Robert J. Maxwell

Systematic protocol for the accumulation of fatty acid data from multiple tissue samples: Tissue handling, lipid extraction and class separation, and capillary gas chromatographic analysis

A systematic procedure was developed for detailed fatty acid profiling of both neutral and polar lipid fractions isolated from hundreds of related bovine muscle and adipose tissue samples. A regimen was established for a nonbiased handling of tissue samples, which included their handling in a predetermined random order. Lipid class separation was accomplished concomitantly during the extraction of the tissues by a selective dry column method, which allowed a detailed analysis of minor but important polyunsaturated fatty acids associated with the polar fraction. Neutral lipids were derivatized to fatty acid methyl esters (FAME) by a literature procedure. However, to protect against lysis of plasmalogens in the polar fraction, a modified nonacidic esterification procedure was developed. FAME profiles were obtained on a program-mable high resolution capillary gas chromatograph (GC). Run programs for unattended GC operation and data storage are described. By this overall procedure, the quantitation and peak identification were obtained for major and minor fatty acid constituents from bovine tissue in a manner that prepares for valid statistical interpretation of the resulting data.

Equilibrium solubilities of β-carotene in supercritical carbon dioxide

Equilibrium solubilities of pure crystalline β-carotene in supercritical CO2 have been measured using an LDC Analytical SPA extractor1. The apparatus features a recirculating loop with an in-line UV detector. Quantitation by UV absorbance permits the detection of lower solubilities than can normally be obtained gravimetrically. The values reported were obtained at temperatures from 40 to 70°C and pressures from 200 to 500 bar. The measured mole fractions ranged from 9.0×10−9 at 40°C and 275 bar to 1.9x10−6 at 70°C and 400 bar. Solubilities are also reported for β-carotene in mixtures of CO2and 1 wt.% ethanol, 1 wt.% methanol and 1 wt.% methylene chloride, at 70°C and various pressures. Addition of the cosolvent increased the solubility in every case, with the largest increase occurring in a mixture of CO2 and 1 wt.% ethanol. Pure component properties for β-carotene were estimated, and the experimental data were correlated to a modified Peng-Robinson equation of state. The model predictions compare well with the experimental data.

Supercritical-fluid extraction of fungal lipids using mixed solvents: Experiment and modeling

Abstract Polyunsaturated fatty acids, notably eicosapentaenoic acid (EPA), have been purported to have beneficial physiological activity, including the prevention of arthritis and cardiovascular disease. A possible source of these fatty acids are filamentous fungi (e.g., Saprolegnia parasitica ). In this work, lipids are extracted directly from the fungal mycelia using supercritical CO 2 and CO 2 mixed with 10 wt % ethanol. Extractions are performed at temperatures from 40 to 60 °C and pressures from 205 to 680 bar. The recovery of lipid increases with increasing pressures and higher recoveries are obtained when a mixture of CO 2 with 10 wt % ethanol is used as the solvent (e.g., 89% recovered with 10% ethanol vs. 48% for 100% CO 2 ). The more polar CO 2 mixture is a better solvent since it is able to extract both the neutral and the polar lipid fractions. An unsteady extraction model which can give reliable representation of the entire extraction curve is presented. Mass transfer coefficients are computed using the experimental data, and these coefficients are correlated as a function of the interstitial velocity.

Determination of flumequine and oxolinic acid in fortified chicken tissue using on-line dialysis and high-performance liquid chromatography with fluorescence detection

Abstract Isolation of the fluoroquinolones, flumequine and oxolinic acid, from fortified chicken liver was achieved using liquid–liquid extraction, aqueous on-line dialysis and trace enrichment. Dialysis, trace enrichment and column switching were performed using the ASTED system. Separation of the isolated compounds in the tissue extracts was performed using reversed-phase HPLC and fluorescence detection. This procedure yielded excellent mean recoveries at the 50, 25, 10 and 5 ng/g spiking levels for flumequine (94–96%; 4–9% R.S.D.) and at the 25 and 5 ng/g spiking levels for oxolinic acid (98–99%; 4–6% R.S.D.). Clean chromatograms were obtained, allowing detection of 5 ng/g flumequine and 2.5 ng/g oxolinic acid to be easily made. Due to its lower organic solvent consumption, automation and on-line capabilities, this method may be a suitable replacement for conventional chemical extraction techniques for drug residues in animal tissues.

Automated multi-residue isolation of fluoroquinolone antimicrobials from fortified and incurred chicken liver using on-line microdialysis and high-performance liquid chromatography with programmable fluorescence detection.

Isolation of the quinolones, sarafloxacin (SAR), oxolinic acid (OXA), and flumequine (FMQ), from fortified chicken liver tissues, and SAR incurred chicken liver tissues was achieved by combined liquid-liquid extraction and aqueous on-line microdialysis using the automated trace enrichment of dialysates (ASTED) system. Analysis of tissue isolates after ASTED clean-up was performed using reversed-phase HPLC and programmable fluorescence detection. Overall recoveries of SAR, OXA and FMQ from samples fortified over a concentrations range of 1-100 ppb were 94, 97 and 87% with overall inter-assay variability of 4.2, 4.1 and 3.6%, respectively. Chicken liver samples incurred with SAR at three concentration levels also were tested by the ASTED method. The method exhibited high peak resolution (3.4-4.2 on average), a high signal-to-noise ratio, and demonstrated good precision. The ASTED-HPLC method overall had a lower limit of detection (LOD) of 0.2 ppb, and a limit of quantitation (LOQ) of 1 ppb.

Supercritical fluid extraction of methyltestosterone, nortestosterone and testosterone at low ppb levels from fortified bovine urine.

A multi-residue supercritical fluid extraction (SFE) method is proposed for the isolation of nortestosterone, testosterone and methyltestosterone from bovine urine. Prior to SFE, bovine urine was hydrolyzed and then fortified with the three steroids at 100 ng/ml and 50 ng/ml each for HPLC analysis and 25 ng/ml and 12.5 ng/ml each for GC-MS analysis. The samples then were mixed with an adsorbent material, placed in an SFE extraction vessel prepacked with a 3-ml SPE column containing neutral alumina and the testosterones were extracted from the urine matrix using unmodified supercritical CO2 at 27.2 MPa and 40 degrees C. The steroids were retained in-line on the neutral alumina sorbent in the SPE column while co-extracted artifactial material was trapped off-line after CO2 decompression. After SFE, the SPE column was removed from the extraction vessel, and the trapped steroids were eluted from the neutral alumina sorbent with 3 ml of a methanol-water mixture. Eluates were used directly without post-SFE clean-up either for HPLC analysis (detection limit 50 ng/ml) or for GC-MS analysis (detection limit 5 ng/ml after steroid derivatization). The multi-residue SFE recoveries (n=6) for nortestosterone, testosterone and methyltestosterone from hydrolyzed bovine urine by GC-MS analysis were 90.8+/-6%, 93.9+/-3% and 92.5+/-5%, respectively for each steroid at the 12.5 ng fortification level.

Effect of gamma irradiation at various temperatures on air and vacuum packed chicken tissues II. Fatty acid profiles of neutral and polar lipids separated from muscle and skin irradiated at 2–5°C

Abstract Chicken muscle and skin were separately irradiated in air and under vacuum packaging at 0, 1, 3, 6 and 10 kGy using 137Cs (dose rate = 0.1 kGy/min). Lipids were isolated as neutral and polar subclasses from muscle samples and as total lipid extracts from skin. Lipids were converted to fatty acid methyl esters, analyzed by capillary gas chromatography and the data compiled as fatty acid profiles by statistical computer analysis. Normalized reports were assembled from this data for the three lipid extract types. Only negligible changes in fatty acid profiles were observed for the neutral lipids of muscle and for the fatty acyl residues of skin lipids. Minor changes of interest, however, were observed for the polyunsaturated fatty acyl residues in the polar lipid fractions of muscle tissue, especially at higher irradiation doses (6 and 10 kGy). Comparisons were made between these results and those of an earlier study were similar tissues were irradiated at -20°C. No new fatty acyl residues or other artifacts due to γ-irradiation were found in detectable amounts by gas chromatography in any of the lipid fractions isolated.

Multiresidue supercritical fluid extraction method for the recovery at low ppb levels of three sulfonamides from fortified chicken liver

A supercritical fluid extraction (SFE) method is proposed for the recovery of three sulfonamides from chicken liver. Samples were extracted at 680 bar and 40 degrees C using unmodified carbon dioxide and were collected free of co-extracted artifactual material on an in-line neutral alumina sorbent bed. High recoveries of sulfamethazine (SMZ), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX) were obtained from chicken liver samples fortified at levels from 1000 to 50 ppm.

Rapid and sensitive method for the quantitation of non-polar lipids by high-performance thin-layer chromatography and fluorodensitometry

Abstract Non-polar lipids were separated by high-performance thin-layer chromatography on silica gel plates and detected by use of a new reagent that induced fluorescence in the separated components. Developed thin-layer plates are dipped into a solution of sulfuric acid—ethanol—hexane (1:35:64, v/v), heated and the lipid classes are quantified by fluorescence densitometry. This technique allowed detection of certain standard lipids at the 5-ng level, is well suited for the rapid and efficient analysis of large numbers of samples and offers distinct advantages over other in situ fluorescence inducing methods. The method was successfully applied to the analysis of the non-polar lipids that occur in enzymatically hydrolyzed beef tallow.

A rapid, quantitative procedure for measuring the unsaponifiable matter from animal, marine, and plant oils

A simple, rapid, quantitative procedure for measuring the unsaponifiable matter (USM) in animal, marine and plant oils is described. Saponification is achieved by grinding the oil with potassium hydroxide pellets followed by a short heating period. The resultant soap mixture is then ground together with Celite powder, transferred to a glass column, and the USM is eluted with small amounts of dichloromethane. Good agreement between duplicate determinations was obtained for all oils examined. Values for the USM of 21 different fats and oils are given. Comparison of some of these values was made with those obtained with Official Methods.