and yellowtails.

Development of a gas chromatographic method for fungicide cymoxanil analysis in dried hops.

An analytical method for detecting cymoxanil [2-cyano-N-[(ethylamino)carbonyl]-2-(methoxyimino)acetamide] residues in dried hops was developed utilizing liquid-liquid partitioning, automated gel permeation chromatography (GPC), solid phase extraction (SPE) cleanup, and gas chromatography (GC). Method validation recoveries from dried hops were 96 +/- 12, 108 +/- 11, and 136 +/- 8% over three levels of fortification (0.05, 0.5, and 1.0 ppm, respectively). The hop samples from three field sites, which were treated with cymoxanil, had residue levels ranging from 0.146 to 0.646 ppm. The detection limit and the quantitation limit of the method developed in the present study were 0.022 and 0.050 ppm, respectively.

Gas chromatographic-mass spectrometric method for the analysis of dimethomorph fungicide in dried hops.

An analytical method for the determination of dimethomorph [(E,Z)-4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine] residues in dried hops was developed utilizing liquid-liquid partitioning, automated gel permeation chromatography (GPC), Florisil and aminopropyl solid phase extraction (SPE) column cleanups, and gas chromatography (GC) with mass selective detection (MSD). Method validation recoveries from dried hops ranged from 79 to 103% over four levels of fortification (0.1, 1.0, 5.0, and 20 ppm). Control and dimethomorph-treated hop samples collected from three field sites had residue levels of <0.10 and 4.06-17.32 ppm, respectively. The method was validated to the limit of quantitation at 0.10 ppm. The limit of detection for this method was 0.045 ppm.

Automated Gel Permeation Chromatographic Clean-up of Human Adipose Tissue for Multiresidue Analysis of Organochlorine Compounds

Human adipose tissue samples have been analysed for organochlorine pesticides and polychlorinated biphenyls (PCBs). Following a micro liquid-solid extraction, clean-up is performed by automated gel permeation chromatography. The GPC separation provides clean extracts, allowing subsequential analysis by capillary gas chromatography-electron capture detection without additional clean-up.

Multiresidue Method for Determination of Organophosphorus Insecticide Residues in Fatty Processed Foods by Gel Permeation Chromatography

A multiresidue method for quantitative determination of 39 organophosphorus compounds (parent pesticides and their major metabolites) in 7 fatty processed foods is described. Samples are extracted with methylene chloride and cleaned up by automated gel permeation chromatography with a Biobeads SX3 column and a methylene chloride-cyclohexane (15 + 85) eluant. Organophosphorus compounds are quantitated by capillary gas-liquid chromatography with flame photometric detection using OV-1701 and DB-5 columns. Average recoveries from samples fortified at 0.025-1 mg/kg ranged from 50.6% for dichlorvos to 185% for malaoxon. Determination limits were between 0.005 and 0.040 micrograms/mL. Results were confirmed by gas chromatography/mass spectrometry with selected-ion monitoring.

A Rapid Multiresidue Screen for Organophosphorus, Organochlorine, and N-Methyl Carbamate Insecticides in Plant and Animal Tissues

A multiresidue screen for the quantitative determination of 43 organophosphorus, 17 organochlorine, and 11 N-methyl carbamate insecticides in 10 g of plant or animal tissues is described. The insecticides are extracted with 5% ethanol in ethyl acetate (v/v). Samples with high lipid content are cleaned up by automated gel permeation chromatography with a 30% ethyl acetate in hexane (v/v) eluant and in-line silica gel minicolumns. Highly pigmented samples are cleaned up with class-specific solid-phase extraction columns. The concentrated extracts are analyzed by selective detection with gas chromatography or liquid chromatography. Recovery of 71 insecticides ranged from 77 to 113%. Analysis of fortified bovine liver (n = 5) resulted in an average recovery of 96 +/- 4% at the 0.5 to 0.05 micrograms/g level. Analysis of fortified alfalfa hay (n = 5) resulted in a mean recovery of 94 +/- 4% at the 0.06 to 0.5 micrograms/g level, and analysis of fortified fresh tomatoes (n = 5) resulted in an average recovery of 97 +/- 3% at the 0.06 to 0.5 micrograms/g level. Method detection limits ranged from 0.02 to 0.5 micrograms/g for the compounds studied with a nominal 10 g sample.

Non-aqueous gel permeation chromatography of wheat starch in dimethylacetamide (DMAC) and LiCl: extrusion-induced fragmentation

Automated gel permeation chromatography (GPC) with application of the universal calibration concept was used to investigate the mechanism of extrusion-induced starch fragmentation in wheat. High and low protein flours were subjected to twin-screw extrusion and the effects of moisture, die temperature, screw speed, mass flow rate and protein content on starch structure and textural properties were investigated by non-aqueous GPC in dimethylacetamide (DMAC) and LiCl. This solvent system allowed for complete dissolution of the native and extruded starches. The use of refractive index and viscosity detectors enabled application of Mark-Houwink calculations to obtain quantitative size profiles of unprocessed and processed starch, and information describing branching patterns of the starch. Fragmentation was most pronounced in amylopectins of MW 10 7–10 8, which yielded fragments of MW 10 5–10 7. Of the operating parameters investigated, low die temperature and low moisture content led to extensive fragmentation. Methylation analysis showed only modest changes in linkage distributions, with little or no increase in terminal glucose indicating few fragmentation points relative to the total number of glycosidic linkages present. The lack of dextrins or oligosaccharides suggests that fragmentation occurs primarily in the B chains of amylopectin. Interactions between die temperature and moisture content were shown to significantly effect the hardness, cohesiveness, springiness, gumminess and chewiness of the extruded flours.

Gas chromatographic determination of organophosphorus insecticides and their dialkyl phosphate metabolites in liver and kidney samples

A method is described for the quantitative determination of organophosphorus insecticides and six dialkyl phosphate metabolites in animal tissues. The procedure relies on solvent extraction, automated gel permeation chromatography cleanup, and gas chromatographic analysis with flame photometric detection. Tetrabutylammonium hydroxide is utilized as the derivatization reagent for dialkyl phosphate metabolite analysis. Method detection limits ranged from 0.02 to 0.05 ppm for the compounds studied. The method was applied to the analysis of liver and kidney samples of pigeons exposed to diazinon and parathion. Residue levels were confirmed by mass spectrometry, using selected ion monitoring.

Rapid multiresidue procedure for the determination of pesticides in anhydrous lanolin and lanolin-containing pharmaceutical preparations utilizing gel permeation chromatography cleanup with gas chromatographic and mass spectrometric techniques

The proposed multiresidue method has been used to quantify residues of 17 different pesticides found in anhydrous lanolin and lanolin-containing pharmaceutical preparations. Lanolin wares are separated from the residues using automated gel permeation chromatography (GPC). Resultingeluates are injected directly into gas chromatographs using a variety of wide-bore capillary columns. A flame photometric detector in the phosphorus mode (FPD/P) is used to quantify organophosphorus pesticides. Organochlorine residues are determined through the use of electrolytic conductivity detection (ELCD)

Multiresidue Screen for Organophosphorus Insecticides Using Gel Permeation Chromatography—Silica Gel Cleanup

A multiresidue screen for quantitative determination of 43 organophosphorus insecticides in 5 g of plant and animal tissues is described. The organophosphorus insecticides are extracted with methanol-dichloromethane (10 + 90, v/v) and cleaned up using automated gel permeation chromatography with hexane-ethyl acetate (60 + 40) eluant and in-line silica gel minicolumns. Concentrated extracts are analyzed by gas chromatography with flame photometric detection. The method recovers 43 organophosphorus insecticides in the range of 72 to 115%. Analysis of fortified bovine liver (n = 5) shows an average 95.9 +/- 4.8% recovery at the 0.05 micrograms/g level and 93 +/- 3.8% at the 0.5 micrograms/g level. Analysis of fortified bovine rumen content (n = 5) shows an average 98 +/- 4.2% recovery at the 0.1 micrograms/g level and 98.7 +/- 2.8% at the 1 micrograms/g level. Method detection limits ranged from 0.01 to 0.05 micrograms/g for the compounds studied using a nominal 5 gram sample.

Rapid Quantitative Measurement of Extrusion‐Induced Starch Fragmentation by Automated Gel Permeation Chromatography

AbstractGel permeation chromatography with application of the universal calibration concept was found to be a rapid and effective method for quantification of extrusion‐induced starch fragmentation. This method (Timpa. J.D., J. Agr. Food Chem. 39 (1991), 270–275) employs dimethyl acetamide (DMAC) with lithium chloride to solubilize samples and automated gel permeation chromatography for fractionation. Instrumentation was equipped with a viscometer and refractometer, data was interpreted by application of the universal calibration concept. These results confirm that the largest starch molecules in corn meal (107 to 108 Daltons) were slightly prone to fragmentation. Fragments produced were primarily in the weight range of 104–107 Daltons. In addition, some smaller fragments (ranging between 5,000 and 7.500) were also generated by extrusion.

A Displacement Pump Procedure to Load Extracts for Automated Gel Permeation Chromatography

Automated gel permeation chromatography (GPC) effectively separates lipids from pesticides in sample extracts that contain fat. Using a large syringe to load sample extracts manually onto GPC models having 5 mL holding loops is awkward, slow, and potentially hazardous. Loading with a small-volume displacement pump, however, is convenient and fast (ca 1 loop every 20 s). And more importantly, the analyst is not exposed to toxic organic vapors because the loading pump and its connecting lines do not leak in the way that a syringe does.