High-performance liquid chromatographic procedure for the determination of di-(2-ethylhexyl)phthalate in human blood specimens. Problems of variable-extraction yield and the use of standard addition for calibration

A high-performance liquid chromatographic procedure was developed for the determination of di-(2-ethylhexyl)phthalate (DEHP) concentrations in human whole blood samples. The solvent extraction of DEHP was found to be highly variable between samples obtained from different subjects (coefficient of variation of 30.4%). The recovery of DEHP following extraction with ethyl acetate was negatively correlated with serum lipid content, as expressed by the sum of serum cholesterol and triglyceride concentrations (r = -0.864). The technique of standard addition of DEHP allowed a single-point calibration of DEHP extractability in individual blood samples, and provided an accurate estimation of DEHP concentration (coefficient of variation of approximately 6% in replicate samples). The potential for intersample variability in the solvent extraction of other highly lipid-soluble compounds should be considered.     

Simple Methodology for the Quantitative Analysis of Fatty Acids in Human Red Blood Cells

In the last years, there has been an increasing interest in evaluating possible relations between fatty acid (FA) patterns and the risk for chronic diseases. Due to the long life span (120 days) of red blood cells (RBCs), their FA profile reflects a longer term dietary intake and was recently suggested to be used as an appropriate biomarker to investigate correlations between FA metabolism and diseases. Therefore, the aim of this work was to develop and validate a simple and fast methodology for the quantification of a broad range of FAs in RBCs using gas chromatography with flame ionization detector, as a more common and affordable equipment suitable for biomedical and nutritional studies including a large number of samples. For this purpose, different sample preparation protocols were tested and compared, including a classic two-step method (Folch method) with modifications and different one-step methods, in which lipid extraction and derivatization were performed simultaneously. For the one-step methods, different methylation periods and the inclusion of a saponification reaction were evaluated. Differences in absolute FA concentrations were observed among the tested methods, in particular for some metabolically relevant FAs such as trans elaidic acid and eicosapentaenoic acid. The one-step method with saponification and 60 min of methylation time was selected since it allowed the identification of a higher number of FAs, and was further submitted to in-house validation. The proposed methodology provides a simple, fast and accurate tool to quantitatively analyse FAs in human RBCs, useful for clinical and nutritional studies.     

Simple Methodology for the Quantitative Analysis of Fatty Acids in Human Red Blood Cells

In the last years, there has been an increasing interest in evaluating possible relations between fatty acid (FA) patterns and the risk for chronic diseases. Due to the long life span (120 days) of red blood cells (RBCs), their FA profile reflects a longer term dietary intake and was recently suggested to be used as an appropriate biomarker to investigate correlations between FA metabolism and diseases. Therefore, the aim of this work was to develop and validate a simple and fast methodology for the quantification of a broad range of FAs in RBCs using gas chromatography with flame ionization detector, as a more common and affordable equipment suitable for biomedical and nutritional studies including a large number of samples. For this purpose, different sample preparation protocols were tested and compared, including a classic two-step method (Folch method) with modifications and different one-step methods, in which lipid extraction and derivatization were performed simultaneously. For the one-step methods, different methylation periods and the inclusion of a saponification reaction were evaluated. Differences in absolute FA concentrations were observed among the tested methods, in particular for some metabolically relevant FAs such as trans elaidic acid and eicosapentaenoic acid. The one-step method with saponification and 60 min of methylation time was selected since it allowed the identification of a higher number of FAs, and was further submitted to in-house validation. The proposed methodology provides a simple, fast and accurate tool to quantitatively analyse FAs in human RBCs, useful for clinical and nutritional studies.

     

One-step cleanup for PAH residue analysis in plant matrices using size-exclusion chromatography

A new one-step cleanup procedure, based on size-exclusion chromatography (SEC), usable for the extracts from accelerated solvent extraction (ASE), Soxhlet extraction, or ultrasonic extraction (USE), is described. The method is suitable for the determination of polycyclic aromatic hydrocarbons (PAHs), especially from very complicated plant matrices (e.g. pine needles, deciduous leaves, mosses). The main improvement compared with previous conventional procedures is that analyte peaks barely overlap with matrix peaks in the chromatograms and that it is a very rapid and simple one-step procedure with clearly improved analytical performance. Essential advantages of this SEC procedure are the sharper GC-MS chromatograms for the PAH fraction at retention times between 9.2 and 12.0 min, distinctly separated substance peaks resulting in better analysis, shorter running times, and lower solvent consumption.     

WEFTA interlaboratory comparison on total lipid determination in fishery products using the Smedes method

Lipid determination by the Smedes method was tested in an interlaboratory trial performed by nine laboratories from seven countries belonging to the West European Fish Technologists Association Analytical Methods Working Group. Five samples of fish and fishery products with different lipid contents, including two blind duplicates, were distributed among the participants. All laboratories applied a slightly modified Smedes method, which included extraction of lipids by cyclohexane and isopropanol, transfer of lipids to the cyclohexane phase by addition of water, phase separation by centrifugation, and gravimetric lipid determination. The results indicate that the RSD for reproducibility (RSD(R)) was between 4.11 and 6.31% for samples with moderate (7%) and high (14%) lipid content, depending on the sample. Larger SDs among the laboratories were obtained for a cod sample with low lipid content of 0.5%. The method is judged to be suitable as a routine method for lipid determination in fish and fishery products.     

Microwave-assisted extraction and accelerated solvent extraction with ethyl acetate-cyclohexane before determination of organochlorines in fish tissue by gas chromatography with electron-capture detection

Focused open-vessel microwave-assisted extraction (FOV-MAE), closed-vessel microwave-assisted extraction (CV-MAE), and accelerated solvent extraction (ASE) were used for extraction before determination of organochlorine compounds (polychlorinated biphenyls, DDT, toxaphene, chlordane, hexachlorobenzene, hexachlorocyclohexanes, and dieldrin) in cod liver and fish fillets. Wet samples were extracted without the time-consuming step of lyophilization or other sample-drying procedures. Extractions were performed with the solvent mixture ethyl acetate-cyclohexane (1 + 1, v/v), which allowed direct use of gel-permeation chromatography without solvent exchange. For FOV-MAE, the solvent mixture removed water from the sample matrix via azeotropic distillation. The status of water removal was controlled during extraction by measuring the temperature of the distillate. After water removal, the temperature of the distillate increased and the solvent mixture became less polar. Only the pure extraction solvent allowed quantitative extraction of the organochlorine compounds. For CV-MAE, water could not be separated during the extraction. For this reason, the extraction procedure for wet fish tissue required 2 extraction steps: the first for manual removal of coextracted water, and the second for quantitative extraction of the organochlorine compounds with the pure solvent. Therefore, CV-MAE is less convenient for samples with high water content. For ASE, water in the sample was bound with Na2SO4. The reproducibility for each technique was very good (relative standard deviation was typically <10%); the slightly varying levels were attributed to deviations during sample cleanup and the generally low levels.     

Quantitative determination of folic acid in multivitamin/multielement tablets using liquid chromatography/tandem mass spectrometry

Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M + H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M - H]- FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (> or =3 orders of magnitude), limits of detection (0.02 ng on-column) and limits of quantification (0.06 ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was > or =95% (based on the manufacturer's assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%.     

Marine macroalgae analyzed by mass spectrometry are rich sources of polyunsaturated fatty acids

Algae from cold water (Canada) and warm water (China) were analyzed for their total lipid content, and for their fatty acid (FA) composition and content. The major findings are that FA from Canadian algae are generally richer in polyunsaturated FA (PUFA), with a higher n-3/n-6 FA ratio, and a higher degree of total unsaturation. The 18 C, 4 double bonds FA (18 : 4 stearidonic acid, morotic acid as synonym) was detected in greater amounts in cold water samples. The high levels of total PUFA, and especially of n-3 FA in Canadian algae, suggests their possible utilizations for nutritional purposes.     

两次双水相萃取结合高效液相色谱法选择性分离唾液蛋白质的初步研究（英文）

双水相萃取是一种新型的液-液萃取技术,具有方法简单,易操作,成本低,易放大,条件温和,可保持蛋白质活性等明显优势,特别适用于生物样品的前处理和组分分离。本文建立了15% PEG-4000/8% NaH₂PO₄双水相体系,通过两次双水相萃取结合高效液相色谱法（HPLC）分离了唾液中的多种蛋白质。经过双水相萃取,对上、下两相中的蛋白质进行色谱的梯度洗脱分析。50 min内蛋白质的色谱峰可分为10组,根据其在上、下两相的分配规律还可划分为6个组分区。结果表明,两次双水相萃取结合HPLC可以实现唾液中的蛋白质的选择性分离。该法为复杂生物样品中的蛋白质多维度、选择性分离和分析提供了新的思路。     

An efficient diethyl ether-based soxhlet protocol to quantify faecal sterols from catchment waters

A study was conducted to evaluate the efficiency and reproducibility of a diethyl ether-based soxhlet extraction procedure for faecal sterols occurring from catchment waters. Water samples spiked with a mixture of faecal sterols were filtered and analytes were extracted using the diethyl ether-based soxhlet method and the Bligh and Dyer chloroform extraction process. For diethyl ether-based soxhlet extraction procedure, solvent extracts were saponified with 100 microL of 10% KOH in methanol (100 degrees C/120 min) and then acidified with 60 microL of 6M HCl. Lipid contents were extracted by ethanol (0.5 mL) from the saponification products. The lipid extracts were then reacted with 100 microL of bis(trimethyl)trifluoroacetamide (BSTFA) containing 1% trimethyl chlorosilane (100 degrees C/60 min) to form the trimethylsilyl (TMS) derivatives. The derivatised extracts were then analyzed by gas chromatography-mass spectrometry. For sterol concentrations ranging from 35 to 175 microg mL(-1), the soxhlet-based extraction process yielded the following recovery efficiencies for coprostanol (101%), epicoprostanol (97%), cholesterol (97%), dihydrocholesterol (97%) and 5alpha-cholestane (111%), whereas the Bligh and Dyer process yielded recoveries of 32, 41, 0, 36 and 51%, respectively. The results suggested that the diethyl ether-based soxhlet extraction method was more efficient and reproducible than the Bligh and Dyer chloroform extraction process for the analyses of trace levels of faecal sterols from water samples. Moreover, it was revealed that the diethyl ether-based soxhlet extraction method used less solvent and was logistically easier.     

Determination of chloride, sulfate and nitrate in groundwater samples by ion chromatography

Groundwater is a significant source of water for both domestic and agricultural use in some regions of the Maracaibo lake basin in Venezuela. Chemically suppressed ion chromatography with a Dionex Model 2000i/sp, lonpac AS11, ASRS-I system was used for the analysis of major inorganic anions in groundwater samples. About 50 samples of groundwater, taken over several months in three different locations, were analyzed after filtration and sometimes dilution. In all the samples, the separation between the peaks of chloride, nitrate and sulfate showed good resolution (symmetrical peaks, not broadened), even when the chloride concentration was as high as 850 mg l(-1) and reproducibility (RSD) was -2%. No other peaks (i.e. fluoride, nitrite and phosphate) were observed at selected experimental conditions. With the chosen parameters, the method is well-suited for the routine determination of these anions in groundwater samples, giving results in less than 10 min (including column clean-up). With an appropriate combination of detector output ranges (300 and 1,000 microS), only one set of calibration solutions was needed for all samples. In the Sierra Maestra location, the groundwater samples, were significantly different in total anion levels. Mean total chloride plus sulfate concentrations (approximately 525 mg l(-1)) were about 100 times higher than in the other sites. Some water quality implications of these groundwater samples are also discussed.     

Rheological properties and structures of cationic surfactants and fatty alcohol emulsions: effect of surfactant chain length and concentration

 Rheological and optical properties of cetyltrimethylammonium chloride (CTAC)/fatty alcohol (FA), behenyltrimethylammonium chloride (BTAC)/FA and CTAC/FA/hydroxyethyl cellulose (HEC) emulsions have been studied with particular emphases on the effects of FA content, the difference in the chain length of the hydrophobic groups between CTAC and BTAC, and the addition of a water soluble polymer, HEC. The effects of the FA content are to accelerate the structure development during the aging period and to increase the storage modulus, the yield stress, and the zero-shear-rate viscosity in the three emulsion systems investigated. At a low FA content of 2% w/w, lamellar and vesicular aggregates and isolated multilamellar vesicles can be observed in the CTAC/FA and BTAC/FA emulsions, respectively. At a high FA content of 6% w/w or with an excess of FA present, networklike structures and sunflower-like structures form, respectively, instead, inducing a higher entanglement storage modulus and a higher yield stress relative to those emulsions with a low FA content. The effect of adding HEC to the CTAC/FA emulsion is to reduce the entanglement storage modulus and the yield stress, consistent with the optical observation that the presence of the polymer disrupts the formations of lamella and vesicular aggregates and network structures. Received: 27 July 2000 Accepted: 28 November 2000     

Determination of Phenolic Antioxidants in Spilled Aviation Fuels by Gas Chromatography-Mass Spectrometry

Summary A gas chromatography-mass spectrometric assay method was developed for the simultaneous determination of 2,6-di-tert-butylphenol (DTBP) and 2,4-dimethyl-6-tert-butylphenol (DMTBP) in aviation fuel. Extraction and purification were achieved with a solid phase extraction procedure using silica gel. Elution was performed with 30 mL of methylene chloride: pentane (2:3) following washing with 10 mL of n-pentane. The extract was concentrated to about 100 L and analyzed by GC-MS (SIM). The peaks had good chromatographic properties by using a semi-polar column (Innowax) and the extraction of these compounds from samples gave recoveries of about 87% for DTBP and about 75% for DMTBP with small variations. Method detection limits were 5.0 ng mL^–1 for DBMP and 7.0 ng mL^–1 for DMTBP. The method may be useful for spilled fuel type differentiation between kerosene and JP-8.     

Simultaneous determination of five polyether ionophores using liquid chromatography with one-step fluorescent derivatization

We present a selective method for simultaneous determination of five polyether ionophores such as salinomycin (SAL), monensin (MON), narasin (NAR), semduramicin (SEM) and lasalocid (LAS) in aquatic samples using a liquid chromatography with one-step fluorescent derivatization of 2-(4-hydrazinocarbonyl-phenyl) 4,5-diphenylimidazole (HCPI) and 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride hydrochloride (DIB-Cl). Fluorescent one-step derivatization for SAL, MON, NAR and SEM using HCPI and for LAS using DIB-Cl was monitored by an LC/fluorescence detector (E(x), 340 nm; E(m), 465 nm). Chromatographic separation was performed on a TSK-GEL ODS-120T (4.6 × 150 mm, 3 μm) column using a mobile phase of 0.1% formic acid in acetonitrile and 0.5 mM ammonium formate in water (70/30, v/v). The limits of detections were 0.01 μg/mL (50 pg) for LAS, 0.05 μg/mL (250 pg) for SAL, NAR and SEM, and 0.1 μg/mL (500 pg) for MON, respectively. The recoveries for water samples were indicated to be the range of 79.6 ± 6.4 - 99.0 ± 4.4% with associated precision values (between-day for 3 days) for repeatability. Based on solid-phase extraction, the limit of quantitation values indicated 0.1 ng/mL for SAL, MON, NAR and SEM, and 0.01 ng/mL for LAS in water samples.     

Cholesterol modulated antibody binding in supported lipid membranes as determined by total internal reflectance microscopy on a microfabricated high-throughput glass chip

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (X(CHOL)). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant K(d), the maximum fluorescence parameter F(infinity), and binding cooperativity n. As X(CHOL) increased from 0 to 0.50, K(d) exhibited a minimum of approximately 4 microM and n reached a maximum of approximately 2.2 at X(CHOL) approximately 0.20. However, F(infinity) appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of approximately 0.08 also at X(CHOL) approximately 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of X(CHOL) approximately 0.20. Interestingly, for X(CHOL) approximately 0.40, NS reached a maximum of approximately 0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.     

Removal of sodium dodecyl sulfate from protein samples prior to matrix-assisted laser desorption/ionization mass spectrometry

Sodium dodecyl sulfate (SDS) is widely used for protein solubilization and for separation of proteins by SDS polyacrylamide gel electrophoresis (SDS-PAGE). However, SDS interferes with other techniques used for characterization of proteins, such as mass spectrometry (MS) and amino acid sequencing. In this paper, we have compared three procedures to remove SDS from proteins, including chloroform/methanol/water extraction (C/M/W), cold acetone extraction and desalting columns, in order to find a rapid and reproducible procedure that provides sufficient reduction of SDS and high recovery rates for proteins prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). A 1000-fold reduction of SDS concentration and a protein recovery at approximately 50% were obtained with the C/M/W procedure. The cold acetone procedure gave a 100-fold reduction of SDS and a protein recovery of approximately 80%. By using desalting columns, the removal of SDS was 100-fold, with a protein recovery of nearly 50%. Both the C/M/W and the cold acetone methods provided sufficient reduction of SDS, high recovery rates of protein and allowed the acquisition of MALDI spectra. The use of n-octyl-beta-D-glucopyranoside in the protein sample preparation enhanced the MALDI signal for protein samples containing more than 2 10(-4)% SDS, after the C/M/W extraction. Following the cold acetone procedure, the use of n-octylglucoside was found to be necessary in order to obtain spectra, but they were of lower quality than those obtained with the C/M/W method, probably due to higher residual amounts of SDS.     

Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500 ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.     

燃煤电厂煤及其燃烧副产物中砷形态分析

燃煤电厂煤中砷（As）的形态在燃烧过程中不可避免地会发生转化。煤及其副产物中砷的形态与人体健康和环境安全密切相关，亟待鉴别。然而目前针对煤燃烧相关产物中砷形态的前处理手段和分析方法尚缺乏。本研究采用高效液相色谱-氢化物发生-原子荧光光谱法（HPLC-HG-AFS）成功测定了电厂煤、粉煤灰和石膏中砷的形态，优化了仪器参数、提取试剂和前处理方法（超声和微波辅助）。优化后，无机砷的分离时间缩短至7 min，As（Ⅲ）和As（V）的检出限分别为1.8 ng/g和4.6 ng/g。砷形态的高效提取剂为1.0 mol/L 磷酸和0.1 mol/L 抗坏血酸的混合溶液。微波辅助（2000 W、80 ℃、40 min）和超声辅助（40 kHz、20 ℃、40 min）分别是煤/粉煤灰和石膏样品中砷形态的最佳提取方法。在微波和超声波辅助提取条件下，As（Ⅲ）/As（V）的回收率分别为95.8%/104.5%和90.6%/89.7%。样品分析结果表明，煤中砷主要以As（V）形式存在，As（Ⅲ）所占比例很小，而在粉煤灰和石膏中只观察到As（V）。该研究揭示了As（Ⅲ）向As（V）的转化是气态砷捕获的关键，可以为控制电厂砷排放提供科学依据。     

Lipids from the Marine Alga <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>

The composition of lipids and fatty acids from the red alga Gracilaria verrucosa, for which a high content of 20:4n-6 acid is typical, was studied. The principal lipids were digalactosyldiacylglycerides, phosphatidylcholines (PC), monogalactosyldiacylglyderides (MGDG), and sulfoquinovosyldiacylglycerides, the fraction of each was approximately the same. Sphingophospholipids, inositephosphoceramides, were identified among the polar lipids. Each lipid class differed in the ratio of fatty acids (FA). The FA of all glycerolipids contained 20:4n-6 acid but its concentration was greatest in MGDG and PC, 67.2% and 56.5% of the acid mass.__________Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 230–232, May–June, 2005.     

Determination of arsenic species in marine organisms by HPLC-ICP-OES and HPLC-HG-QFAAS

Separation and quantification of six arsenic species have been performed in cod, tuna and mussel samples by high performance liquid chromatography (HPLC) using inductively coupled plasma-optical emission spectrometry (ICP-OES) and hydride generation-quartz furnace atomic absorption spectrometry (HG-QFAAS) as detection techniques. It has been shown that arsenic extraction with a water-methanol (11) mixture is sufficiently quantitative for the cod and tuna, in which arsenic is mainly present as arsenobetaine (about 90% of total As extracted). In contrast, only 60% of the element is extracted from the mussels and the chromatograms obtained reveal the presence of an unknown compound. Detection limits are in the g ml^–1 range for the HPLC-ICP-OES technique (quantification of arsenobetaine and arsenocholine) and in the ng ml^–1 range for the HPLC-HG-QFAAS system (quantification of arsenite, arsenate, monomethylarsonic and dimethylarsinic acids).