Determination of D-chiro-Inositol in tartary buckwheat using high-performance liquid chromatography with an evaporative light-scattering detector

A simple method for rapid determination of d- chiro-inositol (DCI) (a potent mediator of insulin metabolism) contained in tartary buckwheat ( Fagopyrum tataricum L. Gaench) was developed on the basis of high-performance liquid chromatography coupled to an evaporative light scattering detector (ELSD). DCI was separated from an extract of tartary buckwheat on an Alltech Prevail Carbohydrates ES 5 mum column. During postcolumn detection, DCI was detected by ELSD. The detection limit was 100 ng. This method was sensitive enough for determining DCI in tartary buckwheat and its related products.     

Improved normal-phase high-performance liquid chromatography procedure for the determination of carotenoids in cereals

Besides the health benefits associated with whole-grain consumption, cereals are recognized sources of health-enhancing bioactive components such as carotenoids, which are a group of yellow pigments involved in the prevention of many degenerative diseases and which have been used for a long time as indicators of the color quality of durum wheat and pasta products. This work reports a fast, sensitive, and selective procedure for the extraction and determination of carotenoids from cereals and cereal byproducts. The method involves sample saponification and extraction followed by normal-phase high-performance liquid chromatography, allowing the separation of the main carotenoids pigments of cereals, especially lutein and zeaxanthin. An application of the established method to various species of cereals and cereal byproducts is also shown. The highest carotenoid levels were found in maize (approximately 11.14 mg/kg of dry weight), which contains high amounts of beta-cryptoxanthin (2.40 mg/kg of dry weight), and, among the cereals considered, has the highest content of zeaxanthin (6.43 mg/kg of dry weight) and alpha+beta-carotene (1.44 mg/kg of dry weight). With the exception of maize, lutein is the main compound found (from 0.23 to 2.65 mg/kg of dry weight in oat and durum wheat, respectively). Moreover, whereas alpha+beta-carotene and zeaxanthin are principally localized in the germ, lutein is equally distributed along the kernel.     

Improved high-performance liquid chromatography (HPLC) method for qualitative and quantitative analysis of allantoin in Zea mays

A high-performance liquid chromatography (HPLC) method for the qualitative and quantitative analysis of allantoin in silk and seed of Zea mays has been developed. Allantoin separation in crude extract was achieved using a C 18 column and phosphate buffer solution (pH 3.0) as a mobile phase at ambient temperature at a flow rate of 1.0 mL/min and detected at 210 nm. The results showed that the amount of allantoin in samples was between 14 and 271 mg/100 g of dry plant material. A comprehensive validation of the method including sensitivity, linearity, repeatability, and recovery was conducted. The calibration curve was linear over the range of 0.2-200 microg/mL with a correlation coefficient of r2>0.999. Limit of detection (LOD, S/N=3) and limit of quantification (LOQ) values of the allantoin were 0.05 and 0.2 microg/mL (1.0 and 4.0 ng) respectively. The relative standard deviation (RSD) value of the repeatability was reported within 1.2%. The average recovery of allantoin added to samples was 100.6% with RSD of 1.5%.     

Simplified extraction of ginsenosides from American ginseng (Panax quinquefolius L.) for high-performance liquid chromatography-ultraviolet analysis

Four methods were tested for extraction and recovery of six major ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) found in roots of American ginseng (Panax quinquefolius): method A, sonication in 100% methanol (MeOH) at room temperature (rt); method B, sonication in 70% aqueous MeOH at rt; method C, water extraction (90 degrees C) with gentle agitation; and method D, refluxing (60 degrees C) in 100% MeOH. After 0.5-1 h, the samples were filtered and analyzed by high-performance liquid chromatography (HPLC)-UV. A second extraction by methods C and D was done, but 85-90% of ginsenosides were obtained during the first extraction. Lyophilization of extracts did not influence ginsenoside recovery. Method D resulted in the highest significant recoveries of all ginsenosides, except Rg1. Method C was the next most effective method, while method A resulted in the lowest ginsenoside recoveries. Method B led to similar recoveries as method C. All methods used one filtration step, omitted time-consuming cleanup, but maintained clear peak resolution by HPLC, and can be used for quantitative screening of ginsenosides from roots and commercial ginseng preparations.     

Improved solid-phase extraction procedure in the analysis of paralytic shellfish poisoning toxins by liquid chromatography with fluorescence detection

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.     

Improved automated extraction and separation procedure for soil lipid analyses

Analysis of soil lipids may contribute to an improved understanding of atmosphere to soil carbon fluxes, soil organic matter source differentiation and pollutant accumulation. Soil lipids, mostly originating from plants and microorganisms, have traditionally been analysed by non‐automated extraction and separation methods, which produce several lipid fractions, operationally defined by polarity. Here we present a combination of fast, automated and reproducible techniques, adopted from organic geochemical studies, for preparative separation of individual soil lipid fractions with increasing polarity. These techniques involve commercially available instruments, including accelerated solvent extraction and a two‐step automated medium‐pressure liquid chromatography procedure. The method yields eight lipid fractions consisting of five fractions fully amenable to gas chromatography/mass spectrometry (GC/MS) (aliphatic hydrocarbons, aromatic hydrocarbons, ketones, alcohols, carboxylic acids), and three fractions of highly polar or high molecular weight compounds (bases, very long‐chain wax esters (C40₊), high polarity compounds) that were not measurable with GC/MS under standard conditions. We tested the method on five agricultural soils. Results show that (i) mass recoveries for the individual fractions are reproducible, (ii) within individual fractions compound distribution patterns are reproducible, as demonstrated for alkanes and carboxylic acids, and (iii) individual fractions represent distinct and clean compound classes, free of interfering substances detectable by GC/MS. Thus, automated separation can be a fast, effective and reproducible procedure for fractionation of complex mixtures of soil lipids into clean compound classes, directly suitable for a variety of molecular (e.g. GC/MS) and isotopic characterizations (e.g. gas chromatography coupled with isotope ratio monitoring mass spectrometry or accelerator mass spectrometry).     

Use of anion-exchange membrane extraction for the high-performance liquid chromatographic analysis of mustard seed glucosinolates

A new one-step extraction using anion-exchange membranes for the HPLC determination of glucosinolates in mustard seeds is reported. The exchange of glucosinolates on the membranes was studied using sinigrin in solutions and sinigrin added as an internal standard to seeds of yellow mustard. By varying time of extraction, membrane size, and sample size, the optimal conditions for maximum glucosinolate recovery were determined and the following procedure was adopted: 0.2 g of ground mustard seeds are heated in 20 mL of boiling water for 5 min. After cooling, samples are transferred to plastic centrifuge tubes, 9-cm(2) membranes are added, and suspensions are shaken on a mechanical shaker for 2.5 h. Glucosinolates are then eluted from the membranes with 25 mL of 1 N KCl by shaking again for 2.5 h. Using this procedure, the sinigrin extraction from solutions and from mustard seeds was linear with 80% recovery. Seeds of yellow, brown, oriental, and Indian mustard were analyzed by this procedure; excellent reproducibility, with coefficients of variation in the range 1.0-4.3% were obtained. This method offers a simple and inexpensive alternative to complicated and tedious procedures for glucosinolate isolation/purification required for chromatographic determinations.     

Improved high-performance liquid chromatography method to determine theobromine and caffeine in cocoa and cocoa products

At present, the commonly used HPLC method for the analysis of caffeine and theobromine contents in aqueous cocoa extracts employs direct application of the extracts on the column. This practice gradually reduces the efficiency of the column and shortens its life. Also, this method gives inflated values due to interfering substances and difficulty in achieving baseline resolution. In the improved method, the interfering cocoa pigments are effectively removed by passing the aqueous extract through a Sep-pak C(18) cartridge. Subsequent injection on a C(18) reverse-phase column employing acetonitrile and water (20:80) as the mobile phase reduces the analysis time without affecting either resolution of the peak or the accuracy of caffeine and theobromine determination or achieving baseline resolution. Therefore, this method is ideally suited for rapid routine analysis of cocoa and its products.     

A simple and economic method for simultaneous determination of 11 antibiotics in manure by solid-phase extraction and high-performance liquid chromatography

Purpose

A simple and highly efficient economic method for the analysis of 11 antibacterial drugs including two tetracyclines, three quinolones, four sulfonamides, chloramphenicol and tylosin, in livestock manure, was developed using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC).

Materials and methods

The analytes were successively extracted by EDTA-McIlvaine solution and organic solvent mixture. The extracts were degreased with n-hexane and cleaned through SPE on a hydrophile-lipophile balance (HLB) cartridge. All compounds were determined on a C18 reverse phase column with gradient elution.

Results and discussion

Recoveries calculated from spiked samples of animal manures ranged from 62.65 to 99.16 % for 11 antibiotics with relative standard deviations of less than 10.0 %. Limits of detection ranged from 0.1 to 1.9 μg kg^?1, and limits of quantification ranged from 0.3 to 5.9 μg kg^?1.

Conclusions

The results show that SPE-HPLC is an inexpensive and practical method for rapid detection of multiple antibiotics in animal manure.

     

Analyses of glycolipids from fish, shellfish, and sea snake lipids by high-performance liquid chromatography.

To determine the existence of glycolipids (neutral glycosphingolipid and glycoglycerolipid) in sea snake, round frigate mackerel, sardine, sea urchin, and abalone, we performed silica gel chromatography and high-performance liquid chromatography (HPLC) using an Aquasil-SS column and a C(8)-reversed phase silica gel column. HPLC with a UV absorption detector was used to analyze neutral glycosphingolipid. These chromatograms showed typical peaks in round frigate mackerel lipid, in sea snake crude fat, in abalone intestine lipid, and in sea urchin intestine lipid. UV-HPLC was also used to analyze glycoglycerolipid. These chromatograms indicated a large peak in round frigate mackerel lipid and a small peak in purified sardine oil. In addition, we observed the same peaks in the glycolipid fraction of round frigate mackerel muscle lipids and sea snake crude fat using a differential refractometer detector. The results of this study suggest that the peaks are neutral glycosphingolipid or glycoglycerolipid and that neutral glycosphingolipid and glycoglycerolipid may have specific physiological functions in each living creature.     

Rapid isolation and purification of 1-cyano-2-hydroxy-3-butene (crambene) from Crambe abyssinica seed meal using immiscible solvent extraction and high-performance liquid chromatography

1-Cyano-2-hydroxy-3-butene (crambene) is a nitrile found in cruciferous vegetables that causes significant upregulation of quinone reductase and glutathione S-transferases in vivo and in vitro, making it a likely candidate as a cancer chemopreventive compound. To investigate further the putative anticarcinogenic mechanisms of crambene, a compound of the highest possible purity is vital. Therefore, a rapid and effective method of purification of crambene is necessary to continue studies of its beneficial health effects. A rapid method to isolate and purify natural crambene from either Crambe abyssinica (crambe) seed or commercially processed crambe seed meal was developed using immiscible solvent extraction followed by high-performance liquid chromatography. Use of this methodology eliminated the need for time-consuming and relatively inefficient column chromatography, improved extraction efficiency, and resulted in higher purity than previously used methodologies. Elimination of trace amounts of fatty acid residues, unachievable with previous methodologies, also was accomplished.     

Improved liquid chromatographic determination of riboflavin in milk and dairy products

Reported here is a simple liquid chromatographic (LC) method for the determination of riboflavin in milk (liquid, evaporated, and dry), yogurt, and cheese. The method involves passing liquid samples or filtrates of semisolid and solid samples through a C18 cartridge. Retained riboflavin is then eluted with an aliquot of 50% methanol in 0.02M acetate buffer of pH 4. A volume of the eluate is injected into the LC system consisting of a C18 column, a solvent of water-methanol-acetic acid (65 + 35 + 0.1, v/v) with a flow rate of 1 mL/min, and a UV detector set at 270 nm. The method is precise and accurate and compares favorably with the present AOAC method. Moreover, it involves fewer sample preparation steps and has a total analysis time of less than 1 h.     

Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.     

Comparison of solvent extraction and microwave extraction for release of dimethyl sulfide from cereals and canola

Natural levels of dimethyl sulfide (DMS) in newly harvested wheat, barley, paddy, and canola were determined by gas chromatography using a flame photometric detector in sulfur mode. The two methods involved determination of DMS in the headspace of cereal or oilseed samples (1) after extraction with microwaves and (2) after a traditional approach using 25% KBr solution. Quantitative results from each method were similar, and therefore both methods are suitable for the determination of DMS in grains and oilseeds. However, the microwave procedure has several advantages; for example, results are obtained very quickly, and only a small amount of sample is required.     

Analysis of polar lipids in the serum from rats fed shiitake by liquid chromatography-mass spectrometry/mass spectrometry

Consumption of a shiitake mushroom diet has been reported to have effects on serum phospholipids. However, much less is known about the effect on serum polar lipids including lysophospholipids and free fatty acids. In the present study, the effects of a shiitake diet were evaluated on the basis of identification and quantification of individual polar lipid components in rat serum using liquid chromatography-mass spectrometry/mass spectrometry. By comparison with standards and published data, 50 lysophospholipids and 32 free fatty acids were identified, and the concentrations of 27 polar lipids in rat serum were determined. Shiitake diets decreased the levels of all individual polar lipid components in the serum of male rat. The total level of serum polar lipids in males fed 4% shiitake diets (1365.71 mol/L) was significantly lower than that of the control (2270.26 mol/L). However, shiitake diets did not significantly affect the levels of serum polar lipids in female rats.     

A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods

A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a high-performance liquid chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization. Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods, and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid phase extraction column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by MS, (13)C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the polyglutamate forms of 5-MTHFA to the monoglutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver), the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these two procedures, it was observed that the mean relative standard error for 5-MTHFA was 12 (range, 2-27%) and 11% (range, 5-25%) for the HPLC and MS methods, respectively. FA was detected in only three of the samples, and the values obtained for it by either method were similar. This is the first paper that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrixes. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrixes.     

Multiresidue method for the analysis of pesticide residues in fruits and vegetables by accelerated solvent extraction and capillary gas chromatography

An analytical procedure using accelerated solvent extraction and capillary gas chromatography with electron capture and flame photometric detections was developed to simultaneously determine residues of different pesticides in fruits and vegetables. Single laboratory validation of the method was carried out for 28 compounds selected from eight pesticide classes, in blank and fortified samples of fresh pear, cantaloupe, white potato, and cabbage. The method had to meet specific established validation criteria for regulatory purposes applicable to our laboratory. At each of the two fortification levels studied, 24 of the 28 pesticides gave recoveries of more than 70% with a coefficient of variation of less than 10%. With respect to existing procedures, the method showed acceptable limits of detection (from 0.0019 to 0.14 microg/g depending on the pesticide and matrix) while minimizing environmental concerns, time, and labor.     

Application of solid/liquid extraction for the gravimetric determination of lipids in royal jelly

Gravimetric lipid determination is a major parameter for the characterization and the authentication of royal jelly quality. A solid/liquid extraction was compared to the reference method, which is based on liquid/liquid extraction. The amount of royal jelly and the time of the extraction were optimized in comparison to the reference method. Boiling/rinsing ratio and spread of royal jelly onto the extraction thimble were identified as critical parameters, resulting in good accuracy and precision for the alternative method. Comparison of reproducibility and repeatability of both methods associated with gas chromatographic analysis of the composition of the extracted lipids showed no differences between the two methods. As the intra-laboratory validation tests were comparable to the reference method, while offering rapidity and a decrease in amount of solvent used, it was concluded that the proposed method should be used with no modification of quality criteria and norms established for royal jelly characterization.     

Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin

Pressurized liquid extraction (PLE) was used to extract anthocyanins from the freeze-dried skin of a highly pigmented red wine grape with six solvents at 50 degrees C, 10.1 MPa, and 3 x 5 min extraction cycles. Temperature (from 20 to 140 degrees C in 20 degrees C increments) effects on anthocyanin recovery by acidified water and acidified 60% methanol were also studied. Acidified methanol extracted the highest levels of total monoglucosides and total anthocyanins, whereas the solvent mixture (40:40:20:0.1 methanol/acetone/water HCl) extracted the highest levels of total phenolics and total acylated anthocyanins. Acidified water extracts obtained by PLE at 80-100 degrees C had the highest levels of total monoglucosides, total acylated anthocyanins, total anthocyanins, total phenolics, and ORAC values. Acidified methanol extracts obtained by PLE at 60 degrees C had the highest levels of total monoglucosides and total anthocyanins, whereas extracts obtained at 120 degrees C had the highest levels of total phenolics. High-temperature PLE (80-100 degrees C) using acidified water, an environmentally friendly solvent, was as effective as acidified 60% methanol in extracting anthocyanins from grape skins.