Determination of ergosterol levels in barley and malt varieties in the Czech Republic via HPLC

Ergosterol is considered to be a suitable indicator of mold infestation in barley and malt. In this study ergosterol levels in different varieties of barley and malt produced in the Czech Republic were determined. A modified high-performance liquid chromatography (HPLC) method was statistically processed, validated (Effivalidation program), and applied to 124 samples of barley and malt. Ergosterol was isolated by extraction and saponification, and the quantification was performed using HPLC with diode array detection. The content of ergosterol ranged between the limit of detection (LOD) and 36.3 mg/kg in barley and between the LOD and 131.1 mg/kg in malt. Ergosterol is presumably connected with metabolites generated when barley grain is attacked by pathogens, and such barley often shows a high overfoaming (gushing) value. However, it was found that the content of ergosterol does not correlate with the degree of beer gushing.     

Determination of ergosterol in ganoderma spore lipid from the germinating spores of Ganoderma lucidum by high-performance liquid chromatography

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the separation and determination of free ergosterol in ganoderma spore lipid (GSL) extracted from the sporoderm-broken germinating spores of Ganoderma lucidum. Sodium hydroxide in methanol was added for the hydrolysis of ergosteryl esters to determine the total content of ergosterol in GSL by HPLC. A 0.04 M concentration of sodium hydroxide in reaction mixtures was appropriate for the complete hydrolysis of ergosteryl esters without a significant loss of ergosterol during saponification. In addition, the ergosterol content in four commercial GSL softgel supplements from four different firms was determined. The results showed that the ergosterol content in these samples had significant differences. Ergosterol content may be a suitable marker for evaluating the quality of GSL products.     

Analysis of ergosterol in single kernel and ground grain by gas chromatography-mass spectrometry

A method for analyzing ergosterol in a single kernel and ground barley and wheat was developed using gas chromatography-mass spectrometry (GC-MS). Samples were saponified in methanolic KOH. Ergosterol was extracted by "one step" hexane extraction and subsequently silylated by N-trimethylsilylimidazole/trimethylchlorosilane (TMSI/TMCS) reagent at room temperature. The recoveries of ergosterol from ground barley were 96.6, 97.1, 97.1, 88.5, and 90.3% at the levels of 0.2, 1, 5, 10, and 20 microg/g (ppm), respectively. The recoveries from a single kernel were between 93.0 and 95.9%. The precision (coefficient of variance) of the method was in the range 0.8-12.3%. The method detection limit (MDL) and the method quantification limit (MQL) were 18.5 and 55.6 ng/g (ppb), respectively. The ergosterol analysis method developed can be used to handle 80 samples daily by one person, making it suitable for screening cereal cultivars for resistance to fungal infection. The ability for detecting low levels of ergosterol in a single kernel provides a tool to investigate early fungal invasion and to study mechanisms of resistance to fungal diseases.     

Modified microwave-assisted extraction of ergosterol for measuring fungal biomass in grain cultures

Ergosterol is a measure for fungal biomass. The recovery rates using a previously described microwave-assisted-extraction (MAE) method for ergosterol analysis tended to be low for grain cultures (pure culture in sterilized 40% moisture content grain) inoculated with Fusarium graminearum . An improved MAE method for measuring ergosterol in grain cultures was developed and compared. Modification to the original MAE included alterations in duration of microwave exposure and extraction solvents. Four autoclaved grains (wheat, rice, barley, and corn) were inoculated with F. graminearum or spiked with ergosterol at concentrations from 0.88 to 100 microg/g and extracted with both methods. The ergosterol recovery rates were significantly different (p < 0.05) for the two methods in assaying both the spiked and grain culture samples. The modified method provided greater recovery rates than the previously reported MAE method for the spiked samples and F. graminearum grain cultures.     

High pressure liquid chromatographic determination of polynuclear aromatic hydrocarbons in oysters.

A high pressure liquid chromatographic (HPLC) procedure is described for determining 13 polynuclear aromatic hydrocarbon (PNA) compounds in oysters at the 2 ppb level. These compounds are extracted from shellfish with acetonitrile and partitioned into petroleum ether; the petroleum ether is removed and the residue is saponified. The aromatic compounds are isolated by passing the saponifeid residue through silica gel and further purified and fractionated by muStyragel gel permeation chromatography. The in-ividual PNAs are then quantitatively determined by using a reverse phase HPLC column coupled to fluorescence, spectrophotometric, and 254 nm absorbance detectors in series. Recoveries from spiked samples generally were greater than 80%.     

Seasonal variation of provitamin D2 and vitamin D2 in perennial ryegrass (Lolium perenne L.)

Ergosterol (provitamin D(2)) is converted to vitamin D(2) in grass by exposure to UV light. Six varieties of perennial ryegrass (Lolium perenne L.) were harvested four times during the season, and the contents of vitamin D(2) and ergosterol were analyzed by a sensitive and selective liquid chromatography tandem mass spectrometry method. Weather factors were recorded, and a principal component analysis was performed to study which factors were important for the formation of vitamin D(2). The results suggest that a combination of weather factors is involved and that the contents of ergosterol and vitamin D(2) change more than a factor of 10 during the season. These results demonstrate that grass potentially can be a significant source of vitamin D for grazing animals and animals fed on silage and hay.     

Screening method for vitamins A and D in fortified skim milk, chocolate milk, and vitamin D liquid concentrates

Vitamin A was determined in fortified chocolate milk and skim milk; vitamin D was determined in fortified chocolate milk, skim milk, and vitamin D concentrates, using reverse phase high pressure liquid chromatography (HPLC). The sample is saponified, extracted with hexane, and chromatographed in an HPLC system on a 10 micron Vydac TP reverse phase C18 column, using acetonitrile-methanol (9+1) as the mobile phase. For 6 replicates, the recoveries of vitamins A and D, using this procedure, were 99 and 98%, respectively.     

High-pressure liquid chromatography of benzo(a) pyrene and benzo (ghi) perylene in oil-contaminated shellfish.

A high-pressure liquid chromatographic procedure is described for the determination of benzo(a) pyrene and benzo(ghi) perylene. These polynuclear aromatics are extracted with acetonitrile and partitioned into petroleum ether, the petroleum ether is removed, and the residue is saponified. The compounds are purified and isolated by passing the residue through a silica gel column and a high-pressure liquid chromatographic column, and detected by their ultraviolet absorption. Recoveries of standards through the procedure averaged 104%.     

Ergosterol Levels and Mould Colony Forming Units in Swedish Grains of Food and Feed Grade

Abstract

Although ergosterol is considered to be a suitable indicator of mould growth in cereal grains, there are few reference values available for Scandinavian conditions. We have determined the ergosterol levels in Swedish grain of different origins: cleaned food-grade wheat from a commercial mill, feed-grade cereals (oats and barley) with different odours and cereals (winter wheat, “American wheat”, triticale and rye) from various field trials conducted in south-central Sweden in 1990. Specific objectives were to elucidate the relationships between ergosterol levels and numbers of mould colony forming units (CFU) and between ergosterol and grain odour.

Ergosterol levels in the food-grade wheat ranged between 2.4 and 2.8 μg/g DW, and between 3.0 and 5.6 μg/g DW in the field trial cereals, while values in most of the feed grain samples ranged from 8–15 μg/g DW. The levels agree with other published data for European grains.

A positive correlation was found between numbers of colony-forming units and ergosterol concentration. The degree of correlation was higher when numbers of CFU were determined on dichloran-glycerol 18% agar with a low water activity (aw = 0.95) than on malt extract agar (aw = 0.99). There was no agreement between ergosterol levels and grain odour, since even samples described as having a fresh smell had high ergosterol levels. However, the highest level (33 μg/g DW) was found in a sample with a pronounced musty odour, and the lowest (1.1 μg/g DW) in a sample that smelled as if it had been heat damaged.     

Ergosterol content in ericaceous hair roots correlates with dark septate endophytes but not with ericoid mycorrhizal colonization

The relationship between ergosterol content in ericaceous hair roots and ericoid mycorrhizal (ErM) colonization versus dark septate endophytic (DSE) hyphal colonization was examined in a dwarf shrub-dominated subarctic mire in Northern Sweden. Ergosterol content in hair roots did not correlate with ErM colonization in corresponding root samples. However, a significant positive relationship was found between hair root DSE hyphal colonization and ergosterol content. This is the first study to demonstrate that ergosterol cannot be used as a colonization indicator for ErM in hair roots growing under natural conditions. It also suggests the possibility of using ergosterol as an estimate of DSE hyphal colonization in ericaceous dwarf shrubs. This study has implications for the interpretation of results in field studies where ergosterol was used as a sole proxy for ErM colonization.     

Determination of vitamin D3 in liquid multivitamin preparation, using reverse phase, solid phase extraction liquid chromatography

A method is described for the determination of vitamin D3 in a liquid multivitamin preparation by liquid chromatography. Samples are purified on a disposable reverse phase extraction (SPE) column with a mobile phase of methanol-2-propanol (97 + 3) and are analyzed on a Zorbax ODS (5 micron) column with an acetonitrile-2-propanol-water (90 + 8 + 2) solvent system. Vitamin D3 is completely resolved from other interfering compounds within approximately 21 min and is detected with a UV detector at 254 nm. A mean of 98.5% of theory with a coefficient of variation of 3.8% was found for determination of vitamin D3 in a commercial preparation.     

Liquid chromatography and fluorescence detection of vitamin A in animal feeds, finished feeds, and premixes

A simple liquid chromatographic method for vitamin A (retinol) in animal feeds is described. The feed is saponified, diluted to minimize interferences, and extracted into petroleum ether with a single step. The analysis is sensitive and specific with liquid chromatography and a fluorescence detector. The minimum level of detection is 15 ng/mL, which is equivalent to 10,000 units/lb vitamin A. The method includes a stable and reproducible standardization of vitamin A that is used to calibrate standard peak heights in terms of units retinol/mL. Guarantees of 10,000 units/lb up to premix levels can be analyzed with good recoveries and precision.     

Modified liquid chromatographic method for determination of gentian violet in animal feed

A modified liquid chromatographic method is described for the determination of Gentian Violet (GV) in animal feed. The reliable detection limit is 0.5 ng (reference standards), and 1 ppm GV was reliably determined in feed. The calibration curve was linear between 1 and 40 micrograms/mL. The method, developed in a study by the National Center for Toxicological Research, was modified to use methanol-water (9 + 1) instead of benzene-methanol as the eluting solution in the column cleanup. GV is extracted from feed with methanol-1N HCl (99 + 1), cleaned up on a Sephadex LH-20 column to remove any remaining interferences, separated on a Nova-Pak C18 column fitted with a precolumn filter, and determined at 588 nm. The identity of GV is confirmed by thin-layer chromatography (Rf = 0.47) by comparison with a reference standard. Average recoveries from 3 sets of 5 feed samples containing 2.5, 5.0, and 10.0 ppm GV were 115, 95, and 102%, respectively.     

Liquid chromatographic determination of vitamin D in milk and infant formula

Vitamin D2 or vitamin D3 is determined by liquid chromatography (LC) in milk and infant formula. Vitamin D is extracted from the saponified sample, passed through an amino-cyano LC cleanup column to remove major interferences, and quantitated using normal phase LC. Within-day precision is 4.5% relative standard deviation (RSD); the overall method RSD (reflecting technician-to-technician, day-to-day, and within-day variability) is 7.7%. Overspike recoveries averaged 97% for milk, 98% for milk-based infant formula, and 93% for soy-based infant formula. The performance of the method is compared with that of the official AOAC vitamin D method (rat bioassay). The method is applicable to the determination of vitamin D in milk and in the major milk- and soy-based infant formulas available in the United States. The method can quantitate (but not distinguish) either vitamin D2 or vitamin D3. The method is applicable to milk and infant formula samples containing between 100 and 1500 IU vitamin D/L. Sample throughput is between 4 and 8 replicates per day.     

Postcolumn derivatization method for determination of reducing and phosphorylated sugars in chicken by high performance liquid chromatography

A postcolumn derivatization method is described for determination of reducing sugars and phosphorylated reducing sugars from chicken meat and other foods using high-performance liquid chromatography (HPLC). Reducing sugars are extracted with ethanol/water, separated on a Kromasil amine-bonded column by isocratic analysis using acetonitrile/water as the mobile phase, and, after postcolumn reaction with tetrazolium blue, are determined by the resulting absorbance at 550 nm. Phosphorylated sugars are first dephosphorylated using alkaline phosphatase and then determined by the same method.     

Rapid liquid chromatographic determination of patulin in apple juice

A rapid method is described for the quantitative determination of patulin in apple juice. The mycotoxin is extracted from the sample with ethyl acetate and the extract is cleaned up by extraction with a sodium carbonate solution. Patulin is determined by reverse phase liquid chromatography using a muBondapak C18 column and a 254 nm ultraviolet detector. The lower detection limit in patulin standard solution is 0.32 ng and recovery is greater than 75%.     

Residue methodology for AMDRO fire ant insecticide (AC 217,300) in pasture grass and crops

Residue methodology is described for the determination of AC 217,300 residues in pasture grass and crop samples. After extraction and subsequent cleanup on an XAD-2 column, residues of AC 217,300 are determined by liquid chromatography (LC), using a reverse phase paired-ion chromatographic system and detection at 300 nm. The method has a validated limit of sensitivity of 0.05 ppm with corresponding control values for the commodities analyzed of less than 0.01 ppm. Apparent residues over 0.05 ppm can be confirmed by either gas chromatography with an electron capture detector (GC-EC) or gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI). The direct GC-NICI method circumvents the need for sample cleanup on the XAD-2 column, and offers a greatly simplified procedure that is useful for screening samples. Recoveries of AC 217,300 from the commodities analyzed have been satisfactory with all methods of analysis.     

Determination of reserpine and rescinnamine in Rauwolfia serpentina preparations by liquid chromatography with fluorescence detection

A method is presented for the determination of reserpine and rescinnamine in Rauwolfia serpentina powder or tablets by liquid chromatography (LC) with fluorescence detection. The sample is dispersed in CH3OH, 0.5N H2SO4 is added, and the mixture is extracted with five 30 mL portions of CHCl3. The extracts are separated from interfering materials on a Celite-0.1N NaOH column, and the eluates are collected in 50 mL CH3OH. After complete removal of the CHCl3, reserpine and rescinnamine are determined by liquid chromatography on a normal phase column with CH3OH as the mobile phase. Because reserpine and rescinnamine produce a single peak, chromatograms are obtained at different wavelengths. Reserpine is determined at an excitation wavelength of 280 nm and an emission wavelength of 360 nm. Rescinnamine is determined at an excitation wavelength of 330 nm and an emission wavelength of 435 nm. Recovery studies were conducted at 2 different levels to simulate 100 and 50 mg Rauwolfia serpentina tablets. Samples of Rauwolfia serpentina powders and tablets were also examined, and the results were compared with those obtained by the current AOAC official method. The proposed method is applicable to the analysis of ground composites and individual tablets.     

Analysis of free and esterified ergosterol in tomato products

A new extraction and chromatographic procedure to quantify free and esterified ergosterol in tomato products was devised. The extraction solution was composed of a dichloromethane/methanol mixture in a 2:1 (v/v) ratio. This extraction solvent allowed for higher ergosterol recovery from tomato products (an average of 25% more) compared to hexane, which is frequently employed for ergosterol extraction. Both free and esterified ergosterol were determined by HPLC reverse-phase chromatography employing a Nova-Pak C-18 column (300 x 3.9 mm), filled with 4 mm average particle size and a guard column of the same material. The elution was performed at a flow rate of 1 mL. min(-1) with a linear gradient of solvent A (methanol/water, 80:20, v/v) and solvent B (dichloromethane). The gradient, starting at sample injection, was from 0 to 50% B for 20 min for the free ergosterol analysis and additional 15 min at 50% B to analyze the ergosterol esters. This technique has proven to be more sensitive for ergosterol determination than other reported chromatographic procedures. Moreover, ergosterol esters, extracted from various fungal sources, separated well and were easily quantified.     

Liquid chromatographic determination of vitamin K1 trans- and cis-isomers in infant formula

The normal phase liquid chromatographic (LC) method for determination of trans- and cis-isomers of vitamin K1 (phylloquinone) in infant formula described here uses an Apex silica column, isocratic elution, and UV absorption detection at 254 nm. Vitamin K1 is extracted quantitatively from the product matrix by pretreating the as-fed liquid with concentrated ammonium hydroxide and methanol, and then extracting it with a 2:1 mixture of dichloromethane and isooctane. The extract is cleaned up by silica open-column chromatography and concentrated for LC analysis. For trans-vitamin K1, the method precision is less than or equal to 3.3% RSD (relative standard deviation), and the spike recovery is 98 +/- 4%. For cis-vitamin K1, the precision is less than or equal to 12% RSD, determined at levels near the detection limit, and the spike recovery is 95 +/- 9%. The detection limit is 0.3 ng for both isomers at signal/noise = 3.