Automated Solid-phase Extraction System Research Articles

A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma.

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre- and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1. Method validation parameters are presented, and the method was positively verified by analyzing control and PTU-treated rat plasma samples (time points day 7, 14, and 28) for their total TH content. The obtained results were compared to published results by using a radioimmunoassay method. The automated SPE system ensures a consistent unified sample preparation, and this method overall showed sufficient specificity and accuracy to detect the given analytes in rat plasma. For the preparation of 50 μL of rat plasma, the following LOQs were established: 0.020 nM for T1, 0.029 nM for T2, 0.023 nM for rT3 and T3, and 3.22 nM for T4. This method is suitable to assess the identification of mechanisms leading to adverse effects, such as disturbed TH metabolism and regulation.

Analytical quality by design using a D-optimal design and parallel factor analysis in an automatic solid phase extraction system coupled to liquid chromatography. Determination of nine PAHs in coffee samples

Optimizing a multi-residue analysis when using an automatic SPE (solid phase extraction) system and complex matrices becomes a difficult problem because of the large number of experimental factors that can influence the recovery of the analytes. Furthermore, in most cases, the conditions of the factors that enhance the response of one analyte are in conflict with those suitable for some others.In this work, AQbD (Analytical Quality by Design) is applied to the development of an analytical procedure based on automatic SPE coupled to HPLC-FLD in the determination of nine polycyclic aromatic hydrocarbons (PAHs) in coffee samples.Focussing on the SPE, the elution volume, the dry time, and volume in the wash stage, and the organic solvent (at two, three, three, and four levels, respectively) were considered.The first problem is to handle these four factors (control method parameters, CMPs) at different levels to optimize responses (critical quality attributes, CQAs). This task has been carried out using a D-optimal design that, starting from a full factorial design of four factors with 72 experiments, reduced this number to 19, maintaining the precision of the estimates, saving time and costs in the laboratory.The second problem is related to the choice of CQAs to apply the AQbD methodology. A complex matrix such as coffee contains impurities that interferes with the target analytes and may even coelute in the chromatographic determination. A PARAFAC decomposition allows avoiding this problem and uses the “second order advantage” to unequivocally identify each analyte. Then, the obtained sample loadings were used as responses. Specifically, each CQA is the difference between spiked and blank coffee samples. All these CQAs must be maximized.Once the experimental data were obtained, two alternatives were posed: on the one hand, the classical optimization based on the estimation of the effects of CMPs on the CQAs, and on the other hand, applying the AQbD methodology to construct the design space that allows to increase the knowledge of the automatic SPE system.Because the experimental domain of CMPs is discrete and the SPE system performs differently for each analyte, it is not possible to obtain the maximum of all CQAs at the same factor levels. Therefore, the design space of the CMPs is obtaining through the Pareto front of the non-dominated values of CQAs.The nine PAHs selected were phenanthrene (PHE), anthracene (ANT), fluoranthene (FLN), pyrene (PYR), chrysene (CHR), benzo[a]anthracene (BaA), perylene (PER), benzo[b]fluoranthene (BbF) and benzo[a]pyrene (BaP). European regulations amending foodstuff set maximum levels for BaP and the sum of the content of four compounds (PAH4): BaP, BaA, BbF and CHR.

Rapid pretreatment for multi-sample analysis of advanced glycation end products and their role in nephropathy.

Advanced glycation end products (AGEs), produced by the Maillard reaction between carbohydrates and proteins, may be involved in diabetes and its complications. Accurate quantification of AGEs in vivo can demonstrate the relation between AGEs and pathological conditions, but it is not widely used in clinical practice because of the multiple pretreatment steps before analyses. We developed a fully automated solid-phase extraction system (FSPES) to simplify rate-limiting pretreatment using a cation exchange column. We applied this device to evaluate AGEs in nephropathy. Among the standard samples, we used arginine, lysine, N‍ε-(carboxymethyl)lysine (CML), N‍ω-(carboxymethyl)arginine (CMA), N‍ε-(carboxyethyl)lysine (CEL), and N‍δ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) for FSPES. We analyzed the coefficient of variation (CV) by mass spectrometry. FSPES performed column operations rapidly at a pressure three times higher compared with the conventional method. FSPES stably performed pretreatment. CV results for CML, CMA, CEL, and MG-H1 measurements in bovine and human serum were the same as those in the conventional pretreatment. Among the AGE structures we measured, CML and CEL increased with the decline in kidney function. The CML and CEL levels of patients with nephropathy were significantly higher than those in normal subjects. Thus, FSPES is useful for clarifying the relation between AGEs and various pathological conditions.

Simultaneous determination of seven bisphenol analogues in surface water by solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry

For the simultaneous determination of seven types of bisphenol analogues (BPs) in surface water, a method combining solid-phase extraction (SPE) with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed. The extraction efficiency was improved by optimizing the parameters that affect it, including the sorbent types of SPE cartridge, pH, enrichment factor, flow rate, rinsing and elution volume. On the basis of the single factor experiments, an attempt to obtain the result of optimizing by response surface methodology was made, and the optimal parameters were as follows: at a flow rate of 8 mL/min, 1000 mL of water sample was run through the hydrophilic-lipophilic balance cartridge, rinsed with 10% methanol, eluted with 9 mL methanol, and concentrated with nitrogen using an automatic SPE system. The analytes were separated on an Acquity UPLC BEH C18 column using gradient elution with a mobile phase of acetonitrile and 0.05% (v/v) ammonia, and then analyzed using UPLC-MS/MS with multiple reaction monitoring in the negative-ion mode, with an 8-minute chromatographic run time. In the range of 1.0–500 μg/L, seven BPs showed good linearity with the correlation coefficient greater than 0.998. The recovery ranged from 82.3 to 96.7%, and the range of matrix effect was 89.2 to 107%. These results indicated excellent recovery and minimal matrix effect. The limits of detection were 0.05–1.5 ng/L, the relative standard deviation ranged from 2.9 to 8.5%. Applying the developed method, seven BPs in Luoma Lake and its inflow rivers were analyzed. Only five BPs were detected in Luoma Lake and four BPs were detected in inflow rivers, and the concentrations of the detected BPs were determined. BPA and BPF were the predominant BPs, and the concentration of BPA was the highest.

An Automatic Solid Phase Extraction and Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry for Determination of Seven Microcystins at Ultra-Trace Levels in Surface Water

A method was developed for the detection of seven microcystins (microcystin-LR, RR, YR, LA, LY, LW and LF) in surface water using automatic solid-phase extraction (A-SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The automated solid-phase extraction system was used to extract microcystins (MCs) from water samples. UPLC-MS/MS was used to determine MCs concentrations in just 5 min. Method detection limits were from 0.3 to 0.9 ng/L, microcystin recoveries ranged from 83.8% to 114%, and the relative standard deviation (RSD) varied from 5.6% to 12.5%. This analytical approach was found to be simple, highly sensitive, accurate, which required little manual operation. Additionally, to validate this analytical method, A-SPE+UPLC-MS/MS was applied to characterize the concentration of MCs in Taihu Lake, Wuxi, China.

Development and application of gas chromatographymass spectrometry method for quantitive determination of volatile N-nitrosamines in smoked meat products

The article presents the results of experimental studies on the development of highly sensitive and selective chromatography-mass spectrometry technique for the determination of 9 N-nitrosamines in food samples (sausage products) using distillation and an automatic solid-phase extraction system on Coconut cartridges for sample preparation. In the elaborated conditions of sample preparation (distillation and solid-phase extraction) and chromatography-mass spectrometric analysis, we achieved a high recovery and efficiency of the separation of nine N-nitrosamines. The quantitation limit was at level of 0.0002 mg/kg with maximum error not exceeding 19%. The complex use of the distillation of N-nitrosamines with the addition of potassium hydroxide in combination with the optimal elution scheme for solid-phase extraction and concentrating the distillate into a Coconut carbon cartridge of 6 ml ensures the recovery of N-nitrosamines from the food product sample (sausage products) up to 93.2-100%. The process of approbation of the chromatography-mass spectrometric method in the samples of food products (sausage products) of various manufacturers revealed the content of N-nitrosamines in the concentration range 0.00029±0.000055÷0.350±0.05 mg/kg. The conducted studies of the content of the sum of highly toxic N-nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine) made it possible to disclose that in sample No. 5 the maximum allowable concentration was exceeded by 47 times, in samples No. 2 and 16, to 57.5 and 22.9 times and in sample No. 4 to 88 times, respectively.

METHODOLOGICAL SUPPORT OF CONTROL OF THE CONTENT OF N-NITROZODIPHENYLAMINE IN FOOD MEAT AND MEAT/PLANT PRODUCTS

The methodical methods used in the practice of the development and application of methods for the determination of chemical compounds in food products for practical instrumental studies used in conducting laboratory studies of food safety are considered. An algorithm for the development of a chromatography-mass spectrometric technique for the determination of one of the group of highly toxic, carcinogenic N-nitrosamines (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidineamin, N-nitrosomorpholinamine, N-nitrosodipropylamine, N- nitrosopiperidinamine N-nitrosoperidineamine and N-nitrosodiphenylamine in homogenized canned meat and meat products (canned meat) is supposed. The algorithm included experimental development of chromium parameters of chromatography-mass spectrometric determination of an analyte, the selection of optimum conditions of sample preparation of samples of canned meat to minimize the influence of the matrix: the transesterification reaction of fatty acids, organic solvent extraction, solid phase extraction, study of extraction completeness by means of the method “introduced - found”, establishment of metrological characteristics of the process of the measurement. The high sensitivity and selectivity of the chromatography-mass spectrometric determination of N-nitrosodiphenylamine in canned meat samples with a lower limit of 0.0002 mg/kg and a maximum error of not more than 23% was achieved owing to the use of a set of methodical techniques - the selection of optimal chromatographic analysis conditions: capillary column series HP-FFAP 50m • 0,320 mm • 0,50 μm, temperature programming of the column: initial temperature of 50ºС, the temperature rise up to 120ºС at a speed of 8ºC/min; from 120º to 185° C at a rate of 12° C min and from 185º to 240° C at a rate of 25°C/min with exposition at a final temperature of 5 minutes; mode of operation of the mass spectrometric detector: selective ion monitoring (SIM) for three characteristic ions of the analyzed compound of 167, 168, 169 m/z. Using the reaction of transesterification of fatty acids contained in canned meat, potassium methylate, removal of the formed esters from samples of canned meat with an organic solvent (hexane), concentration of N-nitrosodiphenylamine in the aqueous layer on cartridges of an automatic solid-phase extraction system provides the extraction of N-nitrosodiphenylamine from the test samples by 99.94%. In the process of approbation of the chromatography-mass spectrometric method, the content of N-nitrosodiphenylamine in the concentration range 0.030 ± 0.011 ÷ 3.89 ± 0.83 mg/kg was found in samples of homogenized canned food meat and meat products (canned meat) from various manufacturers. The highest concentration of N-nitrosodiphenylamine C = 3.89 ± 0.83 mg/kg was found in the sample of canned meat «beef + chicken».

Determination of polycyclic aromatic hydrocarbons in environmental waters from southern Spain by using a continuous solid-phase extraction system and gas chromatography-mass spectrometry

Environmental contextPolycyclic aromatic hydrocarbons are widespread carcinogenic compounds resulting from incomplete combustion of fossil fuels. We report a robust analytical method suitable for detecting these compounds at trace levels in various types of environmental waters. The method allows for accurate monitoring of the levels and behaviour of these priority environmental pollutants. AbstractHuman exposure to polycyclic aromatic hydrocarbons (PAHs) is a major challenge for the scientific community. The European Commission and the European Food Safety Agency have declared PAHs as priority pollutants, and sanctioned their quantification and monitoring in water and foods, owing to their carcinogenic, teratogenic, and mutagenic properties. We report a method for determining sixteen PAHs in various types of environmental water samples collected from various sources and places in the south of Spain. The target compounds were preconcentrated to a reduced volume (350 µL) with an automatic solid-phase extraction system and determined by gas chromatography–mass spectrometry. Optimising the operational variables, such as the type and nature of sorbent, and analytical variables resulted in a very low limit of detection (0.01–0.3 ng L−1), high accuracy and precision (RSD < 7.5 %), and recoveries of 86–102 % from 200 mL of sample. The method was applied to a variety of environmental water samples, many of which were found to contain PAHs at different levels, depending on the nature and origin the sample. In any case, such levels were lower than the maximum tolerated limits except for sea and waste water. In the case of waste water, a comparison between influent and effluent from the waste water treatment plant was performed to assess the effect over the environment after their treatment, where a reduction of the presence of PAHs in the samples was observed.

Determination of N-nitrosodiphenylamine in meat canned food for children by the method of chromato-massspectrometry

This study demonstrates the results obtained from the GC/MS experimental determination of low concentrations of N-nitrosodiphenylamine in meat canned baby food in the concentration range of 0.027-3.89 mg/kg. The perfect conditions of sample preparation (extraction with organic solvent and solid phase extraction) as well as the application of the chromatography-mass spectrometry allowed us to detect N-nitrosodiphenylamine in samples of the meat canned baby food with high selectivity in concentrations ranged from 0.016 to 5 mg/kg when an error of 23% was assumed. The use of the reaction of transesterification of fatty acids by potassium methylate, the removal of the ester generated from the samples of canned meat by organic solvent (hexane), concentrating of N-nitrosodiphenylamine in the aqueous layer on the cartridges of an automatic solid-phase extraction system provided 99.94% extraction of N-nitrosodiphenylamine from the canned meat samples. The experiment has made evident the presence of N-nitrosodiphenylamine in the samples of canned meat (beef + chicken) with the help of mass-spectrometry method in the mode of full ion scanning using the AMDIS automatic mass-spectral identification system.

RapidFire Mass Spectrometry with Enhanced Throughput as an Alternative to Liquid-Liquid Salt Assisted Extraction and LC/MS Analysis for Sulfonamides in Honey.

The use of veterinary drugs in honey bees for the prevention of infectious disease is ever increasing due to the spread of colony collapse disorder around the world. The United States Food and Drug Administration is concerned about the presence of these drugs residues in honey as they often lead to health concerns or potential antibiotic resistance. Currently there is a need for a rapid screening method for the detection of veterinary drugs in honey. Herein is a method that utilizes automated solid-phase extraction that is directly coupled to a mass spectrometer for the quantitative screening of 12 different regulated sulfonamides in honey. Identification of the residues was performed using tandem mass spectrometry (MS/MS) with a triple quadrupole mass spectrometer. The acquisition method developed for this analysis can extract with the automated solid-phase extraction system and analyze a single sample on the mass spectrometer in approximately 20 s, with minimal sample preparation. A target testing limit of 10 ng/g in honey for the sulfonamides was used based upon action limits set for other food commodities regulated by the United States Food and Drug Administration. A complete method validation procedure was conducted to evaluate the effectiveness of this quantitative screening method.

Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood.

Deaths among drug addicts are frequently caused by intoxication with methadone and/or morphine. These drugs are often used in combination with other drugs, such as buprenorphine. In addition, methadone is generally used as a mixture of R- and S-enantiomers. To date, a method for separation and quantitation of these specific drugs has not been developed. The aim of this study was to develop a sensitive enantioselective method for quantitation of morphine, its active metabolite morphine 6-glucuronide, buprenorphine, and R- and S-methadone, in a single analytical run. Whole blood samples were diluted with 0.5 mol/L ammonium carbonate buffer and extracted on a Bond Elut C18 solid-phase extraction column with an automatic solid-phase extraction system. Chromatographic separation was performed on a chiral alpha-1-acid glycoprotein column with an acetonitrile/ammonium acetate buffer (10 mmol/L, pH 7.0, 22:78 v/v) mobile phase. The whole blood concentrations of the drugs were quantified by mass spectrometry using their stable isotope-labeled compounds as internal standards. The method was validated with respect to specificity, linearity, precision, limits of detection, and quantification and matrix effects. The precision (coefficient of variation) was below 15%, and the accuracy was between 90 and 115%. This method will be useful for routine analyses in forensic laboratories where blood samples are frequently analyzed for drugs of abuse. In some cases, sudden death from methadone overdose is caused by the enantiomeric form of the methadone, which makes the enantiomer separation capability of this method important.

Validation of an automated solid-phase extraction method for the analysis of 23 opioids, cocaine, and metabolites in urine with ultra-performance liquid chromatography-tandem mass spectrometry.

The aim of this work was to automate a sample preparation procedure extracting morphine, hydromorphone, oxymorphone, norcodeine, codeine, dihydrocodeine, oxycodone, 6-monoacetyl-morphine, hydrocodone, ethylmorphine, benzoylecgonine, cocaine, cocaethylene, tramadol, meperidine, pentazocine, fentanyl, norfentanyl, buprenorphine, norbuprenorphine, propoxyphene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine from urine samples. Samples were extracted by solid-phase extraction (SPE) with cation exchange cartridges using a TECAN Freedom Evo 100 base robotic system, including a hydrolysis step previous extraction when required. Block modules were carefully selected in order to use the same consumable material as in manual procedures to reduce cost and/or manual sample transfers. Moreover, the present configuration included pressure monitoring pipetting increasing pipetting accuracy and detecting sampling errors. The compounds were then separated in a chromatographic run of 9 min using a BEH Phenyl analytical column on a ultra-performance liquid chromatography-tandem mass spectrometry system. Optimization of the SPE was performed with different wash conditions and elution solvents. Intra- and inter-day relative standard deviations (RSDs) were within ±15% and bias was within ±15% for most of the compounds. Recovery was >69% (RSD < 11%) and matrix effects ranged from 1 to 26% when compensated with the internal standard. The limits of quantification ranged from 3 to 25 ng/mL depending on the compound. No cross-contamination in the automated SPE system was observed. The extracted samples were stable for 72 h in the autosampler (4°C). This method was applied to authentic samples (from forensic and toxicology cases) and to proficiency testing schemes containing cocaine, heroin, buprenorphine and methadone, offering fast and reliable results. Automation resulted in improved precision and accuracy, and a minimum operator intervention, leading to safer sample handling and less time-consuming procedures.

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z′ value 0.8) and provided several tractable hit series for further investigation.

Gas Chromatographic Determination of N-Nitrosamines, Aromatic Amines, and Melamine in Milk and Dairy Products Using an Automatic Solid-Phase Extraction System

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.

LC–MS/MS method for the determination of nine antidepressants and some of their main metabolites in oral fluid and plasma: Study of correlation between venlafaxine concentrations in both matrices

In this paper, a fast, sensitive and selective LC–MS/MS method is described for the simultaneous determination of amitriptyline, imipramine, clomipramine, fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram and venlafaxine, as well as some of their main metabolites (nortriptyline, desipramine, norclomipramine and norfluoxetine), in oral fluid and plasma. The sample (0.2 mL) was extracted with an automated solid-phase extraction system (ASPEC XL), using mixed mode OASIS MCX cartridges. Chromatographic separation was performed in a Sunfire C18 IS column (20 mm × 2.1 mm, 3.5 μm), using a gradient of acetonitrile and ammonium formate (pH 3; 2 mM) as mobile phase, which allowed the elution of all the compounds in less than 5 min. The method has been fully validated in both specimens. This method was initially applied to the analysis of oral fluid and plasma samples from patients on antidepressant treatment in order to assess for which compounds it was likely to find a good correlation between both matrices. The best results were obtained for venlafaxine, so the study was extended for this compound, comparing the ratio between oral fluid and plasma concentrations ( R OF/PL) in five patients on venlafaxine treatment when both samples were collected simultaneously on four different occasions. An important inter and intraindividual variability was found in oral fluid concentrations for 150 mg dose (mean = 287.5 ng/m, range 58.8–531.2 ng/mL) and for 75 mg dose (mean = 186.3 ng/mL, range = 82.1–289.2 ng/mL). R OF/PL was calculated for each patient on the four different occasions, showing also a high variability (CV = 24.2–69.6%).

Simultaneous analysis of Thebaine, 6-MAM and six abused opiates in postmortem fluids and tissues using Zymark ® automated solid-phase extraction and gas chromatography–mass spectrometry

Opiates are some of the most widely prescribed drugs in America and are often abused. Demonstrating the presence or absence of opiate compounds in postmortem fluids and/or tissues derived from fatal civil aviation accidents can have serious legal consequences and may help determine the cause of impairment and/or death. However, the consumption of poppy seed products can result in a positive opiate drug test. We have developed a simple method for the simultaneous determination of eight opiate compounds from one extraction. These compounds are hydrocodone, dihydrocodeine, codeine, oxycodone, hydromorphone, 6-monoacetylmorphine, morphine, and thebaine. The inclusion of thebaine is notable as it is an indicator of poppy seed consumption and may help explain morphine/codeine positives in cases where no opiate use was indicated. This method incorporates a Zymark ® RapidTrace™ automated solid-phase extraction system, gas chromatography/mass spectrometry, and trimethyl silane (TMS) and oxime-TMS derivatives. The limits of detection ranged from 0.78 to 12.5 ng/mL. The linear dynamic range for most analytes was 6.25–1600 ng/mL. The extraction efficiencies ranged from 70 to 103%. We applied this method to eight separate aviation fatalities where opiate compounds had previously been detected.

Determination of malachite green residues in rainbow trout muscle with liquid chromatography and liquid chromatography coupled with tandem mass spectrometry

A method for the determination of malachite green and its major metabolite leucomalachite green in rainbow trout muscle is reported with limits of detection of 0.8 and 0.6 μg kg−1, respectively. Residues were extracted with an acetonitrile–acetate buffer mixture and partitioned into methylene chloride. Clean-up of the extracts was performed on alumina and propylsulfonic acid solid-phase extraction columns using the automated solid-phase extraction system. The chromatographic separation of malachite green and leucomalachite green was achieved on a Chromspher 5B column using an acetonitrile–acetate buffer mobile phase. Leucomalachite green was converted to malachite green by post-column oxidation before spectrophotometric detection at 600 nm. The mean recoveries of malachite green and leucomalachite green from control rainbow trout muscle spiked at 2–50 μg kg−1 were 65% (range 63.4–65.9%, relative standard deviation 3.9–16.1%) and 74% (range 58.3–82.6%, relative standard deviation 3.3–11.4%), respectively. Qualitative confirmation of the determined residues was performed with liquid chromatography coupled with tandem mass spectrometry detection with limits of detection of 2.5 and 1 μg kg−1 for malachite green and leucomalachite green, respectively.

Liquid chromatographic determination of natural and synthetic colorants in lyophilized foods using an automatic solid-phase extraction system.

Five synthetic and five natural colorants were determined in lyophilized dairy products and fatty foods using an automatic method based on lixiviation and a solid-phase extraction process that includes cotton and RP-C(18) columns for the sequential retention of synthetic colorants and natural colorants, respectively. The lyophilization of the sample coupled with the separation procedure provides clean extracts despite the complexity of the matrices studied. In addition, the lyophilization process preserves the sample for at least 2 months without changes in the concentrations of the colorants. Identification and determination of synthetic and natural colorants were carried out using a liquid chromatograph equipped with a diode array detector. The detection limits achieved for all of the colorants (0.03-75 microg/g of lyophilized sample) allowed their determination within the limits established by the European Union, with good precision (approximately 4.5%). In addition, colorants spiked to different foods provided average recoveries (spiked at three concentration levels in four types of dairy samples and in three types of fatty foods) near 94 +/- 4%.

Evaluation of an automated solid-phase extraction system for the enrichment of organochlorine pesticides from waters

An automatic method for the determination of organochlorine pesticides (OCPs) in water samples was developed. The analytes are preconcentrated onto a customized RP-C 18 column and subsequently eluted with 80 μl of ethyl acetate. Gas chromatography with electron capture detection is used for their separation and selective detection. Detection limits for 10 ml sample volumes range from 0.01 to 0.1 ng ml −1, with standard deviations between 4 and 6%. The average recovery at a fortification level of 2 ng ml −1 is 92%. The method was used to screen OCPs in natural waters collected near agricultural areas and also to tap waters. Positive findings were confirmed by gas chromatography with mass spectrometric detection. Finally, the adsorption/degradation of OCPs in natural pond water containing a high level of suspended matter was examined; some OCPs were found to be degraded and irreversibly adsorbed on suspended matter present in the water.

Multiresidue screening of pesticides in fruits using an automatic solid-phase extraction system.

About 20 pesticides were determined in lyophilized fruits using a semiautomatic multiresidue method, based on solid-phase extraction (SPE) with a silica column. The lyophilization of the sample, besides the SPE procedure selected, provided clean extracts despite the complexity of the matrixes studied. In addition, the lyophilization process allows sample preservation for at least three months without changes in the concentrations of the pesticides. Determination and quantitation of organochlorine and pyrethroid residues was carried out using a gas chromatograph equipped with an electron capture detector (GC-ECD), and a mass spectrometric detector (GC-MS) was used for confirmation purposes. Organochlorine pesticides provided average recoveries (spiked at three concentration levels in eight different fruits) near 93 +/- 4%, being lower (89 +/- 8%) for pyrethroids as a consequence of their higher degradation and interaction with the sample matrix. On the other hand, the detection limits achieved for all pesticides (0.5-8 ng per g of lyophilized fruit) allow their determination at the MRLs established by the European Union, with good precision ( approximately 5%). Finally, from the 100 different fruits screened, only 10 positive responses were obtained, which were further confirmed by GC-MS.