A capillary electrophoresis laser-induced fluorescence method for analysis of potato glycoalkaloids based on a solution-phase immunoassay. 2. Performance evaluation

Glycoalkaloids (GAs) occur naturally in potatoes and are toxic to humans and animals. The objective of the present study was to evaluate the performance of a solution-phase immunoassay coupled to capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection for the determination of total glycoalkaloids in potatoes. The immunoassay was based on a competition between potato glycoalkaloids and fluorescently labeled solanidine. Reaction products were separated in the capillary zone electrophoresis mode. A calibration curve of signal vs log[GA] was linear from 50 to 400 nM. The CV for duplicate and day-to-day analyses averaged 5.7% and 12%, respectively. Spike recoveries ranged from 85 to 97% for spike levels ranging from 43 to 170 microg/g fresh potato. Potato samples with GA concentrations ranging from <40 to >200 microg/g were successfully analyzed, indicating that immuno-CE-LIF is a rapid alternative to traditional ELISA and HPLC methods.     

Rapid and sensitive quantification of amino acids in soil extracts by capillary electrophoresis with laser-induced fluorescence

A growing body of research is arguing that amino acids are key components of the soil nitrogen cycle. For example, we now know that many plants can take up intact amino acids, even in competition with soil microbes. Our growing recognition of the importance of amino acids is not matched by knowledge of the amounts and type of amino acids in the soil, certainly not in comparison with our encyclopaedic knowledge of inorganic N. The primary reason that less is known about amino acids than inorganic N is that measuring the amounts of individual amino acids with conventional chromatographic techniques is slow and typically requires extensive sample clean-up if KCl extracts are analysed. The aim of this study was to develop capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) as a more rapid alternative for measuring individual amino acids in KCl extracts of soil. The CE-LIF method separated 17 common amino acids within 12 min, with detection limits between 7 and 250 nM. One molar KCl extracts could be analysed without any sample clean-up or de-salting, and spike and recovery tests indicated that the complex matrix of soil extracts did not affect quantification. Further evidence of the suitability and robustness of the method came from the repeated analysis (n=5) of the same soil KCl extract. The relative standard deviation of migration times for replicate analyses were <0.2% while relative standard deviations for peak areas were <5%. To demonstrate application of the CE-LIF method to real world problems it was used to analyse amino acids in 1 M KCl extracts from a sub-alpine grassland and a Eucalyptus regnans forest. The most abundant amino acids were Ala, Gly and Arg. Other amino acids present at smaller concentrations or in a minority of samples were Asn, Cit, GABA, Glu, His, Phe, Leu, and Lys. The proposed CE-LIF method offers significant advantages over chromatographic methods via its rapidity, reproducibility and, most importantly, its ability to analyse crude KCl extracts.     

Development of a capillary electrophoresis-based immunoassay with laser-induced fluorescence for the detection of carbaryl in rice samples

A capillary electrophoresis-based competitive immunoassay (CEIA) with a laser-induced fluorescence (LIF) detector for the determination of carbaryl was developed. The method was based on the competitive reactions between fluorescently labeled carbaryl tracer (Ag*) and free carbaryl (Ag) with a limited amount of anticarbaryl antibody (Ab), and the relative amounts of each were separated and determined by capillary electrophoresis (CE) with an LIF detector. Using CEIA, equilibrium was reached in 30 min, and the analytical results were obtained within a further 8 min. The linear range and the detection limit for carbaryl were 0.16-50 ng/mL and 0.05 ng/mL, respectively. The sensitivity of this CEIA with an LIF detector was almost 14 times greater than that of ELISA, which used the same immuno-reagents. The method was also applied to the analysis of carbaryl in rice with rapid and simple sample pretreatment. The method is thus proposed as a fast and sensitive assay for the detection of carbaryl.     

Assay of riboflavin in sample wines by capillary zone electrophoresis and laser-induced fluorescence detection

To routinely assay the concentration of riboflavin (RF) in wines, a rapid and sensitive method was developed and evaluated. The method is based on a simple sample preparation, capillary zone electrophoretic separation and laser-induced fluorescence detection (CZE-LIF). Sample preparation required only dilution and filtration. Under optimized conditions, the limit of detection of riboflavin was 0.5 micro g/L, using a hydrodynamic sample introduction of 10 s at 54 mbar. The method was fully validated: the recovery of RF in wines was >95%. The concentrations of RF within the three sample types of Italian wines investigated here ranged from 69 to 151 micro g/L with a mean value (+/-SD) of 112 +/- 25 micro g/L, from 74 to 193 micro g/L with a mean value of 115 +/- 45 micro g/L, and from 156 to 292 micro g/L with a mean value of 226 +/- 40 micro g/L, for white, rosé, and red wines, respectively. Such an accurate and highly sensitive CZE-LIF method represents a powerful improvement over previous methods in terms of sensitivity, simplicity, and efficiency. It is well suited to satisfy the demands for accurate and sensitive detection with minimal sample preparation and cleanup.     

New capillary electrophoresis method for the determination of furosine in dairy products

A new capillary electrophoresis (CE) method was established for the quantitative determination of furosine in dairy products. Sample preparation and suitable electrophoretic conditions allowed accurate and reproducible quantitation of furosine in dairy products. Sample preparation consisted of drying hydrolyzed samples, redissolving them in 0.2 M NaOH, and purifying them by solid-phase extraction. The electrophoretic separation was carried out in an uncoated capillary maintained at 30 degrees C using 0.1 M phosphate buffer containing the additive hexadecyl trimethylammonium bromide (HDTAB, 1.2 mM) (pH 7.0) under 10 kV voltage and reverse polarity. Coefficients of variation of less than 2.25% for migration time and 5.80% for peak areas indicated that the technique was reproducible. The calibration curve followed a linear relationship with a highly significant (p < 0.01) coefficient of multiple determination (R (2) = 0.997). The limit of quantitation was 0.5 ppm, a concentration that corresponds to 4.5 mg/100 g of protein in milk samples. Furosine concentration (mg/100 g of protein) ranges of different dairy products (raw, pasteurized, UHT, and evaporated milks and yogurt) agreed with ranges previously reported. Therefore, the CE method presented is a suitable technique for the routine assessment of furosine in dairy products.     

Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence

The simultaneous detection of Staphylococcus aureus, Listeria monocytogenes, and Salmonella spp. has been approached by a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF). As compared to slab gel electrophoresis, the use of CGE-LIF improved from 10- to 1000-fold the sensitivity of the multiplex PCR analysis, allowing the detection of 2.6 x 10(3) cfu mL(-1) of S. aureus, 570 cfu mL(-1) of L. monocytogenes, and 790 cfu mL(-1) of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu mL(-1) of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The CGE-LIF method is shown to be reproducible, providing relative standard deviation (RSD) values lower than 0.8% for analysis time and lower than 5.8% for peak areas. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way.     

Detection of genetically modified maize by the polymerase chain reaction and capillary gel electrophoresis with UV detection and laser-induced fluorescence

In this paper, the possibilities of capillary gel electrophoresis (CGE) to detect transgenic maize in flours are shown. The method is based on the extraction and amplification by the polymerase chain reaction (PCR) of a specific DNA fragment from transgenic maize and its subsequent analysis by CGE with UV detection or laser-induced fluorescence (LIF). Some useful considerations regarding the optimization of DNA extraction and amplification conditions are given. Also, a comparison is established between the two CGE protocols for DNA detection based on ultraviolet absorption (CGE-UV) and LIF (CGE-LIF). The requirements, advantages, and limitations of both CGE methods are discussed. To our knowledge, this is the first paper on the use of CGE-LIF to detect transgenic food.     

Detection and differentiation of several food-spoilage lactic acid bacteria by multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence

In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria. To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed. The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C. divergens, C. gallinarum, and C. maltaromicum). The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested. These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained. The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges. Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively.     

Separation and determination of diesel contaminants in various fish products by capillary gas chromatography.

A semiquantitative capillary column gas chromatographic method is described for the determination of diesel fuel contamination in various canned seafood products. The diesel contaminants are separated from the fish sample by steam distillation, with little carry-over of interfering intrinsic materials such as fish oils. The diesel fuel is extracted from the condensate with n-hexane, and the extract is analyzed on an SPB-1 fused silica capillary column. The efficiency of recovery of diesel fuel added to canned seafood at levels of 40-400 ppt ranged from 72 to 102%. With the additional step of concentrating the hexane extract, the sensitivity of this procedure may be increased at least 10-fold. This procedure can detect the differences among diesel fuel grades No. 1, 2, and 5, and variations within diesel grade No. 2, and thus may be useful in determining the type of petroleum contaminants present in various canned fish products.     

Separation of olive proteins combining a simple extraction method and a selective capillary electrophoresis (CE) approach: application to raw and table olive samples

A simple extraction method was developed to extract proteins from olive samples based on chloroform/methanol extraction followed by a protein precipitation with cold acetone. Then, a capillary electrophoresis (CE) method was carried out using an acid buffer (1 M formic acid at pH 2) to ensure a positive net charge for proteins and a neutral charge for potential interferents as polyphenols. The method developed was applied to raw and table olive samples. Interestingly, raw olive samples showed differences in protein profiles depending upon the botanical variety of olives and their geographical region. Protein profiles obtained for table olives also showed differences according to the sample treatment. Thus, a signal reduction in the electropherograms obtained for black olives was observed in comparison to those achieved for treated green olives. In this work, the use of protein profiles was demonstrated to be a powerful tool for studying variations among olive samples.     

A rapid sample screening method for authenticity control of whiskey using capillary electrophoresis with online preconcentration

The present study aimed to develop a methodology using capillary electrophoresis for the determination of sinapaldehyde, syringaldehyde, coniferaldehyde, and vanillin in whiskey samples. The main objective was to obtain a screening method to differentiate authentic samples from seized samples suspected of being false using the phenolic aldehydes as chemical markers. The optimized background electrolyte was composed of 20 mmol L(-1) sodium tetraborate with 10% MeOH at pH 9.3. The study examined two kinds of sample stacking, using a long-end injection mode: normal sample stacking (NSM) and sample stacking with matrix removal (SWMR). In SWMR, the optimized injection time of the samples was 42 s (SWMR42); at this time, no matrix effects were observed. Values of r were >0.99 for the both methods. The LOD and LOQ were better than 100 and 330 mg mL(-1) for NSM and better than 22 and 73 mg L(-1) for SWMR. The CE-UV reliability in the aldehyde analysis in the real sample was compared statistically with LC-MS/MS methodology, and no significant differences were found, with a 95% confidence interval between the methodologies.     

Development of a capillary electrophoresis–mass spectrometry method for small peptides in the soil solution

Small peptides are being investigated for their role in ecosystem cycling and plant uptake of organic N, but little is known of molecular forms in the soil solution. The aim of this study was to develop a capillary electrophoresis–tandem mass spectrometry procedure for profiling small peptides in the soil solution. Capillary electrophoresis–mass spectrometry was capable of separating and detecting a range of small peptide standards. Adequate recovery (>90%) of standard peptides spiked into samples of soil solution indicated that separation and detection were robust and not significantly affected by the sample matrix. The method was applied to samples of soil solution from grassland mesocosms filled with clay-loam soil from an abruptic lixisol. Soil solution (ultrafiltered <3 kDa) contained at least 298 putatively identified peptides with most being smaller than 600 Da. Less than 5% of small peptides contained basic amino acids, which may reflect their preferential adsorption to the soil stationary phase versus peptides comprised of acid or neutral amino acids. Capillary electrophoresis–mass spectrometry of small peptides is robust and has already yielded novel findings with its first proof of concept measurements on the soil solution.     

Extraction parameters and capillary electrophoresis analysis of limonin glucoside and phlorin in citrus byproducts.

Limonin glucoside (LG) and phlorin were extracted from citrus fruit tissues and assayed by capillary electrophoresis (CE). LG was determined in dried [1.20 +/- 0.10 mg of dry weight (dw)] and wet peel residues (1.16 +/- 0.04 mg of dw), orange juice finisher pulp (0.58 +/- 0.03 mg of dw), dried grapefruit seeds (2.70 +/- 0.15 mg of dw), and 50 degrees Brix molasses (2225 +/- 68 mg/L). Phlorin was purified from orange peel residue and grapefruit albedo, and concentrations were determined in some citrus products. Phlorin and LG were extracted from residues with water/pectinase or with water solutions of methanol and ethanol. Efficient LG extraction from grapefruit seeds (2.40 +/- 0.15 mg/g) was achieved with 50-65% methanol, solvent polarity P' approximately equal to 7-8. Extracts were purified and concentrated by adsorptive resins and HPLC to obtain 95% pure compounds of LG and phlorin. CE analysis did not require extract purification beyond filtration. LG and phlorin migrated as anions in electropherograms containing peaks representing other citrus flavonoids and limonoid glucosides.     

1,3-beta-glucan quantification by a fluorescence microassay and analysis of its distribution in foods

The objective of this study was to establish analytical approaches to quantify 1,3-beta-glucan (1,3-beta-G) in foods. Six food categories including legumes, cereals, tubers, vegetables, fruits, and mushrooms and 17 total items were tested. An extraction procedure was designed to prepare food cold-water soluble, hot-water soluble, cold-alkaline soluble, and hot-alkaline soluble fractions. A fluorescence microassay based on aniline blue dye, which bound specifically to 1,3-beta-G, was developed to measure its content in the food fractions. Curdlan was used as standard to construct the 1,3-beta-G calibration curve, and a linear correlation within a 14 microg/mL concentration range was obtained. This microassay displayed selectivities among various 1,3-beta-G species. Biologically active ones such as pachyman and yeast glucan possessed much stronger fluorescent signals than others such as laminarin and barley glucan. Possible fluorescent interference from food proteins was estimated. This assay tolerated up to 50% of bovine serum albumin in 10 microg/mL curdlan. Analysis of the four food fractions showed that besides the well-known lentinan-containing shiitake, popular foods such as celery, chin-chian leaves, carrot, and radish contained nearly 20% 1,3-beta-G in their total sugar. Soybean dry weight contained 0.8% 1,3-beta-G, which was twice the amount compared to shiitake. Snow mushroom dry weight had the highest 1,3-beta-G content, at 2.5%, and was rich in both water (0.67%) and alkaline soluble (1.87%) forms. In conclusion, this dye-binding fluorescence microassay in conjunction with the extraction procedure can be applied in the prescreening of potential foods rich in functional 1,3-beta-G.     

基于熵权法和TOPSIS法优化马铃薯钾肥种类和滴灌量组合

[目的]探寻滴灌施肥条件下实现沙土马铃薯高产优质和水肥高效的管理方式,为陕北马铃薯滴灌水钾管理提供科学依据.[方法]以'青薯9号'为试验材料,于2020年马铃薯生长季,在陕北榆林风沙区设置W1?(60％?ETc,198.4?mm,ETc为作物需水量)、W2?(80％?ETc,246.2?mm)和W3?(100％?ETc...     

High-performance liquid phase separation of glycosides. 5. Determination of individual glucosinolates in cabbage and rapeseed by laser-induced fluorescene capillary electrophoresis via the enzymatically released isothiocyanate aglycon.

A capillary electrophoresis (CE) method was developed for the profiling and determination of individual glucosinolates (GSs) via their isothiocyanate degradation products upon myrosinase digestion. The resulting isothiocyanates, the structures of which are reflective of the parent GS's, were then converted to their corresponding amines via base hydrolysis or reaction with 1, 2-benzenedithiol. Subsequently, the amines were fluorescently labeled to allow their sensitive detection by laser-induced fluorescence (LIF). The CE method involved the use of in situ charged micelles for the separation of isothiocyanates and their corresponding fluorescently labeled amines by micellar electrokinetic capillary chromatography (MECC). The term "in situ charged micelles" refers to micelles formed by complexing the polar hydroxyl groups of glycosidic surfactants with borate. The MECC method with on-column LIF detection was applied to the determination of GSs in white cabbage, rapeseed leaves, and rapeseed roots.     

Improved cleanup for liquid chromatographic analysis and fluorescence detection of aflatoxins M1 and M2 in fluid milk products

A rapid method is described for extraction and cleanup of raw and processed milk for determination of aflatoxins M1 and M2 by using a C18 Sep-Pak/silica gel cleanup column combination. Aflatoxins are separated by normal phase liquid chromatography and their concentrations are determined by fluorescence detection in a silica gel-packed flow cell. Recoveries ranged from 99 to 103% with coefficients of variation less than 2% for M1 levels of 0.117-1.17 ng/mL added to raw milk. Similar recoveries were obtained for M2. The coefficient of variation for analysis of 5 subsamples of naturally contaminated milk was less than 1%. Agreement with the official method is satisfactory. Each sample requires less than 25 mL solvent and 10 min actual handling time. Sample chromatograms show no interferences in the M1-M2 elution region and no late-eluting peaks, which permits spacing injections at 13-20 min intervals. Aflatoxin levels as low as 0.03 ppb may be determined by this procedure. Extracts have also been analyzed by thin layer chromatography.     

Simultaneous confirmatory analysis of different transgenic maize (zea mays) lines using multiplex polymerase chain reaction-restriction analysis and capillary gel electrophoresis with laser induced fluorescence detection

A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control.