Cathie Ménard

Liquid chromatographic determination of domoic acid in shellfish products using the paralytic shellfish poison extraction procedure of the association of official analytical chemists

Domoic acid, the recently discovered toxic substance found in contaminated mussels from an area in eastern Prince Edward Island (Canada) was extracted from mussel tissue using the procedure of the Association of Official Analytical Chemists for paralytic shellfish poisons. This involved a 5-min boiling of the sample with 0.1 M hydrochloric acid then cooling and centrifuging. An aliquot of the supernatant was diluted ten to one-hundred times with water, filtered and analysed by reversed-phase liquid chromatography with a mobile phase consisting of acetonitrile-water (12:88) at pH 2.5 and an absorption wavelength of 242 nm. The detection limit was about 0.5 mg/kg domoic acid in seafood samples. The technique was successfully applied to a variety of commercially purchased shellfish and shellfish products.

Determination of microcystins in blue-green algae, fish and water using liquid chromatography with ultraviolet detection after sample clean-up employing immunoaffinity chromatography

Anti-microcystin LR immunnoaffinity cartridges were evaluated for their ability to selectively remove microcystins from extracts of blue-green algae, fish and water samples for subsequent analysis by liquid chromatography with UV absorbance detection at 238 nm. Blue-green algae and fish samples were extracted with 75% methanol in water. A portion of the extract was diluted and passed through an immunoaffinity cartridge. Water samples were applied directly to the cartridge. The cartridge was rinsed with water and 25% methanol in water. The microcystins were eluted with 80% methanol in water containing 4% acetic acid. It was found that the cartridges were effective in isolating the microcystins from blue-green algae, fish and water samples, resulting in extracts that were clean enough to enable direct LC-UV detection down to approximately 0.03 microg/g in the blue-green algae and fish samples, and as low as 0.02 ng/ml for water samples. The cartridges were found to have a capacity of approximately 200 ng each for a mixture of microcystins RR, YR, LR and LA, or as much as 525-800 ng for individual compounds. Recoveries trough the complete analytical procedure ranged from 64 to 115% (all values) with an overall average of approximately 80% at spiking levels of 0.5-4.0 microg/g for the microcystins in blue-green algae. The average recoveries (n=8) from spiked (0.1-0.5 microg/g) fish samples were 73% for RR, 79% for YR, 81% for LR and 77% for LA, while from the spiked (2.0-0.04 ng/g) tap and river water samples (n=6), recoveries were 78% for RR, 86% for YR, 94% for LR and 89% for LA.

Use of immunoaffinity chromatography as a simplified cleanup technique for the liquid chromatographic determination of phenylurea herbicides in plant material

Immobilized polyclonal antibodies were evaluated for the cleanup of extracts of several food samples (carrots, celery, corn, grapes, onions, potatoes and strawberries). The antibodies were generated in the rabbit after inoculations with an antigen prepared from the urea herbicide, isoproturon. The antibodies were immobilized onto the surface of activated silica particles and packed into disposable plastic syringe barrels and used in the same general manner as cartridges for standard solid-phase extraction. They displayed substantial cross-reactivity with six other urea herbicides which permitted them to be used as a multi-urea herbicide cleanup procedure. Reversed-phase liquid chromatography with UV detection at 244 nm was the only equipment required for the quantitation. Methanolic extracts of the plant tissue samples were concentrated and then diluted with water before passage through the immunoaffinity (IA) cartridge. The cartridge was washed and the herbicides eluted with 70% methanol in water for analysis by HPLC. The cleanup provided by the IA cartridge enabled the direct detection and quantitation of the herbicides at a concentration level of 25 ng/g in potatoes and carrots. An additional cleanup step using a strong anion-exchange solid-phase extraction cartridge (SPE-SAX) was required for determination of the herbicides in grape, onion, celery, corn and strawberries at levels of 25 ng/g. With the combined SPE-SAX and IA cleanup, the detection limits in the plant material examined were about 2–5 ng/g depending upon the herbicide. At 25 ng/g spiking levels, recoveries through the complete procedure for monuron, chlortoluron, isoproturon and durion averaged 103±10% (n=6 for each herbicide); for chloroxuron (80±5%, n=6); chlorbromuron (65±12%, n=6); linuron (37±15%, n=6). The only organic solvent used was methanol mainly for the initial sample extraction and in the LC mobile phase. No organic-aqueous partitions or adsorption chromatography employing organic solvents were required.

Evaluation of immunoaffinity chromatography as a replacement for organic solvent clean-up of plant extracts for the determination of triazine herbicides by liquid chromatography

A method employing immunoaffinity chromatography for sample clean-up has been developed for a number of triazine herbicides. The approach eliminates the requirement for solvents such as hexane, dichloromethane, acetone and other solvents commonly used for organic-aqueous partitions or adsorption chromatographic clean-up of sample extracts. The only solvent used was methanol, (the least toxic, most environmentally friendly and least expensive of those mentioned) for the initial sample extraction and clean-up. Acetonitrile was used in the liquid chromatographic mobile phase, although methanol proved to be satisfactory for all traizines except for atrazine which coeluted with simetryn and simazine which coeluted with metribuzin. An immunoaffinity cartridge employing immobilized polyclonal antibodies generated against atrazine but which cross-reacted with several other triazines, was used for the clean-up of extracts of apple, carrot, celery, corn, potato and peas. The triazines atrazine, cyanazine, simazine, propazine, terbutylazine, simetryn and prometone were studied. The method involved extraction of the triazines from the plant material with methanol followed by concentration of an aliquot of the extract with subsequent dilution in aqueous phosphate buffered saline (PBS). The PBS mixture was cleaned up sequentially using a strong anion-exchange solid phase extraction cartridge (SPE-SAX) followed by the immunoaffinity cartridge. Quantitation was carried out using reversed-phase liquid chromatography with UV absorption detection at 220 nm. Atrazine, simazine, propazine and terbutylazine were recovered from extracts consistently above 80% at a spiking level of 25 ng/g each. The average recovery for cyanazine was 71% (n=9), for prometone it was 72% (n=7) and for simetryn it was 65% (n=9). Detection limits were estimated to be 2–10 ng/g depending upon the triazine and the plant tissue analyzed. One immunoaffinity cartridge was used for 49 plant extracts with no loss of activity or effectiveness for extract clean-up. A combination of two immunoaffinity cartridges (one not recognizing the triazines and one recognizing them) was evaluated for the selective clean-up of several of the plant extracts. This approach compared well with the SPE-SAX-immunoaffinity cartridge combination. Spiked (25 ng/g of each triazine) extracts of celery and corn were readily confirmed by GC-mass spectrometry.

Melamine in infant formula sold in Canada: occurrence and risk assessment.

An analytical method incorporating simple liquid extraction followed by mixed mode cation exchange/reversed phase solid phase extraction and liquid chromatography-tandem mass spectrometry was developed and validated for the analysis of melamine (MEL) in liquid and powdered infant formula. The method used two different MEL stable isotope labeled internal standards to monitor analyte recoveries and to account for matrix effects. The method is sensitive (limit of quantitation of 4 ng/g), accurate, and precise (during validation, recoveries corrected by internal recovery standard averaged between 92 and 104% for all fortification levels and matrices). The method was used to analyze 94 samples of infant formula purchased from major retailers in Ottawa, ON, Canada, to examine whether or not Canadian infants are exposed to background levels of MEL. MEL was detected in 71 of the 94 products analyzed at concentrations ranging from 4.31 to 346 ng/g (median = 16 ng/g). A comparison of estimated dietary exposures to the recently recommended World Health Organization toxicological reference value for melamine suggests that the presence of low levels of MEL in infant formula purchased in Canada does not represent a health risk.

Determination of clenbuterol in beef liver and muscle tissue using immunoaffinity chromatographic cleanup and liquid chromatography with ultraviolet absorbance detection

Clenbuterol, a beta-agonist, was determined in samples of beef liver and muscle. The method employed an acidic aqueous extraction followed by protein precipitation. The supernatant liquid was passed through a weak cation-exchange cartridge and then through a commercially available immunoaffinity cartridge. Clenbuterol was eluted from the immunoaffinity cartridge with 80% ethanol in water. The eluate was concentrated and analysed directly by reversed-phase liquid chromatography using gradient elution and UV detection at 245 nm. Detection limits were estimated to be 0.3 ng g-1 clenbuterol. A single immunoaffinity cartridge was used for ten sample extracts with no significant loss in capacity. No organic solvents other than ethanol and methanol were employed in the procedure. Recoveries of clenbuterol from samples of beef liver and muscle spiked at 2 and 5 ng g-1 carried through the entire procedure were 63 +/- 11% (range, 53-74%) compared to pure standards. Absolute recoveries of pure standards (30 ng clenbuterol) carried through the same analytical steps were 70 +/- 5% (n = 6), the losses being primarily due to the ion-exchange step.

Determination of melamine, ammeline, ammelide and cyanuric acid in infant formula purchased in Canada by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry-based isotope dilution method was developed for the analysis of the triazine compounds melamine (MEL), ammeline (AMN), ammelide (AMD) and cyanuric acid (CYA) in infant formula samples purchased in Canada in 2008 for the purpose of a combined exposure and risk assessment. Infant formula samples were extracted with 1:1 acetonitrile–water, cleaned up on disposable ion-exchange solid-phase extraction cartridges, and analysed by ultra-high-performance liquid chromatography-tandem mass spectrometry. MEL and CYA were detected in almost all infant formula products: the highest concentrations observed were 0.32 mg kg−1 MEL and 0.45 mg kg−1 CYA. Samples that were relatively high in MEL in this survey tended to be low in CYA, and vice versa. Concentrations of AMN and AMD were very low in all samples. The total of MEL-related compounds (sum of all four analytes) in all samples was below the interim standard of 0.5 mg kg−1 for infant formula products established by Health Canada.

Liquid chromatographic determination of okadaic acid and dinophysistoxin-1 in shellfish after derivatization with 9-chloromethylanthracene

The reagent 9-chloromethylanthracene was evaluated for derivatization of the diarrhetic shellfish poisons, okadaic acid and dinophysistoxin-1 (DTX-1), to form fluorescent products separable by liquid chromatography. The toxins were reacted with the reagent in acetonitrile in the presence of tetramethylammonium hydroxide for 1 h at 90 degrees C. The products were purified by using two silica solid-phase extraction cartridges before being determined by reversed-phase liquid chromatography with fluorescence detection. The results are comparable to those obtained using 9-anthryldiazomethane (ADAM) for okadaic acid and DTX-1 in mussel tissue. Detection limits were estimated to be about 70-100 ng/g hepatopancreas (equivalent to 12-20 ng/g whole tissue) for each toxin.

Determination of marine toxins by liquid chromatography

SummaryDomoic acid, the shellfish toxin discovered in 1987 off the eastern coast of Canada and northern US is determined by isocratic reversed-phase LC with UV detection at 242 nm. Extraction from shellfish tissue is achieved with 0.1 mol/l HCl or water, with minimal cleanup before LC analysis. Detection limits are around 0.5 μg/g. Results can be confirmed by pre-chromatographic derivatization at either the — NH or — COOH moiety. An LC screening method using pre-chromatographic oxidation has been developed for paralytic shellfish poison (PSP, comprised mainly of 12 related compounds). The individual toxins in the PSP family produced single products with periodate oxidation. However, they could not all be separated using a variety of chromatography systems including reversed-phase and ion-pair chromatography with heptane sulfonate or tetrabythyl-ammonium ion. Detection limits were about 0.05 μg/g total PSP toxin in shellfish. Saxitoxin could be detected at 5–10 pg per injection. Comparisons of this method with the mouse bioassay and the post-column technique showed reasonable agreement between results.

Confirmation of domoic acid in shellfish using butyl isothiocyanate and reversed-phase liquid chromatography

A simple chemical confirmatory technique has been developed for domoic acid, a neurotoxic amino acid of marine origin. After extraction with water-methanol, the domoic acid-containing extract is analysed directly by reversed-phase liquid chromatography with UV absorption detection at 242 nm. For confirmation of positive results an aliquot of the extract is evaporated to dryness and reacted with butyl isothiocyanate to form a thiourea derivative which elutes later than underivatized domoic acid. No additional sample cleanup is required in order to carry out the derivatization for conformation of domoic acid at the Canadian 20 micrograms/g guideline level in shellfish. In mussel extract, domoic acid was converted to the thiourea derivative with a yield of 86-91% compared to a pure standard carried through the same reaction. The detection limit for the derivative was about 5-10 micrograms/g of equivalent domoic acid in extracts of mussels, clams or oysters.