Supercritical fluid extraction of pesticides from meat: a systematic approach for optimisation

A method for quantification of pesticide residues in meat and fatty matrices was developed using supercritical fluid extraction (SFE). The SFE method allows selective extraction of residues and subsequent gas chromatography analysis without further clean-up. Quantification was done by GC using nitrogen-phosphorus detection and electron capture detection. Initial method development was made using organophosphorus pesticides (OPPs). The dependence of fat and OPP residue recovery on supercritical fluid density, temperature, flow rate and extraction time was investigated through a reduced factorial design. Since temperature and density were found to have pronounced effect on the recovery of OPPs these extraction parameters were studied using a new arbitrary measure for co-extractivity. An optimisation score was established as relative pesticide recovery subtracted by relative fat recovery. Using this algorithm a response plane was modelled varying the primary factors temperature and density. The applicability of this approach and the algorithm was verified. The polarity range covered by the SFE method was demonstrated using OPPs: chlorpyrifos, chlorpyrifos-methyl, malathion, pirimifos-methyl and prothiofos. Additionally the final method was evaluated using four pesticides that are not OPPs: carbofuran, phorate, procymidone and vinclozolin. All pesticides showed good recovery (78-95%), and limits of detection (0.01-0.03 mg kg-1) and limits of determination (0.01-0.05 mg kg-1) meet the requirements set by the European Council (Directive 96/33/EEC). Compared to traditional methods based on organic solvent extraction, the SFE method is fast, less labour intensive, uses smaller amounts of potentially harmful solvents and has the potential to be fully automated.     

An improved analysis for chlorinated pesticides and polychlorinated biphenyls (PCBs) in human and bovine sera using solid-phase extraction

Chlorinated pesticides and polychlorinated biphenyls (PCBs) remain public health concerns because of their unresolved health impact and their persistence in humans. Current epidemiological studies of cancer, non-Hodgkins lymphoma, and endocrine disruption in National Center for Environmental Health (NCEH) laboratories require exposure assessment of many analytes in thousands of people. Previous methods of analyzing pesticides and PCBs in serum have proven inadequate for timely processing of the number of samples required for epidemiological studies. A new method that involves solid-phase extraction (SPE) and cleanup followed by dual-column gas chromatographic separation and electron capture detection has been developed. Nine surrogate compounds were added to the serum prior to sample workup to provide quality assurance for the SPE steps. These surrogates mimic the chemistry of the analytes in the extraction, cleanup, and gas chromatographic analysis steps. To increase selectivity, extracts were injected onto two gas chromatographs with different capillary columns, a DB-1701 and a DB-5. Recoveries of 17 pesticides, 28 PCB congeners, and one polybrominated biphenyl congener ranged from 40 to 80%. Recoveries from this procedure were found to be similar to those from the previously used liquid-liquid extraction method. Correlation of analyte and surrogate recoveries were compared to examine the ruggedness of the technique. The SPE method was found to provide improved sample throughput by a factor of 15.     

Electron capture gas chromatographic analysis of the amine metabolites of pesticides: derivatization of anilines

A number of amines have been shown to result from metabolism of various pesticides. From an epidemiological standpoint, it may be possible to monitor human exposure to these pesticides through the excretion of their corresponding amines in urine. An investigation has been initiated to develop and apply methods of analysis of amines in human urine. The results of a survey of derivatization techniques involving several substituted anilines are presented. These include conditions for derivatization, utilizing a number of halo- and nitro- substituted reagents; electron capture and gas chromatographic properties of the derivatives; and stability of the derivatives to extraction and column chromatography for purposes of separation and cleanup. The recoveries of anilines from spiked water and urine samples at the 1.0 ppm and 0.1 ppm levels were between 85 and 90%. The advantages and disadvantages of the various derivatives and techniques are discussed and a rationale is presented for the preliminary selection of a particular derivative for application of the analysis of aniline metabolites in urine.     

固相萃取-气相色谱法测定大米中有机氯有机磷农药残留

采用固相萃取方法,结合气相色谱建立了同时检测大米中15种有机氯、有机磷农药残留的分析方法.样品用乙腈提取,氨基固相萃取柱净化,经DB-5石英毛细管柱分离后,直接用气相色谱(GC)检测,外标法定量.结果 表明15种农药在0.01 ～0.2 μg/mL浓度范围内呈现良好的线性关系,相关系数r均大于0.995 1,样品在2个...     

Determination of organochlorine and organophosphorus pesticide levels in imported beef

A simple and efficient cleanup method was established for capillary gas chromatographic determination of 12 organochlorine and 11 organophosphorus pesticides in beef. Extracted fat was subjected to silica gel dry column chromatography and further cleaned up by Florisil minicolumn chromatography for organochlorine pesticide analysis, while partitioning between n-hexane and acetonitrile of the extract and silica gel minicolumn chromatography were employed for the analysis of organophosphorus pesticides. Several samples (imported Australian beef) were analyzed by the proposed method. DDT was detected in 14 (0.01-0.10 ppm). BHC was found in 11 (0.003-0.031 ppm) and dieldrin was demonstrated in 2 (0.004 and 0.008 ppm). Heptachlors and the 11 organophosphorus pesticides investigated were not detected in any of the meat samples.     

Determination of nicotine, cotinine and caffeine in meconium using high-performance liquid chromatography

A high-performance liquid chromatographic method with diode-array detection for the determination of nicotine and its metabolites, cotinine and caffeine, in meconium is described. This method is suitable to assess foetus exposure to tobacco smoke. The analytes were extracted by solid-phase extraction before chromatography. From among 30 meconium samples 11 were positive for cotinine (20-86 ng/g) and 27 for caffeine (10-45 ng/g). No nicotine was present in the samples because of its rapid metabolism into cotinine.     

Detection of thiazide-based diuretics in equine urine by liquid chromatography/mass spectrometry

Thiazide-based diuretics are included in the list of banned drugs in the horse-racing industry. One effect of their misuse is increased urine flow, contributing to dilution of other doping agents. Their determination is essential in ensuring compliance to horse-racing regulation. This study evaluates the feasibility of using liquid chromatography/mass spectrometry (LC/MS) with electrospray and atmospheric pressure chemical ionization interfaces to analyze thiazidic diuretics in equine urine samples. Existing LC and gas chromatography/MS methods are limited in their applicability to thiazide analysis. Sample preparation, analyte extraction, chromatographic separation, ion-source collision induced dissociation, solvent composition, ionization mode, and ion polarity are discussed. The practicality of LC/MS for this analysis is demonstrated with actual equine administration samples collected at specified time intervals. Detection limits were 270 ng/mL for chlorothiazide, 131 ng/mL for hydrochlorothiazide, and 384 ng/mL for trichlormethiazide.     

Direct determination of procainamide and N-acetylprocainamide by capillary zone electrophoresis in pharmaceutical formulations and urine

A method is described for the simultaneous determination of sixteen organochlorine pesticides in drinking water using automated solid-phase extraction followed by high-volume (80 microliters) capillary column gas chromatography using electron capture detection. The fully automated extraction method followed by high-volume injection permits rapid sample analysis compared to previously described procedures since no further pre-concentration of the analytes is necessary after they have been eluted from the octadecyl solid-phase extraction cartridge. The lowest detectable concentrations of the pesticides are between 1-5 ng l(-1), relative recoveries range from 92-105% in tap water spiked at 100 ng l(-1) and the relative standard deviations are in the range 5-12%.     

Isolation of priority polycyclic aromatic hydrocarbons from natural sediments and sludge reference materials by an anti-fluorene immunosorbent followed by liquid chromatography and diode array detection

The selective isolation of PAHs from complex environmental mixtures was accomplished by means of a new methodology based on antigen--antibody interactions. This method consists on the extraction of PAHs from water samples onto an anti-fluorene immunosorbent (IS) followed by liquid chromatography with diode array detection. Environmental sediments and a sludge reference material containing PAHs were analyzed using this methodology in order to validate the performance of the IS for the cleanup procedure of such materials. Sediments were extracted by sonication with dichloromethane/methanol (2:1), and the extracts were brought to a volume of 100 mL of water in order to perform the extraction with the anti-fluorene IS. The reliability of the cleanup achieved by the IS was well demonstrated in the analysis of sediment and sludge complex samples containing the priority PAHs established by the U.S. EPA at concentrations varying from 56 micrograms/kg to 26 mg/kg. Results were compared to those obtained with conventional cleanup procedures showing a better selectivity for PAHs. The chromatograms presented a clear baseline allowing the determination and quantification of PAHs at the ppb level. Using immunosorbents, extraction, trace enrichment, and cleanup were accomplished in only one step.     

Maize and foxtail millet as substantial sources of dietary lead intake

In 1996, 24-h food duplicate samples were collected from two groups of 50 non-smoking women each; one group was in Jinan, the capital city of Shandong Province in China, and the other in a farming village in the Zhangqiu area some 30 km away from the city. The people in the village took significantly more dietary lead (46 micrograms/day) than their counterparts in the city (26 micrograms/day), and blood lead concentrations (35 and 50 micrograms/l for the urban and the rural people, respectively) were in parallel with the dietary lead intake. Search for cereals as the determinants of dietary lead intake and blood lead concentration by multiple regression analysis showed that maize was the most influential source of dietary lead intake among the four common cereals of wheat, rice, foxtail millet (to be called just millet) and maize, whereas millet was the leading determinant of the blood lead level among the four cereals although the influential power was weaker than millet for dietary lead. Lead content in maize (47 ng/g) and millet (47 ng/g) was twice or even more times higher than the levels in wheat (26-30 ng/g) and rice (20-21 ng/g). The significant roles of non-rice/non-wheat cereals such as millet and maize as possible dietary lead sources for farming populations are discussed.     

Reversed-phase high-performance liquid chromatographic determination of enoxacin and 4-oxo-enoxacin in human plasma and prostatic tissue. Application to a pharmacokinetic study

A simple high-performance liquid chromatographic method has been developed for the simultaneous determination of enoxacin and 4-oxo-enoxacin in plasma and prostatic tissue. The work-up procedure involves a liquid-liquid extraction step followed by isocratic chromatography on a reversed-phase analytical column, with ultraviolet absorbance detection (lambda = 340 nm). Using a mobile phase of 20.9% (v/v) acetonitrile buffer (pH 2.1), adequate retention time and separation among the analytes has been obtained using tetrabutylammonium hydroxide included in the eluent. Retention times are 5.2 min for enoxacin, 6.8 min for pefloxacin and 12 min for 4-oxo-enoxacin. For plasma and prostatic tissue, the precision of the assay was below 9%. The percent recovery from the nominal values for accuracy ranged from 94 to 108%. The limits of quantitation were 20 ng/ml for plasma and 50 ng/g for tissue (precision < 18%). The detection limits were 10 ng/ml and 25 ng/g, respectively. The calibration curves were linear from 20 to 1000 ng/ml for plasma and from 50 to 2500 ng/g for tissue. In plasma, the extraction recoveries averaged 52% for enoxacin and 63% for 4-oxo-enoxacin. In prostatic tissue, they were 57 and 76% for the two analytes, respectively. This method has been employed for the determination of enoxacin and 4-oxo-enoxacin in plasma and prostatic tissue samples from patients following repeated oral administration of enoxacin (400 mg twice a day for four days).     

Fully automated determination of pesticides in wine

A fully automated solid-phase extraction gas chromatographic/mass spectrometric (SPE/GC/MS) method was developed for determination of pesticides in wine. All steps from aspiration of infiltrated wine to printout of the integrated chromatogram were performed without human interaction. A dedicated robot performed addition of internal standard, application of wine onto the SPE cartridge, elution of analytes, drying and concentrating of eluate, and passing of concentrate to the GC sampler. All steps were performed in standard liquid chromatography/GC vials, using a minimum of organic solvent. The method permits determination of 21 different pesticides. Individual detection limits were 0.005-0.01 mg/L. The regression coefficients relating to linearity were > 0.99; only 4,4-dichloro-benzphenone and dicofol showed lower coefficients. The recoveries for 17 pesticides ranged from 80 to 115%.     

Methods for the determination of organochlorine pesticide residues in human serum

The effectiveness of solid-phase extraction with Florisil for the determination of 12 organochlorine pesticide residues from human serum was examined. Recoveries greater than 84% and coefficients of variation better than 19% were obtained. Others methods, such as column partition and matrix solid-phase dispersion, were compared. The better method provides quantification limits ranging from 1.08 microg/l for gamma-HCH and 37.5 microg/l for p,p'-DDT when capillary gas-liquid chromatography with electron-capture detection is used for the final determination.     

Expression and estrogen regulation of the HEM45 MRNA in human tumor lines and in the rat uterus

The combination of manual and automated extraction procedures using low sample volumes (5-50 ml) with large-volume oncolumn injection (LVI) (200 microliters) in capillary gas chromatography with flame photometric detection (GC-FPD) has allowed the determination of 16 organophosphorus pesticides in clean water samples at the low ng l-1 level with an important simplification in the sample preparation step. A simple and fast offline liquid-liquid microextraction procedure (2-5 ml water/l ml methyl tert.-butyl ether) has been applied to spiked groundwater samples (containing 0.5 ng of each pesticide) with good recoveries (over 80%) and precision (better than 10%), giving detection limits between 5 and 100 ng l-1 using 200 microliters injections in the GC-FPD system. The application of an inline automated liquid-liquid microextraction-LVI-GC procedure (2 ml water/2 ml methyl tert.-butyl ether: injection of 200 microliters in GC-FPD) using the autosampler ASPEC XL led to lower recoveries (> 50%) as a result of the low efficiency for mixing organic and aqueous phases, although with very satisfactory coefficients of variation (lower than 7%) and detection limits between 20 and 200 ng l-1. Manual and automated solid-phase extraction procedures using the well known C18 cartridges and the new Oasis HLB have been applied to groundwater samples (5-50 ml) spiked with 1 ng of each pesticide. Results obtained for both the manual and the automated procedures were satisfactory (recoveries over 80%) and the limits of detection for 50 ml sample volume ranged from 1 to 6 ng l-1.     

Multiresidue determination of pesticides in apples and pears by gas chromatography-mass spectrometry

This paper describes a rapid, specific and sensitive multiresidue method for the routine analysis of several classes of pesticides used for the treatment of apples and pears, involving a rapid extraction procedure at pH 4.5 with a mixture of acetone-dichloromethane-hexane (50:20:30, v/v/v) and gas chromatography coupled to mass-selective detection, in order to achieve quantitative analysis down to their respective maximum residue limit. Extraction recoveries were between 55 and 98%. Limits of detection and limits of quantitation ranged respectively, from 0.01 to 0.05 mg/kg and from 0.02 to 0.1 mg/kg. Intra-assay relative standard deviation was less than 19% for all compounds. An excellent linearity was observed from these LOQs up to 500 mg/kg. Intermediate (inter-assay) precision and accuracy were satisfactory. The method has been applied to many fruit samples intended for commercialisation.     

Multiresidue method for analysis of pesticides in liquid whole milk

A multiresidue method to analyze liquid whole milk for 59 compounds was developed. The method involves a single extraction and cleanup strategy for many classes of compounds--organophosphorus compounds (OPs), organochlorine compounds (OCs), N-methylcarbamates (MCs), etc. Initial extraction is performed with ethanol-ethyl acetate as solvent and sodium sulfate as drying agent. A portion of the extract is concentrated to an oily consistency, and target analytes are partitioned into acetonitrile. Further cleanup is achieved by sequential solid-phase extractions--octadecyl (C18)-bonded silica cartridges followed by aminopropyl (NH2)-bonded silica cartridges. The solvent is exchanged to acetone for analysis by gas chromatography (GC) with electrolytic conductivity, flame photometric (FPD) or mass spectrometric (MS) detection and to methanol for analysis by liquid chromatography with postcolumn derivatization. Typical limits of detection (LODs; peak-to-peak signal-to-noise ratio > or = 3) were 0.3 ppb for OPs, 0.9 ppb for OCs and MCs, and 9 ppb for mass-selective detection. From the level of quantitation (LOQ = 3.33 x LOD) to 10 x LOQ, linear instrument response was observed for all detectors except the FPD which required a second-order calibration curve. Average recoveries for spikes at the LOQ ranged from 69 to 127% with standard deviations of about 10%. Similar accuracy and precision were observed for fortifications at 5 x LOQ and 10 x LOQ. The method was used to analyze 20 milk samples from various liquid milk processing plants. Incurred residues were confirmed by high-resolution GC/MS and GC/MS/MS.     

Vitamin D3 in plasma: quantitation by mass fragmentography

Combined gas chromatography-mass spectrometry with multiple-ion detection is used to quantitate vitamin D3 in plasma samples. Extensive cleanup of the plasma extract prior to analysis is required before mass fragmentographic analysis is possible, i.e., solvent extraction, digitonin precipitation, Lipidex column chromatography, and derivatization. Low resolution mass fragmentography is performed by monitoring simultaneously the molecular ions of the heptafluorobutyric esters of the all-trans isomer of vitamin D3 and dihydrotachysterol2, which is used as an internal standard. This new method uses 5 ml of plasma and it is specific and sensitive to 600 pg of vitamin D3. This assay shows that in plasma of normal adult subjects there is a vitamin D3 concentration of 5 to 11 ng/ml, lower than those values reported for biological and competitive protein binding assays. Specifically the method described offers potential as an eventual reference method.     

Simultaneous measurement of [18F]FDOPA metabolism and cerebral blood flow in newborn piglets

Three diets containing either borage oil (BO) and southern hemisphere fish oil Marinol (MO), or BO and tuna orbital oil (TO), or a northern hemisphere fish oil (FO) were fed to duplicate groups of turbot (Scophthalmus maximus) of initial mean weight 1.2 g for a period of 12 weeks. The BO/MO and BO/TO diets were enriched in gamma-linolenic (18:3n-6, GLA) and eicosapentaenoic (20:5n-3, EPA) acids, and GLA and docosahexaenoic acid (22:6n-3, DHA), respectively. No differences were observed in final weights or growth rates, either between duplicate tanks or between dietary treatments. Half of the FO-fed fish sampled showed a histopathological lesion indicative of lipoid liver degeneration while the other treatments only showed a slight incidence of the same pathology. The fatty acid compositions of carcass and tissues broadly reflected the dietary input. In general, fish fed the BO/MO diet had increased levels of 18:2n-6, 18:3n-6, 20:3n-6 and 20:5n-3, but a lower level of 22:6n-3, compared to fish fed FO. In fish fed the BO/TO diet, levels of 18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6 were increased while levels of 20:5n-3 and 22:5n-3 were reduced, compared to fish fed FO. Concentrations of thromboxanes B (TXB) and leukotrienes B (LTB), derived from 20:4n-6 and 20:5n-3, were measured in plasma and stimulated blood cells. Levels of TXB2 were greatest in fish fed the BO/TO diet compared to both other treatments, while LTB4 was decreased in fish fed the BO/MO diet compared to both other treatments. In a stress test which involved anaesthesia followed by measurement of recovery times, fish fed the BO/MO diet had significantly lower recovery times compared to fish fed the FO diet.     

Cholesterol oxidation in meat from chickens fed alpha-tocopherol- and beta-carotene-supplemented diets with different unsaturation grades

The production of B-ring and side-chain oxysterols was evaluated in meat from chickens fed diets differing by the kind of oil or fat added. The effect of supplementary levels of natural antioxidants, as alpha-tocopherol and beta-carotene, on the meat cholesterol oxidative stability was also studied. Lard, sunflower and olive oil were used as dietary fat. Raw and cooked meats were analyzed for oxysterols, and cholesterol was also quantified. Oxysterol analyses were carried out by combining the use of solid-phase extraction, thin-layer chromatography, capillary gas chromatography, and capillary gas chromatography-mass spectrometry. Oxysterols were detected within the 0.1-0.5 microg/g range in raw meat. Cooking increased the oxysterol content of the meat, and levels as high as 5 microg/g muscle tissue were observed. B-Ring oxysterols were mainly produced: the alpha- and the beta-epoxycholesterols, the 7alpha- and 7beta-hydroxycholesterols, and the 7-ketocholesterol. The results showed that the meat from the chickens fed the olive oil-based diet containing alpha-tocopherol at 200 mg/kg of diet presented the best cholesterol oxidative stability. A positive effect could not be found for dietary beta-carotene administered at levels of 15 and 50 mg/kg of diet. Furthermore, a significant decrease in the tissue cholesterol content was observed with the olive and the sunflower oil-based diets.     

Measurement of plasma levels of tricylic psychoactive drugs and their metabolites by UV reflectance photometry of thin layer chromatograms

A method has been developed for the determination of perazine, clozapine, imipramine and amitriptyline and their demethylated metabolites in plasma. Other metabolites measured were perazine sulfoxide and the N-oxides of clozapine and perazine, the latter two following their reduction to the parent drugs with ascorbic acid. 10-Hydroxynortriptyline was identified as an amitriptyline metabolite in plasma. The general procedure included extraction of alkalinized plasma samples (3 - 6 g) with benzene or toluene and thin layer chromatography of the extracts, followed by reflectance photometry of the plates at appropriate wave lengths in ultraviolet light. Spots of questionable identity were further characterized by two-dimensional chromatography and by colour reactions. Therecoveries of compounds added in therapeutic concentrations were between 70 and 98 %. The limits of detectability were 5 - 10 ng/g plasma.