Supercritical fluid extraction of macrocyclic lactone mycotoxins in maize flour samples for rapid amperometric screening and alternative liquid chromatographic method for confirmation

A rapid and simple method for the direct screening of macrocyclic lactone mycotoxins (zearalenone, ZON; alpha-zearalenol, alpha-ZOL; and beta-zearalenol, beta-ZOL) in maize flour samples is proposed. The sample screening method comprises supercritical fluid extraction (SFE) and clean-up on Florisil adsorption cartridge of the selected toxic compounds, followed by continuous flow electrochemical detection. Those samples for which the total concentration is close to or above the threshold limit established by legislation (0.200 mg kg(-1)) are subjected to preconcentration on C18 chromatographic material and liquid chromatographic separation for confirmation purposes. This confirmation method allows the determination of ZON, alpha-ZOL and beta-ZOL in the range between 30 and 300 microg kg(-1), with a average relative standard deviation lower than 5.2 in all cases.     

Supercritical fluid extraction as an on-line clean-up technique for determination of riboflavin vitamins in food samples by capillary electrophoresis with fluorimetric detection

An automatic method for the separation and determination of riboflavin (RF) vitamins (RF, flavin mononucleotide and flavin adenine dinucleotide) in food samples (chicken liver, tablet and powder milk) is proposed. The method is based on the on-line coupling of a supercritical fluid extractor (SFE) with a continuous flow-CE system with guided optical fiber fluorimetric detection (CF-CE-FD). The whole SFE-CF-CE-FD arrangement allowed the automatic treatment of food samples (clean-up of the sample followed by the extraction of the analytes), and the direct introduction of a small volume of the extracted plug to the CE-FD system for the determination of RF vitamins. Fluorescence detection introduced an appropriated sensitivity and contributed to avoid interferences of nonfluorescent polar compounds coming from the matrix samples in the extracted plug. Electrophoretic responses were linear within the 0.05-1 microg/g range, whereas the detection limits of RF vitamins were in the 0.036-0.042 microg/g range. The proposed arrangement opens up interesting prospects for the direct determination of polar analytes in complex samples with a good throughput and high level of automation.     

在线净化/固相萃取-高效液相色谱法测定饮用水和环境水体中的百草枯和敌草快

建立了在线净化/固相萃取(SPE)-高效液相色谱(HPLC)快速、准确测定饮用水和环境水体中的两种痕量除草剂百草枯和敌草快的方法。样品用大体积自动进样器注入在线净化小柱并流经固相萃取小柱,通过双梯度高效液相色谱系统中的上样泵实现净化和富集后,通过阀切换将固相萃取小柱切换至分析流路中;用分析泵将待测物从富集柱冲洗至分析柱进行测定。上样泵流速和分析泵流速分别为0.7和0.6 mL/min,采用等度洗脱方式完成两种除草剂的分离和检测。检测波长分别为260 nm (百草枯)和311 nm (敌草快),进样体积为2.5 mL,整个分析时间为16 min。该方法在1.0～20 μg/L范围内线性关系良好,两种除草剂的线性相关系数均大于0.9980,检出限分别为0.10和0.12 μg/L(S/N=3)。该方法前处理简单,快速,可用于饮用水和环境水体中痕量除草剂的测定。     

Screening and confirmation of PAHs in vegetable oil samples by use of supercritical fluid extraction in conjunction with liquid chromatography and fluorimetric detection

A fast, simple, non-destructive method for the direct screening of polycyclic aromatic hydrocarbons (PAHs) in vegetable oil samples is proposed. The method uses a supercritical fluid extraction (SFE) system coupled on-line with a fluorimetric detector to determine PAHs. This special assembly avoids the main problems encountered in the determination of PAHs in complex matrices such as vegetable oils. PAHs are selectively extracted by using silica gel in the thimble and cleaned up by passage through a C18 column. Interferences are preferentially retained by the silica gel during the SFE process while PAHs are adsorbed in the C18 column and the remainder of the matrix is sent to waste. Finally, the C18 column is purged to remove residual CO₂ gas and adsorbed PAHs are recovered by desorption with a solvent. The extracts from positive samples are subsequently analyzed by liquid chromatography (LC) with fluorescence detection. The proposed method allows the confirmation of vegetable oil safety and hence provides a new tool for consumer protection.     

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.     

Supercritical fluid extraction and ultra performance liquid chromatography of respiratory quinones for microbial community analysis in environmental and biological samples

Microbial community structure plays a significant role in environmental assessment and animal health management. The development of a superior analytical strategy for the characterization of microbial community structure is an ongoing challenge. In this study, we developed an effective supercritical fluid extraction (SFE) and ultra performance liquid chromatography (UPLC) method for the analysis of bacterial respiratory quinones (RQ) in environmental and biological samples. RQ profile analysis is one of the most widely used culture-independent tools for characterizing microbial community structure. A UPLC equipped with a photo diode array (PDA) detector was successfully applied to the simultaneous determination of ubiquinones (UQ) and menaquinones (MK) without tedious pretreatment. Supercritical carbon dioxide (scCO(2)) extraction with the solid-phase cartridge trap proved to be a more effective and rapid method for extracting respiratory quinones, compared to a conventional organic solvent extraction method. This methodology leads to a successful analytical procedure that involves a significant reduction in the complexity and sample preparation time. Application of the optimized methodology to characterize microbial communities based on the RQ profile was demonstrated for a variety of environmental samples (activated sludge, digested sludge, and compost) and biological samples (swine and Japanese quail feces).     

Determination of glyphosate in groundwater samples using an ultrasensitive immunoassay and confirmation by on-line solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry

Despite having been the focus of much attention from the scientific community during recent years, glyphosate is still a challenging compound from an analytical point of view because of its physicochemical properties: relatively low molecular weight, high polarity, high water solubility, low organic solvent solubility, amphoteric behaviour and ease to form metal complexes. Large efforts have been directed towards developing suitable, sensitive and robust methods for the routine analysis of this widely used herbicide. In the present work, a magnetic particle immunoassay (IA) has been evaluated for fast, reliable and accurate part-per-trillion monitoring of glyphosate in water matrixes, in combination with a new analytical method based on solid-phase extraction (SPE), followed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS), for the confirmatory analysis of positive samples. The magnetic particle IA has been applied to the analysis of about 140 samples of groundwater from Catalonia (NE Spain) collected during four sampling campaigns. Glyphosate was present above limit of quantification levels in 41% of the samples with concentrations as high as 2.5 μg/L and a mean concentration of 200 ng/L. Good agreement was obtained when comparing the results from IA and on-line SPE-LC-MS/MS analyses. In addition, no false negatives were obtained by the use of the rapid IA. This is one of the few works related to the analysis of glyphosate in real groundwater samples and the presented data confirm that, although it has low mobility in soils, glyphosate is capable of reaching groundwater.     

Supercritical fluid extraction and clean-up of organochlorine pesticides in Chinese herbal medicine

A method involving the simultaneous extraction and clean-up of 13 organochlorine pesticides (OCPs) from Chinese herbal medicines (CHMs) was developed using supercritical fluid extraction (SFE) followed by gas chromatography-electron capture detection and mass spectrometric confirmation. The pesticides in the study consisted of alpha-, beta-, gamma-, and delta-benzene hexachloride, heptachlor, aldrin, heptachlor epoxide, endosulfan I, 4,4'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethene), dieldrin, endrin, 4,4'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane), endosulfan II, 4,4'-DDT (2,2-bis(p-chlorophenyl)1,1,1-trichloroethane), endrin aldehyde, and endosulfan sulfate. A series of experiments was conducted to optimize the final extraction conditions [pure CO2, 250 atm extraction pressure (1 atm = 101,325 Pa), 50 degrees C extraction temperature, 5 min static extraction time, 20 min dynamic extraction time, 2.0-g Florisil sorbent on top of 0.1-g samples, 12-ml n-hexane eluting at 1 ml/min, and a 10-ml extraction vessel]. Florisil sorbent was placed with the sample in the SFE vessel to provide a facile and effective clean-up approach. Mean recoveries of 78-121% with reproducibilities of 5-31% were obtained for the pesticides except for endosulfan II, endosulfan sulfate and endrin aldehyde. The simple and rapid method may be used to determine OCPs in CHMs routinely, and in fact, was used to analyze CHMs sold in Taiwan.     

Determination of tetracyclines in bovine kidney by liquid chromatography/tandem mass spectrometry with on-line extraction and clean-up

A novel, sensitive, high performance liquid chromatography/tandem mass spectrometric (i.e. mass spectrometry/mass spectrometry) method with on-line extraction and clean-up for the screening and confirmation of residues of tetracyclines in kidney has been developed. After liquid extraction of homogenised kidney with McIlvain buffer, an aliquot of the extract is directly injected on the LC/MS/MS system with further extraction and clean-up of the sample on-line. Detection of the analytes was achieved by positive electrospray ionization followed by multiple reaction monitoring. For each tetracycline the collisional decomposition of the protonated molecule to a unique, abundant fragment ion was monitored. The method has been validated for tetracycline, oxytetracycline, chlortetracycline and doxycycline. Calibration curves resulting from spiked blank kidney samples at the 100-1200 microgram/kg level showed good linear correlation. At the level of 600 microgram/kg both within- and between-day precision, as measured by relative standard deviation (RSD), were less than 7%. The limits of detection (LODs) for tetracycline, oxytetracycline, chlortetracycline and doxycyline were 18, 23, 24 and 21 microgram/kg, respectively. The limits of quantification (LOQs) for tetracycline, oxytetracycline, chlortetracycline and doxycyline were 36, 46, 47 and 42 microgram/kg, respectively. The recoveries ranged from 71 to 91%. The procedure provides a rapid, reliable and sensitive method for the determination of residues of tetracyclines in bovine kidney. The advantage of this method over existing methods is its decreased sample preparation and analysis time, which makes the method more suitable for routine analysis.     

固相萃取净化-超高效液相色谱-高分辨质谱法测定尿液中百草枯和敌草快残留

尿液样品中百草枯(PQ)和敌草快(DQ)的检测是理化检验工作的难点。PQ和DQ具有分子极性大和水溶性好等特点,常规反相色谱柱难以保留;现有文献方法多采用亲水相互作用色谱法(HILIC)进行保留,但文献方法需采用高浓度缓冲盐作为流动相,增加了质谱仪的污染。基于上述问题,研究建立了弱阳离子交换(WCX)固相萃取净化-超高效液相色谱-高分辨质谱法(UPLC-HRMS)快速准确测定尿液样品中PQ和DQ残留的检测方法。尿液样品经混合磷酸盐缓冲液(pH=6.86)稀释和WCX固相萃取净化后,在Syncronis HILIC色谱柱(100 mm×2.1 mm, 1.7 μm)上进行梯度洗脱分离,采用正离子电喷雾离子化模式(ESI⁺)和一级全扫描-数据依赖二级质谱扫描模式(Full mass-ddMS²)进行定量分析。研究通过对色谱条件的不断优化,将HILIC模式下流动相中甲酸铵缓冲盐的浓度降低至10 mmol/L,并系统优化了样品前处理过程中影响PQ和DQ准确性的因素。在最优条件下,PQ和DQ线性关系良好(r²>0.998),在4个加标水平下(1.0、20.0、100.0和200.0 μg/L), PQ和DQ的平均加标回收率分别为85.8%~101%和80.3%~86.9%,精密度(RSD)分别为0.8%~5.1%和0.9%~4.2%。方法的检出限(S/N≥3)和定量限(S/N≥10)分别为0.2 μg/L和0.6 μg/L。将建立的方法用于中毒病人临床治疗过程尿液中DQ含量的跟踪监测。该方法具有快速、简便、灵敏和准确等优点,适用于临床中毒病例尿液样品中PQ和DQ的检测。     

Supercritical fluid extraction and clean-up of organochlorine pesticides and polychlorinated biphenyls in mussels

The simultaneous extraction and clean-up of mussel samples followed by gas chromatography with electron-capture detection and mass spectrometric confirmation of 15 organochlorine pesticides (OCPs) and 11 polychlorinated biphenyls (PCBs) is developed using Florisil sorbent in the supercritical fluid extraction cell. The method detection limits vary from 1 to 10 ng/g for OCPs and from 2 to 15 ng/g for PCBs. Mean reproducibilities of 11% and 10% and mean recoveries of 80% and 53%, respectively, for OCPs and PCBs are obtained. The feasibility of the proposed supercritical fluid extraction method was confirmed by analyzing a certified reference material and mussels collected from Taiwan region. The method is simple, rapid and requires only small amounts of samples and solvents. It may serve as a screening protocol for the determination of OCPs and PCBs in mussels on a routine basis.     

Supercritical fluid extraction—Achiral liquid chromatography with circular dichroism detection for the determination of menthone enantiomers in natural peppermint oil samples

A simple and enantioselective method for the determination of menthone enantiomers in peppermint essential oil samples is proposed. The method involves the initial supercritical fluid extraction (SFE) to clean-up and extraction of analytes and their preconcentration on C₁₈ adsorption cartridges followed by achiral liquid chromatographic separation and direct circular dichroism (CD) detection. The calibration curve of the anisotropy factor (g) versus the enantiomeric excess was linear, with a correlation coefficient (R²) of 0.9970. The precision evaluated by UV peak area and CD peak area was suitable both in terms of intra- and inter-day precision (RSD < 5.1% in all cases). The usefulness of the proposed method was demonstrated by analyzing natural and spiked peppermint oil samples. This method has the advantages of being rapid and precise without using an expensive chiral column. It was demonstrated to be suitable for the simultaneous determination of both enantiomers and for assessing the chemical purity of menthone.     

Automated on-line multi-dimensional high-performance liquid chromatographic techniques for the clean-up and analysis of water-soluble samples

The application of an automated on-line multi-dimensional liquid-liquid chromatographic technique for the clean-up and analysis of water-soluble samples was investigated. The use of microparticulate aqueous-compatible steric exclusion columns as the primary separation step coupled to either reversed-phase, normal-phase or ion-exchange columns as the secondary step allowed the direct injection of complex samples without prior clean-up. The entire operation was automatically controlled by a microprocessor-based liquid chromatograph with time-programmable events which allowed precise switching of high-pressure pneumatically operated valves. Both heart-cutting and on-column concentration methods were used. The heart-cutting technique had the advantage of selectivity but lacked sensitivity; more successful was the on-column concentration technique, which, by the concentration of the solute from a larger volume of exclusion column effluent on to the secondary column, gave better sensitivity. The technique was applied to the analysis of theophylline and caffeine in biological fluids, catecholamines in urine, vitamins in a protein food supplement and sugars in molasses and candy bars.     

Determination of eight sulfonamides in bovine kidney by liquid chromatography/tandem mass spectrometry with on-line extraction and sample clean-up

A sensitive, high performance liquid chromatography/tandem mass spectrometric (i.e. mass spectrometry/mass spectrometry; LC/MS/MS) method with on-line extraction and sample clean-up for the screening and confirmation of residues of sulfonamides in kidney is described. The sulfonamides are extracted from homogenized kidney with methanol. After centrifugation of the extract, an aliquot of the extract is directly injected on the LC/MS/MS system with further extraction and clean-up of the sample on-line. Detection of the analytes was achieved by positive electrospray ionization (ESI) followed by multiple reaction monitoring. For each sulfonamide the collisional decomposition of the protonated molecule to a common, abundant fragment ion was monitored. The method has been validated for sulfadimethoxine, sulfaquinoxaline, sulfamethazine, sulfamerazine, sulfathiazole, sulfamethoxazole, sulfadiazine and sulfapyridine. Calibration curves resulting from spiked blank kidney samples at the 10-200 microg/kg level showed good linear correlation. At the level of 50, 100 and 200 microg/kg both within- and between-day precision, as measured by relative standard deviation (RSD), were less than 16%. The limits of detection (LODs) ranged from 5 to 13.5 microg/kg. The recoveries ranged from 78 to 82%. The procedure provides a rapid, reliable and sensitive method for the determination of residues of sulfonamides in bovine kidney. The advantage of this method over existing methods is its decreased sample preparation and analysis time, which makes the method more suitable for routine analysis.     

The use of alternative fluids in on-line supercritical fluid extraction – capillary gas chromatography

A key feature differentiating analytical supercritical fluid extraction (SFE) from conventional liquid extraction is the possibility of varying the solvent strength of a supercritical fluid to achieve selective extractions of specific target compounds, or functional classes of compound, from complex matrices. This can be accomplished by using supercritical fluids other than carbon dioxide, for example, sulfur hexafluoride, nitrous oxide, or sulfur hexafluoride-modified carbon dioxide. The use of these fluids will be demonstrated by the characterization of complex environmental and petroleum matrices by directly coupled SFE – capillary GC. On-line SFE-GC involves the decompression of pressurized extraction fluid directly into the heated, unmodified capillary split injection port of the chromatograph. This paper will also show how, by adjustment of the extraction temperature and pressure, SFE selectivity may be further enhanced.     

Sensitive determination of probenecid in urine samples by reversed-phase liquid chromatography and UV-visible detection using solid-phase extraction techniques for sample clean-up

Summary This study describes a rapid method for the determination of probenecid in human urine by liquid chromatography with UV detection at 254 nm, after clean-up through a C8 solid-phase extraction column. Liquid chromatography was carried out on a C18-bonded phase using an acetonitrile-acetate buffer (pH=4) gradient elution. Ethacrynic acid was used as internal standard. The system has been applied to the determination of probenecid in the 0.10–100.0 g/ml concentration range; the limit of detection was 5 ng/mL.     

Supercritical fluid chromatographic analysis for on-line monitoring of hexane removal from soybean oil miscella using liquid carbon dioxide

Liquid carbon dioxide (L-CO2) can be used to separate hexane from hexane/soybean oil (SBO) mixtures (i.e., miscella). An on-line supercritical fluid chromatographic (SFC) method was developed to monitor this separation. L-CO2 (25 degrees C and 9.31 MPa) was passed through 50 mL of a 25% (w/w) hexane miscella and then directed on-line through a SFC injector. After passing 300-L expanded CO2, the hexane concentrations in the L-CO2 were 0.05% and 0.04% for n-hexane and isohexane, respectively and the residual hexane concentrations in the SBO were 3.8 and 3.3 ppm, respectively. This technique provided real time on-line monitoring of the hexane separation process.