Supercritical fluid extraction (SFE) as a multi-residue extraction procedure for beta-agonists in bovine liver tissue

A supercritical fluid extraction procedure has been developed for the extraction of beta-agonists in bovine liver samples. The method is suitable for compounds of different beta-agonist classes: the substituted aniline-type compounds (e.g. clenbuterol) and the phenolic-type compounds (e.g. salbutamol), including conjugated forms of the latter. The developed procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol and salbutamol residues at the low ppb level. Addition of methanol modifier and removal of sample moisture are necessary for the extraction of more polar analytes such as salbutamol. Method validation incorporating intra- and inter-assays was carried out on fortified liver tissue and showed good recovery and low variation (RSD < 15%). An enzyme hydrolysis procedure was incorporated into the method for the deconjugation of conjugated residues. The developed procedure was shown to be successful for the determination of both clenbuterol and salbutamol in incurred liver tissue.     

Selective detection of underivatized 2,4-dichlorophenoxyacetic acid in soil by supercritical fluid chromatography with ion mobility detection

A simplified procedure was developed for the determination of 2,4-dichlorophenoxyacetic acid (2,4-D) in soils. Soil samples were separated by supercritical fluid chromatography after extraction without derivatization and without the use of column chromatography for cleanup. Interferences in the chromatographic separation were eliminated by using a tunably selective ion mobility detector. An atmospheric pressure ion formed by the free acid was selectively monitored so the detector could monitor 2,4-D in the presence of other electron-capturing compounds. For a randomly chosen soil sample, the level of 2,4-D detected was estimated at 500 ppb.     

The analysis of chlorinated dibenzofurans in municipal fly ash: Supercritical fluid extraction vs Soxhlet

A multiresidue supercritical fluid extraction procedure for chlorinated dibenzofurans (PCDFs) has been optimized as an efficient method for the determination of these compounds in fly ash samples. The results were compared with those obtained by Soxhlet extraction. Extracts from the two procedures were analyzed by gas chromatography–mass spectrometry (HRGC–MS) Statistical analysis of the results confirmed that supercritical fluid extraction provides data with a relative standard deviation of less than 10% while Soxhlet extraction data show a much greater spread.     

Isolation of chloramphenicol from whole eggs by supercritical fluid extraction with in-line collection

Egg consumption, at more than 65 billion per year in the United States, represents a potentially significant source of exposure to drug residues, particularly if the laying hens are treated with antimicrobial compounds or fed a diet containing medicated feed. Residues resulting from the use of chloramphenicol (CAP) is especially problematic if this compound is not used in accordance with national registration, e.g., for the control of Salmonella microorganisms in poultry. The most commonly used methods for the determination of CAP in biological samples require the use of large amounts of organic solvent. As a result, a less solvent intensive supercritical fluid extraction (SFE) method was developed for CAP in whole chicken eggs, and the results were compared with those for a solvent extraction procedure. In the SFE method, the egg sample is extracted with supercritical CO2 (without a modifier) at 10,000 psi (680 bar), 80 degrees C, and an expanded gas flow rate of 3.0 L/min to a total volume of 150 L. The CAP is trapped in-line on a Florisil sorbent bed. The CAP is eluted post-SFE by using the liquid chromatographic mobile phase solvent (water-methanol), and determined on a C8 column with ultraviolet detection at 280 nm. Recovery from eggs fortified at the 10 ppb level (n = 6) was 81.2 +/- 4.3%. To obtain eggs containing incurred CAP, hens were given a single daily dose of 75 mg CAP (orally by gelatin capsule) for 2 consecutive days, and the eggs were collected over a 12-day period. The mean value for "normally incurred" CAP in the eggs (n = 17) analyzed by SFE ranged from none detected to 174.5 ppb, with an overall mean of 60.5 ppb, compared with a mean of 60.4 ppb for the solvent extraction method. No significant difference in results was found between methods. However, the SFE method is more rapid, uses less solvent, and gives recoveries similar to those for the solvent extraction method, making it ideal for regulatory monitoring.     

Evaluation of methods aimed at complete removal of template from molecularly imprinted polymers.

Polymers imprinted with clenbuterol were used to study the influence of various post-polymerization treatments [e.g., thermal annealing, microwave assisted extraction (MAE), Soxhlet extraction and supercritical fluid template desorption] on the bleeding of residual template. The aim of the study was to reduce the bleeding to levels that would allow the use of the materials as affinity phases for extraction of clenbuterol from bovine urine at concentrations below 1 ng ml-1. After treatment, the clenbuterol imprinted polymers were packed into solid-phase extraction columns and the bleeding was estimated by quantifying the amount of template released in 10 ml of methanol-acetic acid (9 + 1 v/v). This was followed by an assessment of selectivity and recovery in comparison with non-treated material. The lowest bleeding level was found after MAE using 100% trifluoroacetic acid for 3 x 20 min at 100 degrees C. The collected eluate contained in this case 3 ng ml-1 of clenbuterol. The same material was subsequently used for the extraction of clenbuterol from spiked bovine urine. The resulting selectivity and recovery were lower compared with those obtained using the untreated material. A milder but still efficient method to reduce the bleeding level was found to be MAE with formic acid. In this case a bleeding level of 14 ng ml-1 was found after only a 1 h extraction time. In a second model system, using a polymer imprinted with L-phenylalanine anilide, the bleeding was reduced to a similar level by extensive on-line washing in good swelling solvents containing acid or base additives and after thermal annealing of the polymers in the dry state.     

Confirmation of eprinomectin, moxidectin, abamectin, doramectin, and ivermectin in beef liver by liquid chromatography/positive ion atmospheric pressure chemical ionization mass spectrometry

A liquid chromatographic (LC) multiresidue screening procedure was developed for determination of eprinomectin, moxidectin, abamectin, doramectin, and ivermectin in beef liver at 0, 25, 50, and 100 ppb levels. A procedure using low resolution LC/atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) was developed with further purification steps added to the quantitative LC method to confirm residues. Acetonitrile extracts of liver, prior to derivatization for LC analysis, were further purified by using a C8 solid-phase extraction cartridge and an alumina-B cartridge. The purified extract was analyzed by injection into an LC/positive ion APCI MS. Identity of the compound was confirmed by comparison of its retention time and relative intensity data with those of a standard or recovery from a fortified control liver sample. Anthelmintic drugs in acetonitrile extracts of liver containing eprinomectin, moxidectin, abamectin, doramectin, and ivermectin at 25 ppb, the lowest level of fortification used in the LC determinative method, were successfully confirmed.     

FT-MIR and Raman spectroscopy coupled to multivariate analysis for the detection of clenbuterol in murine model

A fast and simple screening method for the determination of clenbuterol at the ppb level in a murine model was demonstrated by Mid Infrared (MIR) and Raman spectroscopy in conjunction with multivariate analysis. In order to build the calibration models to quantify clenbuterol in rat meat, mixtures of rat meat and clenbuterol were prepared in a range of 5-10,000 ppb. Partial Least Square (PLS) analysis was used to build the calibration model. The results shown that Mid Infrared and Raman spectroscopy were efficient, but Mid Infrared (R(2) = 0.966 and SEC = 0.27) were superior to Raman (R(2) = 0.914 and SEC = 1.167). The SIMCA model developed showed 100% classification rate of rat meat samples with or without clenbuterol. The results were confirmed with contaminated meat samples from animals treated with clenbuterol. Chemometric models represent an attractive option for meat quality screening without sample pretreatments which can identify veterinary medicinal products at the ppb level.     

Simultaneous analysis of antioxidants and preservatives in cosmetics by supercritical fluid extraction combined with liquid chromatography-mass spectrometry

This study evaluated supercritical fluid extraction (SFE) combined with liquid chromatography-mass spectrometry (LC-MS) to determine trace preservatives and antioxidants including methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol (alpha-t) and alpha-tocopherol acetate (alpha-ta) in cosmetic products. A supercritical fluid extraction procedure was used to isolate four paraben preservatives and four antioxidants from the cosmetic matrix before quantitative analysis. The optimum extraction condition was performed with static extraction for 5 min, then dynamic extraction for 20 min by using carbon dioxide supercritical fluid at 14,000 kPa and 65 degrees C. Methanol was used as collection solvent and the sea sand was chosen as a filling material. The analytes were separated on a C18 reversed-phase column using methanol-water as mobile phase and quantified by measuring its mass spectrometry. The linearity range is from 10 to 20,000 ng/g with RSD values below 18%. Detection limits are achieved at the level of 4.7-142 ng/g. It was successfully applied to the determination of paraben preservatives and antioxidants in cosmetics without tedious pretreatment.     

Determination of major aromatic constituents in vanilla using an on‐line supercritical fluid extraction coupled with supercritical fluid chromatography

An on‐line supercritical fluid extraction coupled with supercritical fluid chromatography method was developed for the determination of four major aromatic constituents in vanilla. The parameters of supercritical fluid extraction were systematically investigated using single factor optimization experiments and response surface methodology by a Box–Behnken design. The modifier ratio, split ratio, and the extraction temperature and pressure were the major parameters which have significant effects on the extraction. While the static extraction time, dynamic extraction time, and recycle time had little influence on the compounds with low polarity. Under the optimized conditions, the relative extraction efficiencies of all the constituents reached 89.0–95.1%. The limits of quantification were in the range of 1.123–4.747 μg. The limits of detection were in the range of 0.3368–1.424 μg. The recoveries of the four analytes were in the range of 76.1–88.9%. The relative standard deviations of intra‐ and interday precision ranged from 4.2 to 7.6%. Compared with other off‐line methods, the present method obtained higher extraction yields for all four aromatic constituents. Finally, this method has been applied to the analysis of vanilla from different sources. On the basis of the results, the on‐line supercritical fluid extraction‐supercritical fluid chromatography method shows great promise in the analysis of aromatic constituents in natural products.     

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.     

A two-step supercritical fluid extraction of polycyclic aromatic hydrocarbons from roadside soil samples

A two-step procedure for the supercritical fluid extraction (SFE) of polycyclic aromatic hydrocarbons from soil samples was developed. The procedure consists of a static supercritical fluid treatment in a closed extraction cell at a high temperature (T=250 or 340degreesC for 20 min) and an SFE with a solvent trapping. During the static phase, the sample is exposed to a supercritical organic solvent (methanol, toluene, dichloromethane, ACN, acetone, and hexane). The solvent penetrates particles of the matrix to substitute strongly bonded molecules and dissolves the analytes in the supercritical phase. At ambient temperature, supercritical fluids became liquid and lost their solvation abilities. Most of the analytes condense on the surface of the particles or on the extraction cell walls without forming strong bonds or penetrating deep into the matrix. Thus, the pretreatment liberates the analytes and they behave similar to those in freshly spiked samples. The common SFE with toluene-modified CO2 as an extraction fluid follows the static phase. With the use of the most suitable extraction phases (toluene, ACN), the extraction efficiency of the combined procedure is much higher (approximately100%). The results of the combined procedure are compared to the SFE procedure of the same untreated sample (difference less than 5%) and to the Soxhlet extraction. The extracts were analyzed using a GC with the flame ionization detection.     

Solid-phase micro extraction (SPME) and headspace derivatization of clenbuterol followed by GC–FID and GC–SIMMS quantification

Solid-phase micro extraction (SPME) and on-fiber derivatization followed by Gas Chromatography coupled with Flame Ionization Detection (GC-FID) or Selected Ion Monitoring Mass Spectrometry (GC-SIMMS) allows for simple yet sensitive quantification for the hexamethyldisilazane derivative of the beta-agonist clenbuterol. Using an 85- micro m polyacrylate fiber, the analysis method is optimized with respect to extraction time, derivatization time and temperature, and solution pH. In addition, the use of a rapid temperature ramping injection port allows for optimization of fiber desorption conditions. Under optimal conditions, the limits of detection for the hexamethyldisilazane derivative of clenbuterol are 1.1 ppb by FID and 0.20 ppb by SIMMS.     

Improved SFE recovery of trace analytes from liver using an integral micrometering valve–SPE column holder assembly

A novel integral restrictor–collector has been designed for use with a conventional supercritical fluid extraction (SFE) apparatus. The assembly reduces the path length between a micrometering valve and collector (a solid phase extraction (SPE) column), obviating the need for the complicated tubing and connectors usually associated with such devices. Also described is a heating-block assembly which encases the micrometering valve and provides uniform heating of the valve during extraction. The valve–SPE column assembly was part of a system used to perform the first reported SFE multi-residue drug recovery from fortified liver. Extractions used carbon dioxide pressurized to 690 bar as the supercritical fluid. Flow rates of expanded gas through the SPE columns were 3–4 L/min with concomitant quantitative trapping of the analytes on the sorbent bed. After SFE the three nitrobenzamide antimicrobial drug residues from the liver were eluted from the SPE columns by off-line analysis. The results demonstrated that losses of trace level analytes in tissue may be significantly reduced by including an integral metering valve-collector assembly as part of the SFE apparatus.     

Determination of clenbuterol in pig liver by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to determine clenbuterol in pig liver using HPLC with coulometric electrode array system, for this compound can be irreversibly oxidized at high potentials by ordinary methods. Investigation into the effect of the pH of mobile phase on the retention factor and peak height of clenbuterol was made. The electrochemical behavior of clenbuterol at graphite electrodes was taken into account. Optimization of different extract conditions was also performed. The samples were pretreated using liquid-liquid extraction based on diethyl ether and the organic layer was evaporated to dryness. The residue was dissolved in mobile phase and monitored by an ESA electrochemical detector. Four electrodes in series were used for quantitation and the potentials of electrodes were set at 450, 600, 650 and 680 mV, respectively. Calibration curve showed good linearity and the detection limit of clenbuterol was 1.2 ng/g. This method developed using HPLC-ECD is reproducible, and sensitive enough for the determination of clenbuterol in pig liver. It is easy to perform.     

Chemiluminescence detection of nine corticosteroids in liver

A method for the determination of nine corticosteroids in bovine liver was developed. The liquid-liquid extraction method consisted in the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract was evaporated to dryness and the residue redissolved in acetonitrile. The removal of fats was performed with n-hexane and the acetonitrile layer was evaporated. Afterwards, analysis of the extracts was performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as reagent. The recovery curves, performed at five spiking levels (different for each corticosteroid), showed that recoveries of at least 70.5% could be obtained from liver. The chemiluminescence detection gave satisfactory results with respect to sensitivity, and the LOQ ranged from 0.13 ppb for prednisolone to 8.40 ppb for betamethasone. The maximum mean RSD was 4.8% and 7.3% for intra- and inter-day assay, respectively.     

A fast immunoassay for the screening of beta-agonists in hair.

Hair has been shown to be an excellent site for the accumulation of clenbuterol residues. Compared with other matrices, hair sampling is very easy and this might result in large numbers of samples. In this study, a simple digestion-extraction procedure was combined with a sensitive clenbuterol ELISA, which resulted in an easy screening procedure suitable for the detection of at least four beta-agonists. Hair from untreated cows (n = 40) resulted in low blank levels (0.9 +/- 0.7 and 0.5 +/- 0.2 ng g-1 for black and white hair, respectively). The detection limits for clenbuterol, bromobuterol, mapenterol and mabuterol were determined as 1-1.5 ng g-1 for white and 3-4 ng g-1 for black hair. The accumulation of mabuterol and mapenterol in black and white hair from treated calves was demonstrated by GC-MS. The screening assay is not suitable for the detection of cimbuterol (owing to the low extraction efficiency) and for salbutamol and terbutaline (owing to the low cross-reactivity of the antibodies used for the ELISA and the low extraction efficiency). Black hair samples from cows treated with clenbuterol were still found to be positive (> 5 ng g-1) at 23 weeks after treatment. The fast screening procedure is a powerful means to detect and track the illegal use of clenbuterol, bromobuterol, mabuterol and mapenterol in animal production.     

The use of nonendcapped C18 columns in the cleanup of clenbuterol and a new adrenergic agonist from bovine liver by gas chromatography-tandem mass spectrometry analysis

More specific official methodology is needed to survey the illegal use of clenbuterol in animal production plus the synthesis of new compounds that currently elude routine analytical methods. The identification of a new adrenergic agonist, N1-(2-(4-amino-3,5-dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide (known as compound A) in animal feed has prompted studies to verify if the existing cleanup procedures developed for clenbuterol are really effective. This study considers the ion-exchange mechanism on cyanopropyl (CN), sulfonic cation exchange (SCX), mixed phase (MPH) (C8 + SCX), and nonendcapped C18 (C18NE) solid-phase extraction (SPE) columns. Results indicate that compound A (by contrast with clenbuterol) is not efficiently retained on the CN, SCX, and MPH SPE columns (recovery <10%). This finding thus leads to the development of a gas chromatography-tandem mass spectrometry procedure based on C18NE SPE that is able to purify both agonists from bovine livers spiked at 0.5, 1.0, and 2.0 ppb with a mean recovery of 93% for clenbuterol and 92% for compound A.     

气相色谱-柱后分流-反吹技术测定含脂羊毛中有机氯和有机磷类杀虫剂残留

建立了同时测定含脂羊毛中20种有机氯和有机磷类杀虫剂残留的气相色谱-柱后分流-反吹检测方法.通过二氧化碳超临界萃取技术提取含脂羊毛样品中的杀虫剂残留并经中性氧化铝固相萃取柱净化,采用气相色谱-柱后分流技术,可以同时得到3个检测器信号(MSD、μECD和NPD),可对两类杀虫剂准确定性和定量分析.同时运用反吹技术,减少样品中高沸点杂质对色谱系统的污染.3个添加水平的回收率为85.6%～120.9%,相对标准偏差在1.2%～17.3%之间.方法干扰小、重现性好、自动化程度高,能够对含脂羊毛中有机氯和有机磷类杀虫剂残留准确地定性定量分析.     

A Unified Sample Preparation System for Extraction of Various Analyte/Matrix Combinations

A supercritical fluid extraction/enhanced solvent extraction system (SFE/ESE) was used to remove polar and non-polar analytes from various matrices. Extraction of environmental pollutants from soil, additives from low density polyethylene, sulfa drugs from animal tissue, and drug from tablet was performed using both SFE and ESE. Results showed that a single instrumental system can be used to perform both ESE with organic solvents and SFE with carbon dioxide-based fluids. Each method has its own unique advantages and applications. The ability to carry out both solvent extraction and supercritical fluid extraction with one system has obvious economical advantages.     

同位素稀释质谱法测定猪肉中克伦特罗含量的国际比对APMP. QM-S6

建立了同位素稀释质谱法（IDMS）测定猪肉中克伦特罗含量的高准确度方法,并应用于亚太计量规划组织（Asia Pacific Metrology Programme,APMP）开展的国际比对“猪肉中克伦特罗含量测定”（APMP. QM-S6）。本研究考察了影响测定结果的喷雾电压值、流动相、色谱柱、提取条件、净化条件等主要因素并进行了优化,并对测定结果的不确定度进行了评定。结果表明:流动相组成以及pH值会影响克伦特罗的质谱响应以及最优的电喷雾电压值;样品溶剂会影响克伦特罗的色谱保留,甲醇溶剂会引起严重的溶剂效应,甚至导致峰分裂;克伦特罗易在固相萃取柱及亲水性滤膜上吸附,且吸附材料和滤膜上吸附的杂质有可能被洗脱,引起基质效应,干扰测定。提取效率最高的方法是采用0.1%（v/v）甲酸乙腈溶液为提取溶剂,通过匀浆进行提取。方法检出限（以信噪比大于3计）为0.2 μg/kg。比对样品中克伦特罗的含量为5.18 μg/kg±0.50 μg/kg（k=2）,比对结果与参考值等效一致,取得国际互认。该方法准确可靠,可为猪肉中克伦特罗的日常检测提供参考。