水果蔬菜中多种杀菌剂的基质固相分散液相色谱测定方法研究

采用基质固相分散(MSPD)代替液液分配和固相萃取,从蔬菜水果中提取、净化10种常用杀菌剂农药残留,用C18硅胶交联剂作为固相吸附剂,乙酸乙酯作为洗脱液,用HPLC/PDA和LC-MS进行分析检测。10种杀菌剂在0.5~5 mg/kg含量的添加回收率在65%~110%之间,相对标准偏差小于10%,使用HPLC、PDA和LC-MS的检出限分别在0.02~0.2 mg/kg和0.002~0.01 mg/kg之间。该方法节省溶剂,提取和净化一步完成,适用于新鲜水果和蔬菜中10种杀菌剂的残留分析。     

高效液相色谱-串联质谱法检测花生中的黄曲霉毒素B_1

应用高效液相色谱-电喷雾串联四极杆质谱联用系统(HPLC-MS/MS),在多反应离子检测方式(MRM)下,对花生中的黄曲霉毒素B1进行检测.对花生中黄曲霉毒素B1的提取、净化、液相分离及串联质谱等相关检测参数进行了优化研究.用V(甲醇):V(水)=6:4提取,OASIS HLB SPE小柱净化,定容过滤.采用V(甲醇):V(水)(含体积分数0.1%甲酸)=7:3为流动相,前级离子313.0,二级离子241.1、269.1,ESI正离子方式检测,在3.3 min出峰.结果表明,在ESI正离子模式下,黄曲霉毒素B1在其线性定量范围0.1～50μg/kg内,相关系数达到0.9999,检出限为0.03μg/kg,最低定量限为0.1μg/kg.低、中、高浓度添加回收率范围为93%～105%.     

Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography

A new and accurate method to quantify ochratoxin A (OA) in table wine has been developed. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection for quantification of the toxin. Wine was diluted with a solution containing 1% polyethylene glycol (PEG 8000) and 5% sodium hydrogencarbonate, filtered and applied to an OchraTest immunoaffinity column. The column was washed with a solution containing sodium chloride (2.5%) and sodium hydrogencarbonate (0.5%) followed by water. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase. Average recoveries of OA from white, rosé and red wine samples spiked at levels from 0.04 to 10 ng/ml ranged from 88% to 103%, with relative standard deviations (RSDs) between 0.2 and 9.7%. Detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The method was applied successfully to 56 samples of red (38), rosé (8), white (9) and dessert (1) wine. The levels of OA ranged from <0.01 to 7.6 ng/ml with red wines more contaminated than rosé and white wines. A good correlation (r=0.987) was found by comparative analysis of 20 naturally contaminated samples using this method and the method of Zimmerli and Dick with better recoveries of OA and better performances for the new method. Several advantages of this method with respect to the actually available methods have been pointed out, with particular reference to red wine which appears to be the most difficult to analyze.     

Determination of polycyclic aromatic sulfur heterocycles (PASH) in diesel fuel by high performance liquid chromatography and photodiode-array detection

An oxidation method (sulfone method) for the determination of polycyclic aromatic sulfur heterocycles (PASH) in diesel fuel is presented. The aromatic fraction of a diesel fuel, isolated by solid phase extraction, is oxidized under controlled conditions with hydrogen peroxide. The oxidation products, mainly methylated dibenzosulfone, are determined and quantified directly, without further clean-up, by HPLC with photodiode-array detection.     

Analysis of zearalenone and α-zearalenol in animal feed using high-performance liquid chromatography

Different extraction and clean-up techniques used before HPLC analysis were compared in order to obtain a reliable method for the quantitative determination of zearalenone (ZEA) and α-zearalenol (α-ZOL) in animal feed. Immunoaffinity clean-up was compared to C₁₈ and Florisil column clean-up. Extracted samples were analysed by reversed-phase HPLC with fluorescence detection (λ_ex=274 nm, λ_em=440 nm). A mobile phase of acetonitrile:water (50:50 (v/v)) and a flow-rate of 1.0 ml min⁻¹ resulted in a good separation between ZEA and α-ZOL. Using immunoaffinity clean-up the linear range was between 25 and 600 μg kg⁻¹ for ZEA and α-ZOL in maize. Intra-laboratory coefficients of variation (CV) (under repeatability conditions) were 9.16% for ZEA and 2.18% for α-ZOL. Recoveries for spiked ZEA and α-ZOL samples ranged from 89 to 110% with CVs between 5.2 and 11.2% (under within-laboratory reproducibility conditions). Using C₁₈ and Florisil solid-phase clean-up, matrix interference was too high. Therefore, naturally contaminated animal feed samples were analysed using the developed HPLC method coupled to the immunoaffinity clean-up.     

Experimental design for extraction and quantification of phenolic compounds and organic acids in white "Vinho Verde" grapes

An experimental design was applied for the optimization of extraction and clean-up processes of phenolic compounds and organic acids from white “Vinho Verde” grapes. The developed analytical method consisted in two steps: first a solid-liquid extraction of both phenolic compounds and organic acids and then a clean-up step using solid-phase extraction (SPE). Afterwards, phenolic compounds and organic acids were determined by high-performance liquid chromatography (HPLC) coupled to a diode array detector (DAD) and HPLC-UV, respectively. Plackett-Burman design was carried out to select the significant experimental parameters affecting both the extraction and the clean-up steps. The identified and quantified phenolic compounds were: quercetin-3-O-glucoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-glucoside, quercetin, kaempferol and epicatechin. The determined organic acids were oxalic, citric, tartaric, malic, shikimic and fumaric acids. The obtained results showed that the most important variables were the temperature (40 °C) and the solvent (acid water at pH 2 with 5% methanol) for the extraction step and the type of sorbent (C18 non end-capped) for the clean-up step.     

液相色谱串联四极杆质谱法同时测定婴幼儿配方食品中甲基香兰素和乙基香兰素

建立了混合阴离子交换固相萃取柱净化,液相色谱串联四极杆质谱法测定婴幼儿配方食品中甲基香兰素和乙基香兰素的方法.样品经水和乙腈提取,CuSO4溶液沉淀蛋白,NaOH调节pH,增加样品溶解性,阴离子交换固相萃取柱净化.目标化合物在梯度洗脱条件下经C18柱分离后采用ESI源负离子多反应监测模式进行检测.分别选取了婴幼儿配方乳...     

High-performance liquid chromatographic determination of secondary cardiac glycosides in Digitalis purpurea leaves

An analytical method for the determination of secondary cardiac glycosides in Digitalis purpurea leaves by high-performance liquid chromatography (HPLC) is described. The procedure consisted of extraction of dry leaf powder with ethanol-chloroform (2:1) and clean-up by Sep-Pak cartridges prior to HPLC analysis. HPLC was performed on an octylsilyl bonded silica column, using acetonitrile-methanol-water (4:4:5) for trisdigitoxosides and acetonitrile-methanol-water (8:30:43) for strospeside; the effluent was monitored by ultraviolet detection (at 220 nm). Quantitation of these cardiac glycosides was carried out by the internal standard method. The amounts of digitoxin, gitoxin, gitaloxin and strospeside per 100 mg of dry leaf powder were estimated to be 22.6, 14.0, 54.7 and 1.9 micrograms, respectively. The method is sufficiently sensitive and reproducible to assay secondary glycosides in Digitalis purpurea leaves.     

An improved residue analytical method for phenylurea herbicides

Summary An improved HPLC method with electrochemical detection is applied to the determination of phenyl urea herbicide residues in crops. The analysis includes extraction and hydrolysis with NaOH, clean-up by steam distillation and partition into methanol/HCl and quantitation of the aniline by HPLC with 2-column switching technique and amperometric detection.     

Miniaturized Salting-Out Assisted Liquid-Liquid Extraction Combined with Disposable Pipette Extraction for Fast Sample Preparation of Neonicotinoid Pesticides in Bee Pollen

As the main source of nutrients for the important pollinator honeybee, bee pollen is crucial for the health of the honeybee and the agro-ecosystem. In the present study, a new sample preparation procedure has been developed for the determination of neonicotinoid pesticides in bee pollen. The neonicotinoid pesticides were extracted using miniaturized salting-out assisted liquid-liquid extraction (mini-SALLE), followed by disposable pipette extraction (DPX) for the clean-up of analytes. Effects of DPX parameters on the clean-up performance were systematically investigated, including sorbent types (PSA, C18, and silica gel), mass of sorbent, loading modes, and elution conditions. In addition, the clean-up effect of classical dispersive solid-phase extraction (d-SPE) was compared with that of the DPX method. Results indicated that PSA-based DPX showed excellent clean-up ability for the high performance liquid chromatography (HPLC) analysis of neonicotinoid pesticides in bee pollen. The proposed DPX method was fully validated and demonstrated to provide the advantage of simple and rapid clean-up with low consumption of solvent. This is the first report of DPX method applied in bee pollen matrix, and would be valuable for the development of a fast sample preparation method for this challenging and important matrix.     

Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry

The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function SPE sorbents was tested. Polymeric sorbents were found to retain most of the toxins. Optimization experiments were carried out to maximize recoveries and the effectiveness of the clean-up. In LC–MS/MS analysis, the observed matrix effects can depend on the chromatographic conditions used, therefore, two different HPLC methods were tested, using either an acidic or an alkaline mobile phase. The recovery of the optimized SPE protocol was around 90% for all toxins studied and no break-through was observed. The matrix effects were determined by comparing signal response from toxins spiked in crude and SPE-cleaned extracts with those derived from toxins prepared in methanol. In crude extracts, all toxins suffered from matrix effects, although in varying amounts. The most serious effects were observed for okadaic acid (OA) and pectenotoxin-2 (PTX2) in the positive electrospray ionization mode (ESI⁺). SPE clean-up on polymeric sorbents in combination with the alkaline LC method resulted in a substantial reduction of matrix effects to less than 15% (apparent recovery between 85 and 115%) for OA, yessotoxin (YTX) in ESI⁻ and azaspiracid-1 (AZA1), PTX2, 13-desmethyl spirolides C (SPX1), and gymnodimine (GYM) in ESI⁺. In combination with the acidic LC method, the matrix effects after SPE were also reduced but nevertheless approximately 30% of the matrix effects remained for PTX2, SPX1, and GYM in ESI⁺. It was concluded that SPE of methanolic shellfish extracts can be very useful for reduction of matrix effects. However, the type of LC and MS methods used is also of great importance. SPE on polymeric sorbents in combination with LC under alkaline conditions was found the most effective method.     

Separation and determination of saturated very-long-chain free fatty acids in plasma of patients with adrenoleukodystrophy using solid-phase extraction and high-performance liquid chromatographic analysis

An improved method was developed for the isolation of very-long-chain free fatty acids (VLCFFAs) in plasma and their separation and determination by high-performance liquid chromatography (HPLC). The method includes sample clean-up using solid-phase extraction, fluorophoric labelling of the FFAs and reversed-phase HPLC separation. The solid-phase extraction was carried out with aminopropyl-bonded phase columns. The FFAs were then derivatized with 9-anthryldiazomethane (fluorescent) reagent and separated by HPLC on an RP-18 column with methanol as the mobile phase. Using this method, the concentrations of C20:0, C22:0, C24:0 and C26:0 were determined in the plasma of five adrenoleukodystrophy (ALD) patients, one obligatory heterozygote, four healthy male volunteers and one child with cerebral leukodystrophy but without any other ALD symptoms. Statistically significant differences were found in the levels of C24 and C26 and in the ratios C24/C22 and C26/C22 in ALD patients and in normal controls. The values were higher in patients with X-ALD. This method therefore provides a rapid and accurate procedure for the laboratory confirmation of X-ALD.     

Automated aflatoxin analysis of foods and animal feeds using immunoaffinity column clean-up and high-performance liquid chromatographic determination

A commercially available system is described for the fully automated clean-up and high-performance liquid chromatographic (HPLC) analysis of aflatoxins in foods and animal feeds. The system marketed primarily for handling solid-phase extraction columns has modified software to facilitate use with immunoaffinity columns. Sample extract clean-up followed by injection onto an HPLC column with post-column iodination and fluorescence detection is carried out completely unattended. A coefficient of variation of 5.1% for aflatoxin B1 analysis was obtained, and the accuracy of the system was demonstrated by the analysis of peanut butter certified reference material.     

Sensitive method for the determination of lipophilic marine biotoxins in extracts of mussels and processed shellfish by high-performance liquid chromatography–tandem mass spectrometry based on enrichment by solid-phase extraction

A solid-phase extraction (SPE) method for the enrichment and clean-up of lipophilic marine biotoxins from extracts of different species of bivalve molluscs and processed shellfish products was developed. Okadaic acid (OA), pectenotoxin2 (PTX2), azaspiracid1 (AZA1) and yessotoxin (YTX) were determined by LC–MS/MS in hydrolyzed and non-hydrolyzed extracts. Applying a concentration factor of 10 the limit of quantification for the four toxins was determined to be 1 μg/kg. An organized in-house ring trial proved transferability of the method protocol and satisfactory results for all four toxins with a relative standard deviation (RSD) of 5–12%. The precision of the whole method including LC–MS detection was determined by processing seven independent extractions analyzed in independent sequences. RSD ranged between 12% and 24%. This SPE method was tested within a concentration range corresponding to the range of the current European Union regulatory limits (up to 160 μg/kg for the OA group), but it would also be applicable to a lower μg/kg range which is important in view of a possible decrease of regulatory limits as proposed by a working group of the European Food Safety Authority. The potential of SPE as a cleaning tool to cope with matrix effects in LC–MS/MS was studied and compared to liquid–liquid portioning.     

Use of solid phase extraction for the isolation and clean-up of a derivatised furazolidone metabolite from animal tissues

A method is presented for the determination of protein-bound residues of furazolidone in animal tissue. The use of furazolidone in food-producing animals has been banned in the EU. Illegal use of furazolidone can be monitored most effectively by testing for bound residues containing the 3-amino-2-oxazolidone (AOZ) moiety which, unlike the parent drug, is stable and can be detected for prolonged periods after cessation of treatment. This paper reports the development of an extraction and clean-up procedure for AOZ from liver using solid phase extraction. The method replaces solvent extraction and provides extensive sample clean-up with removal of approximately 99% of the derivatising agent, 2-nitrobenzaldehyde, which may interfere with the determination. It also offers the advantage of being suitable for automation, thereby increasing throughput of samples. The extraction procedure may be used for HPLC and ELISA screening techniques. The method has been validated in fortified and incurred pig liver samples, yielding mean recovery of AOZ in excess of 60%.     

Determination of Selected Persistent Organochlorine Pollutants in Human Serum by Solid-phase Disk Extraction and Dual-Column Capillary Gas Chromatography with Electron Capture Detection

Abstract

An improved clean-up method by solid-phase disk extraction was developed to isolate and concentrate trace levels of POPs (persistent organochlorine pollutants) in human serum prior to gas chromatography with electron capture detection on two different capillary columns, providing an improved selectivity. An Empore^TM C₁₈ bonded silica extraction disk cartridge is used for the initial extraction and enrichment of the analytes. Subsequent clean-up is achieved by concentrated sulphuric acid and silica gel adsorption chromatography. Recoveries for selected POPs are ranging from 62 to 74% and a good reproducibility (RSD < 14%) is demonstrated. Human samples analysed under these conditions, show a similar relative concentration profile.     

Determination of abamectin and doramectin in sheep faeces using HPLC with fluorescence detection

An analytical procedure for the determination of abamectin and/or doramectin in sheep faeces has been developed. Avermectins were extracted from sheep faeces with acetonitrile, clean-up using solid phase extraction (SPE) and analysed by high performance liquid chromatography (HPLC) with fl uorescence detection after derivatization with N-methylimidazole.The method has a low detection limit (1.0 ng/g of moist sheep faeces), low quanti fi cation limit (2.5 ng/g of moist sheep faeces), good recovery in the range 66.4-80.8% for abamectin and 67.7-85.5% for doramectin as well as good repeatability (>85%). The method is applicable to the study of the time pro fi le of excretion in sheep faeces and also for ecotoxicological studies of both avermectins.     

Determination of oxytetracycline, tetracycline and chloramphenicol antibiotics in animal feeds using subcritical water extraction and high performance liquid chromatography

A rapid analytical method for the determination of oxytetracycline (OTC), tetracycline (TC) and chloramphenicol (CAP) antibiotics in animal feeds has been developed based on subcritical water extraction (SWE) without further sample clean-up followed by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. On extracting target antibiotics from spiked samples, the efficiency of the water extraction device was evaluated in terms of pH and volume of the extractant, temperature and time of the static extraction. The best extraction conditions were obtained by using 5.5 mL of water adjusted to pH 2 with hydrochloric acid as the extractant at 100 °C with 5-min static extraction. After filtration, 20 μL of the aqueous extract was directly injected into the HPLC column. Recoveries between 82.1% and 90.0% with relative standard deviations ranging between 1.6% and 4.8% were achieved from spiked animal feed samples by using this method. Compared with the traditional ultrasonic extraction, this procedure was remarkably more efficient in extracting OTC, TC and CAP, simpler to perform, and there was no use of toxic organic solvents.     

Urinary testosterone measurement by gas chromatography after solid-phase extraction and high-performance liquid chromatography.

A method was developed for the rapid determination of testosterone in urine. The procedure consists of solid-phase extraction (SPE) followed by high-performance liquid chromatographic (HPLC) clean-up before gas chromatographic determination. Recovery was evaluated by adding [3H]testosterone (10(4) cpm) to urine samples; the mean recovery of radioactivity after SPE and HPLC was 82%. Precision was estimated by repeated measurement of testosterone in four different urine samples; the coefficient of variation was 7.9% (95% confidence limits 6.1-11.4%). Accuracy was evaluated by standard addition and dilution assays; a linear relationship was found between the expected and observed values (r2 = 0.982). The method is rapid, effective and suitable for routine analysis.     

Determination of active components of Ginkgo biloba in human urine by capillary high-performance liquid chromatography/mass spectrometry with on-line column-switching purification

Ginkgo biloba is one of the most popular herbal nutritional supplements, with terpene lactones and flavonoids being the two major active components. An on-line purification high-performance liquid chromatography/mass spectrometry (HPLC/MS) method was successfully developed for the quantitative determination of flavonoids and terpene lactones excreted in human urine after ingesting the herbal supplement. Satisfactory separation was obtained using a C18 capillary column made in-house with sample clean-up and pre-concentration achieved using a C18 pre-column with column switching. High selectivity and limits of detection of 1-18 ng/mL were achieved using a selected ion monitoring (SIM) scan in negative ion mode; the on-line solid-phase extraction (SPE) recovery of the active components in Ginkgo biloba determined in this study was greater than 75%.