Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M1 in milk

Using a highly specific antibody against aflatoxin M1, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation of M1 in milk. RIA was sensitive in the range of 5-50 ng per assay but was subject to interference by whole milk. Extraction and cleanup were therefore necessary for the detection of M1 in milk at 0.5 ng/mL. An ELISA procedure was developed by using an aflatoxin M1-carboxymethyl-horseradish peroxidase conjugate as the ligand. Competitive assays revealed that this system was relatively more sensitive for M1 than for B1, and had a much lower degree of cross-reactivity for aflatoxins B2, G1, G2, B2a, and aflatoxicol. As low as 0.25 ng M1/mL in artificially contaminated milk (raw, whole, skim) could be detected by ELISA in 3 h without extraction or cleanup. Because of its simplicity, sensitivity, and specificity, ELISA is the preferred method for monitoring aflatoxin M1 in milk.     

Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.     

Improved cleanup for liquid chromatographic analysis and fluorescence detection of aflatoxins M1 and M2 in fluid milk products

A rapid method is described for extraction and cleanup of raw and processed milk for determination of aflatoxins M1 and M2 by using a C18 Sep-Pak/silica gel cleanup column combination. Aflatoxins are separated by normal phase liquid chromatography and their concentrations are determined by fluorescence detection in a silica gel-packed flow cell. Recoveries ranged from 99 to 103% with coefficients of variation less than 2% for M1 levels of 0.117-1.17 ng/mL added to raw milk. Similar recoveries were obtained for M2. The coefficient of variation for analysis of 5 subsamples of naturally contaminated milk was less than 1%. Agreement with the official method is satisfactory. Each sample requires less than 25 mL solvent and 10 min actual handling time. Sample chromatograms show no interferences in the M1-M2 elution region and no late-eluting peaks, which permits spacing injections at 13-20 min intervals. Aflatoxin levels as low as 0.03 ppb may be determined by this procedure. Extracts have also been analyzed by thin layer chromatography.     

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers.     

Determination of dapsone in muscle tissue and milk using high-performance liquid chromatography-tandem mass spectrometry

A precise and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of dapsone in muscle tissue and milk has been developed. The sample preparation was based on extraction with organic solvent and automated solid-phase extraction (SPE) cleanup. At least three product ions were monitored for the analyte. The method was validated according to the European Decision 2002/657/EC. Estimated analytical limits were 0.0018 ng/g for CCα and 0.0031 ng/g for CCβ in meat and milk. An excellent linear concentration range was observed for both matrices with a correlation coefficient better than 0.997. Recoveries were 105-117% in meat and 101-108% in milk, with satisfactory precision and coefficients of variance (CV) less than 8%. Additionally, a simplified quantification approach was successfully evaluated depending only on the response factor (F) without the use of calibration curve. The developed method provides reliable and sensitive identification and quantification of dapsone in meat and milk.     

Liquid chromatographic determination of zearalenone and alpha- and beta-zearalenols in milk

Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.     

Hyphenation of sorbent extraction and solid-matrix time-resolved luminescence using tetracycline in milk as a model analyte

Hyphenation of sorbent extraction and solid-matrix time-resolved luminescence (TRL) was demonstrated using tetracycline (TC) in milk as a model analyte. The performance of a C18-impregnated silica layer was evaluated as both an extraction sorbent and a TRL substrate. To extract TC, a 10 x 6 mm glass-backed C18 layer was dipped into a 10 mL milk sample for 10 min followed by a 3-min water immersion for cleanup. The sorbent was then spotted with a TRL reagent solution at pH 9 that contained 5 mM europium nitrate and 5 mM EDTA. After a brief desiccation period, TRL was measured directly on the sorbent surface with a commercial fluorescence spectrophotometer. By eliminating the need to elute the analyte from the sorbent, organic solvent was not needed and sample preparation was greatly simplified. The integrated signal showed a linear dependence (R(2) = 0.9938) on TC concentration in the 0-3000 microg/L range. The same protocol was applicable to screening TC in fat-free, 2% low-fat, and whole milk at 300 microg/L, the US. regulatory tolerance level set by the Food and Drug Administration (FDA). This easy, fast, and low-cost screening method is friendly to the environment and particularly suitable for liquid samples.     

Rapid high pressure liquid chromatographic determination of aflatoxin M1 in milk and nonfat dry milk

A modification of the current revised AOAC method, 26.A10-26.A15, is described for the rapid analysis of aflatoxin M1 in milk and nonfat dry milk. The method incorporates chloroform extraction and eliminates the need for column chromatography by using liquid-liquid partition for sample extract cleanup. Quantitation is carried out by using fluorescence detection combined with high pressure liquid chromatography (HPLC) of aflatoxin M1 which has been converted to aflatoxin M2a with trifluoroacetic acid. The method has a detection limit of 0.014 micrograms/L (2 X signal/noise) for whole milk. For 6 samples of naturally contaminated nonfat dry and freeze-dried milk, the modified method gave an average result of 0.698 micrograms/L; the AOAC method gave an average result of 0.386 micrograms/L.     

Gas chromatographic analysis of milk for deoxynivalenol and its metabolite DOM-1

A gas chromatographic method is described for the determination of deoxynivalenol (DON) and its metabolite DOM-1 in milk. Milk samples were extracted with ethyl acetate on a commercially available disposable extraction column, followed by hexane-acetonitrile partitioning. Final purification was accomplished on a reverse phase C-18 cartridge. The trimethylsilyl ether (TMS) derivatives of DON were prepared, chromatographed on an OV-17 column, and quantitated with an electron capture detector. Chromatography of the TMS derivatives of milk extracts was compared to that of the corresponding heptafluorobutyryl derivatives. The limit of detection using TMS derivatives was 1 ng/mL for both toxins with recoveries averaging 82% +/- 9% at 2.5 and 10 ng/mL milk for DON and 85% +/- 6% at 10 ng/mL for DOM-1.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Liquid chromatographic determination of aflatoxin M1 in milk

The official AOAC method for aflatoxin M1 in milk was modified by replacing cellulose column chromatography with cartridge chromatographic cleanup and replacing thin layer chromatographic (TLC) determination with liquid chromatographic (LC) quantitation to yield a new method for bovine and porcine milk. An acetone extract of milk is treated with lead acetate and defatted with hexane, and M1 is partitioned into chloroform as in the AOAC method. Chloroform is removed by evaporation under a stream of nitrogen at 50 degrees C. The residue is dissolved in chloroform, the vessel is rinsed with hexane, and the 2 solutions are applied in sequence to a hexane-activated silica Sep-Pak cartridge. Less polar impurities are removed with hexane-ethyl ether, and M1 is eluted with chloroform-methanol, and determined by C18 reverse phase LC using fluorescence detection. Recoveries of M1 added to bovine milk at 0.25, 0.50, and 1.0 ng/mL were 90.8, 93.4, and 94.1%, respectively. The limit of detection was less than 0.1 ng M1/mL for both bovine and porcine milk.     

Determination of pesticides and PCBs in virgin olive oil by multicolumn solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by ion-trap GC-MS

A multicolumn solid-phase extraction cleanup for the determination of organophosphorus (OP) and organochlorine (OC) pesticides plus PCB congeners in virgin olive oil is presented. The method involves dissolution of the olive oil in hexane, followed by a cleanup system using a diatomaceous earth column (Extrelut-QE) with reversed (C(18)) and normal (alumina) phase SPE columns. Determination of OPs was by GC-NPD, while the OCs and PCBs were analyzed using GC-ECD. Recovery assays for OPs varied from 81.7% to 105.3%, for OCs ranged between 74.3% and 99.4%, while for PCBs were from 60.1% to 119.2%. Quantitation limits ranged from 10 to 25 microg/kg olive oil for OPs, and from 1 to 6 microg/kg olive oil for OCs and PCBs. In the case of positive samples, the confirmation of pesticide identity was performed by ion-trap GC-MS/MS. The applicability of the method was assayed with 19 virgin olive oil samples collected from different olive mills of Aragón (Spain). Only one OP pesticide (acephate) was detected in one sample at a concentration of 10 microg/kg. Organochlorine pesticides were found in 5-47% of samples at very low levels ranging from 1.5 to 5.2 microg/kg. PCBs were found in 20-90% of samples, showing concentrations between 2.3 and 17.3 microg/kg.     

Investigation of sample treatment steps for the analysis of polycyclic aromatic hydrocarbons in ground coffee

Sample treatment procedures were tested for the determination of polycyclic aromatic hydrocarbons (PAHs) in ground coffee. Pressurized liquid extraction (PLE), under different conditions, was combined with several cleanup methods, namely in situ purification, C18-silica solid-phase extraction (SPE), silica SPE, acid digestion, and alkaline saponification. Soxhlet extraction and direct alkaline saponification were also tested. Best results were obtained using PLE with hexane/acetone 50:50 (v/v) under 150 degrees C. Alkaline saponification followed by cyclohexane extraction and silica SPE was required to eliminate interferent compounds. Finally, 11 PAHs could be quantified in ground coffee with limits of detection in the range of 0.11-0.18 microg kg(-1). Application to ground Arabica coffee lots from Colombia revealed the presence of several PAHs, giving an overall toxicity equivalence in the range of 0.16-0.87 microg kg(-1). PAH identification was performed using both high-performance liquid chromatography-diode array detection and gas chromatography coupled to mass spectrometry.     

Determination of chlorinated pesticide residues in foods. I. Rapid screening method for chlorinated pesticides in milk.

A rapid analytical method for determining chlorinated pesticide residues in milk was developed. Thirteen pesticides were almost completely extracted. Ten mL samples of fortified milk were extracted 3 times with 20 mL portions of n-hexane as follows: (A) in the absence of water-soluble solvent; in the presence of (B) 1 mL acetonitrile; (C) 3 mL acetonitrile; (D) 5 mL acetonitrile; (E) 5 mL ethanol; (F) 5 mL acetonitrile and 1 mL ethanol. System F produced the highest pesticide recoveries but the lowest fat extraction, thus eliminating the necessity for liquid-liquid partitioning and minimizing Florisil column cleanup. Pesticide recoveries throughout the procedure were 94--103%. It was noticed, however, that the fat in high fat-containing raw milk is more readily extracted than that in commercial milk.     

Immunoaffinity purification of chlorimuron-ethyl from soil extracts prior to quantitation by enzyme-linked immunosorbent assay

A competitive-indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantify chlorimuron-ethyl in soil. The linear working range of the assay was from 1 to 1000 ng mL(-)(1). The assay had an I(50) value of 54 ng mL(-)(1), with a limit of detection of 2 ng mL(-)(1) and a limit of quantification of 27 ng mL(-)(1). Three soils were extracted using a carbonate buffer (pH 9.0) and the extracts spiked with chlorimuron-ethyl. Because of the effects of coextractants (matrix effects) from soil on the accuracy and precision of the ELISA, immunoaffinity chromatography (IAC) was used to purify chlorimuron-ethyl from soil extracts prior to analysis. The immunoaffinity columns, which had a total binding capacity of 1350 ng of chlorimuron-ethyl mL(-)(1) of immunosorbent, were prepared by binding anti-chlorimuron-ethyl antibodies to protein G Sepharose 4B. Although the matrix effects were largely removed using the affinity column, they could be completely removed by first passing the extract through a column containing epoxy-coupled 1,6-diaminohexane (EAH) Sepharose 4B to remove organic acids prior to IAC. Assay sensitivity was increased 100-fold using IAC to purify and simultaneously concentrate chlorimuron-ethyl from soil extracts. The purification strategy (EAH followed by IAC chromatography) removed matrix effects from all three soils and allowed for the accurate quantitation of chlorimuron-ethyl in soil extracts.     

Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS.

Milk is typically screened for beta-lactam antibiotics by nonspecific methods. Although these methods are rapid and sensitive, they are not quantitative and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five beta-lactam and two cephalosporin antibiotics commonly or potentially used in the dairy industry is described using high-performance liquid chromatography with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin, and ceftiofur. The antibiotics were extracted from milk with acetonitrile, followed by reversed-phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water/methanol gradient containing 1% acetic acid on a C-18 reversed-phase column. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Quantitation was based on the most abundant product ions from fragmentation of the protonated ion for amoxicillin, cephapirin, ampicillin, and ceftiofur and on the fragmentation of the sodium adduct for penicillin G, penicillin V, and cloxacillin. The method was validated at the U.S. FDA tolerance or safe level and at 5 or 2.5 ng/mL for these compounds in bovine milk. Theoretical method detection limits in milk based on a 10:1 signal to noise ratio were 0.2 ng/mL (ampicillin), 0.4 ng/mL (ceftiofur), 0.8 ng/mL (cephapirin), 1 ng/mL (amoxicillin and penicillin G), and 2 ng/mL (cloxacillin and penicillin V) using a nominal sample size of 5 mL.     

Multi-residue analysis of PAH, PCB, and OCP optimized for organic matter of forest soil

Purpose

Analyzing organic pollutants in forest soil is challenging because they are strongly physical and chemical bound to soil organic matter (SOM). Within the framework of a forest soil inventory, an analytical protocol for the determination of polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and organochlorine pesticides (OCP) should be established and validated using one and the same extraction and cleanup procedure. The protocol should be applicable for reliable analysis of a high number of samples in a short timeframe.

Materials and methods

Two different soil samples representative for the humic layer from a typical mixed and coniferous forest soil had been used for the analysis. Three solvents of different polarity, namely cyclohexane (CH), ethylacetate (EA)/CH (1/1, v/v), and acetone (AC)/CH (2/1, v/v), and the six standard extraction techniques (pressurized liquid extraction (PLE), soxhlet extraction, fluidized bed extraction, sonication, shaking, and one-step extraction recommended for analyzing agricultural soil in Germany (VDLUFA 2008)) were compared concerning their extraction efficiency. For additional matrix separation, two different cleanup procedures (gel permeation chromatography (GPC) and solid-phase extraction (SPE) with different sorbents) were tested. Quantification was carried out using gas chromatography combined with mass spectrometry (GC–MS) and two different injection systems (split/splitless injection and programmable temperature vaporizer (PTV) injection). Labeled internal standards, added prior to extraction, were used for method evaluation.

Results and discussion

For the simultaneous extraction of PAH, PCB, and OCP from organic forest soil PLE with acetone/cyclohexane (2/1, v/v) provided the highest extraction efficiency. A two-step cleanup procedure consisting of GPC followed by SPE with silica gel was entirely sufficient for the separation of humic substances without discrimination of analytes. Recovery rates for the different extraction and cleanup steps ranged between 89% and 106%. For quantification, a GC–MS method was developed using two different injection systems and two capillary columns of different selectivity.

Conclusions

By comparing six standard extraction techniques for PAH, PCB, and OCP from forest soil, we obtained the highest extraction efficiency when using PLE with AC/CH (2/1). For sample injection, we achieved best results using an optimized PTV injection system as it highly reduced the breakdown of thermolabile pesticides. Using this combination of technical equipment, it is possible to determine a concentration of the analytes in the trace level range of 1–2 μg kg^?1 in humic soil.     

Evaluation of liquid chromatographic behavior of cephalosporin antibiotics using restricted access medium columns for on-line sample cleanup of bovine milk

Microsample deproteinization of bovine milk was carried out on-line using a series of restricted access medium (RAM) bovine serum albumin (BSA) columns: C8, C18, phenyl, and cyano. The four different columns prepared showed a high percentage of protein exclusion using water as the mobile phase and provided an appropriate retention profile for a series of five cephalosporin antibiotics (cefoperazone, cephacetril, cephalexin, cephapirin, and ceftiofur). Chromatographic conditions such as washing time, buffer pH, and type and percentage of organic modifier were fully evaluated with respect to the protein elution profile and retention of the antibiotic by RAM column. One of these columns was chosen to develop and validate a method for the determination of cefoperazone in bovine milk. The system used in this work was composed of a RAM-BSA phenyl column coupled to a C18 analytical column. The standard curve was linear over the range 0.100-2.50 microg/mL. The limits of quantification and detection were 0.100 and 0.050 microg/mL, respectively. The developed method showed high intermediate precision (CV of 2.37-2.63%) and accuracy (90.7-94.3%) with adequate sensitivity for drug monitoring in bovine milk samples.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.     

Determination of T-2 and HT-2 toxins in cereals including oats after immunoaffinity cleanup by liquid chromatography and fluorescence detection

A reliable method for the determination of T-2 toxin and HT-2 toxin in different cereals, including oats, as well as in cereal products was developed. After extraction with methanol/water (90/10, v/v) and dilution with a 4% NaCl solution, the toxins were purified with immunoaffinity columns, derivatized with 1-anthroylnitrile, separated by HPLC, and determined using fluorescence detection. Due to the unspecific derivatization reagents, validation parameters were matrix dependent: in the range 10-200 microg/kg, recovery rates of 74-120% with relative standard deviations between 0.5 and 20.3% were obtained. On average, the limit of quantitation was shown to be 8 microg/kg for each toxin. For naturally contaminated samples, comparable results were obtained when analysis was performed according to this method without derivatization as well as according to a method based on a SPE cleanup utilizing tandem mass spectrometric detection in both cases. Using aqueous acetonitrile as extractant resulted in incorrectly high toxin concentrations due to water absorption of dry samples and toxin accumulation in the organic phase in the subsequent phase separation of the extractant. Furthermore, when comparing the commercially available immunoaffinity columns for T-2 and HT-2 toxins, significant differences regarding capacity and cleanup performance were observed.