Classification of octadecyl-bonded liquid chromatography columns

The differences in performance characteristics exhibited by columns available under the same octadecyl (C-18) label cause difficulties for the authors and users of official methods. This paper presents a simple scheme for classifying C-18 columns on the basis of the 2 predominant reverse phase retention mechanisms-hydrophobic and silanophilic interactions, which are dependent on the hydrocarbon coverage and the unreacted silanol sites, respectively. Hydrophobic interactions are represented by the ratio of anthracene k:benzene k in an acetonitrile-water (65 + 35) mobile phase. The unreacted silanols are represented by the N,N-diethyltoluamide k:anthracene k ratio with acetonitrile as the mobile phase. Column efficiency, which is largely dependent on particle size and packing technique, is included as a third classification criterion. Twelve brands of C-18 packings were subjected to this test scheme and qualitatively classified into 3 major groups on the basis of 3 criteria:hydrophobicity index, free silanol index, and efficiency. This scheme is intended as an aid in the selection of similar or equivalent columns and in defining the type of column suitable for an analytical method.     

Separation of rotenoids and the determination of rotenone in pesticide formulations by high-performance liquid chromatography.

The rotenoids deguelin, B-dihydrorotenone, dehydrorotenone, rotenone, 6alpha beta, 12alpha beta-rotenolone, and tephrosin were chromatographed on 8-12 mum silica. A mobile phase of chloroform-isooctane (35+65) pumped at a flow rate of 1 ml/min through a 30 cm column was used and the absorbance of the eluate was monitored at 294 nm. Rotenone, B-dihydrorotenone, deguelin, and dehydrorotenone are completely resolved while 6alpha beta, 12alpha beta-rotenolone and tephrosin chromatograph as one peak. This method has potential as a preparative separation technique for rotenoids. Also described is a procedure to quantitatively measure rotenone in pesticide formulations. Samples were extracted with chloroform and chromatographed at a flow of 2.5 ml/min. The method is rapid (rotenone is eluted in 12 min) and reproducible.     

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.     

Monitoring aldehyde production during frying by reversed-phase liquid chromatography.

Acrolein (2-propenal) and other low molecular weight aldehydes (LMWAs) formed by degradation of the frying medium (triglycerides) were monitored by liquid chromatography (LC) during preparation of fried items. LMWA contents of coatings from codfish and of doughnuts and their volatiles that codistill with steam are monitored by trapping the vapors and distillate from the food matrix in a 2,4-dinitrophenylhydrazine solution. The resulting hydrazones are partitioned from the aqueous phase, first into isooctane and then into acetonitrile for LC analysis. The hydrazones are separated and quantified on a C18 reversed-phase column with acetonitrile-water as the mobile phase. LMWAs are confirmed by gas chromatography/mass spectrometry. No difference was found in LMWA content in coatings from fish fillets fried at 182 or 204 degrees C. Cake doughnuts were higher in acrolein content than yeast-raised doughnuts prepared under similar conditions. Freshness of the frying medium, frying time, and batch size did not seem to influence LMWA production from doughnuts. Results indicated that most of the LMWAs formed codistilled with steam during frying rather than remaining with the food item.     

Enantiomeric resolution of chiral pesticides by high-performance liquid chromatography

Successful enantiomeric separation of 10 chiral pesticides by high-performance liquid chromatography (HPLC) using cellulose-tris(3,5-dimethylphenylcarbamate) (CDMPC) chiral stationary phase (CSP) was performed. The mobile phase was n-hexane modified by ethanol, propanol, 2-propanol (IPA), butanol, or isobutanol. The effects of mobile phase composition and column temperature on the separation were investigated. Baseline separation was obtained with ethofumesate, fluroxypyr-meptyl, malathion, benalaxyl, diclofop-methyl, methamidophos, vinclozolin, and lactofen, whereas near baseline separation was obtained with profenofos and acetochlor. Butanol was the best modifier for benalaxyl; isobutanol was the best modifier for lactofen, malathion, diclofop-methyl, and ethofumesate; and IPA was the best modifier for the other five. Better separations were not always at low temperature. The elution orders of the eluting enantiomers were determined by a circular dichroism (CD) detector. The quantitative analysis methods for the enantiomers of ethofumesate, benalaxyl, and diclofop-methyl were established. Validation parameters include linearity, precision, and limit of detection (LOD). The enantiomeric residual analysis procedures in soil and water samples were also developed using acetone extraction and C(18) solid phase extraction. The methods were reliable for residual analysis of the enantiomers.     

Analysis of phenolic acids in barley by high-performance liquid chromatography

Phenolic acids from 30 barley varieties (combination of hulled/hulless/two-row/six-row/regular/waxy) were investigated by HPLC following four different sample treatments: (a) simple hot water extraction, (b) extraction after acid hydrolysis, (c) acid plus alpha-amylase hydrolysis, and (d) acid plus alpha-amylase plus cellulase hydrolysis treatments. The benzoic acid (p-hydroxybenzoic, vanillic, and protocatechuic acids) and cinnamic acid derivatives (coumaric, caffeic, ferulic, and chlorogenic acids) were identified, and some of the phenolic acids were quantified after each above-mentioned treatment. The data indicated that a combination of sequential acid, alpha-amylase, and cellulase hydrolysis treatments might be applicable for release of more phenolic acids from barley.     

Quantification of the group B soyasaponins by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins alphag, betag, and betaa, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 micromol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 micromol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 micromol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.     

Steviol quantification at the picomole level by high-performance liquid chromatography

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.     

Determination of deltamethrin in cattle dipping baths by high-performance liquid chromatography

Deltamethrin (S)-alpha-cyano-3-phenoxybenzyl) (1R,3R)-3-(2, 2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylate is classified as a pyrethroid pesticide that is largely used as an acaricide and scabicide. For bovines, especially, the treatment is done with the aid of dipping baths of the pyrethroid solution. Analytical control of the concentration of deltamethrin in these baths must be done periodically in order to guarantee treatment efficacy. In the proposed procedure, the sample is prepared by centrifugation followed by filtration and measurement by high-performance liquid chromatography (HPLC) with spectrophotometric detection at 275 nm. Separation is done in a Nucleosil C-18 column with acetonitrile-water as the mobile phase. A calibration curve was constructed with external standards, and a detection limit of 0.2 mg L(-)(1) was obtained. In the samples analyzed, only ca. 20% of the total deltamethrin content was found in the solution. The results obtained demonstrate the potential of the described procedure for the determination of deltamethrin in animal baths.     

Quantitative high-performance liquid chromatography analyses of flavonoids in Australian Eucalyptus honeys

Flavonoids of nine Australian monofloral Eucalyptus honeys have been analyzed and related to their botanical origins. The mean content of total flavonoids varied from 1.90 mg/100 g of honey for stringybark (E. globoidia) honey to 8.15 mg/100 g of honey for narrow-leaved ironbark (E. crebra) honey, suggesting that species-specific differences occur quantitatively among these Eucalyptus honeys. All of the honey samples analyzed in this study have a common flavonoid profile comprising tricetin (5,7,3',4',5'-pentahydroxyflavone), quercetin (3,5,7,3',4'-pentahydroxyflavone), and luteolin (5,7,3',4'-tetrahydroxyflavone), which, together with myricetin (3,5,7,3',4',5'-hexahydroxyflavone) and kaempferol (3,5,7,4'-tetrahydroxyflavone), were previously suggested as floral markers for European Eucalyptus honeys. Thus, flavonoid analysis could be used as an objective method for the authentication of the botanical origin of Eucalyptus honeys. Moreover, species-specific differences can also be found in the composition of honey flavonoid profiles. Among these honeys, bloodwood (E. intermedia) honey contains myricetin and tricetin as the main flavonoid compounds, whereas there is no myricetin detected in yapunyah (E. ochrophloia), narrow-leaved ironbark (E. crebra), and black box (E. largiflorens) honeys. Instead, these types of Eucalyptus honeys may contain tricetin, quercetin, and/or luteolin as their main flavonoid compounds. Compared to honeys from other geographical origins, the absence or minor presence of propolis-derived flavonoids such as pinobanksin, pinocembrin, and chrysin in Australian honeys is significant. In conclusion, these results demonstrate that a common flavonoid profile exists for all of the Eucalyptus honeys, regardless of their geographical origins; the individual species-specific floral types of Eucalyptus honey so common in Australia could be possibly differentiated by their flavonoid profile differences, either qualitatively or quantitatively or both.     

Determination of seven organosulfur compounds in garlic by high-performance liquid chromatography

The properties of garlic (Allium sativum L.) are attributed to organosulfur compounds. Although these compounds change during cultivation and storage, there is no report of their simultaneous analysis. Here, a newly developed analytical method with a rapid and simple sample preparation to determine four sulfoxides and three gamma-glutamyl peptides in garlic is reported. All garlic samples were simply extracted with 90% methanol solution containing 0.01 N hydrochloric acid and prepared for analysis. Alliin, isoalliin, methiin, cycloalliin, and gamma-l-glutamyl-S-methyl-l-cysteine were determined by normal-phase HPLC using an aminopropyl-bonded column. gamma-l-Glutamyl-S-(2-propenyl)-l-cysteine and gamma-l-glutamyl-S-(trans-1-propenyl)-l-cysteine were separated on an octadecylsilane column. The overall recoveries were 97.1-102.3%, and the relative standard deviation values of intra- and interday precision were lower than 2.6 and 4.6%, respectively. This newly developed method offers some advantages over the currently accepted techniques including specificity, speed, and ease of use and would be useful for chemical and biological studies of garlic and its preparations.     

Separation of Delta5- and Delta7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.     

Measurement of food flavonoids by high-performance liquid chromatography: A review

The flavonoids are plant polyphenols found frequently in fruits, vegetables, and grains. Divided into several subclasses, they include the anthocyanidins, pigments chiefly responsible for the red and blue colors in fruits, fruit juices, wines, and flowers; the catechins, concentrated in tea; the flavanones and flavanone glycosides, found in citrus and honey; and the flavones, flavonols, and flavonol glycosides, found in tea, fruits, vegetables, and honey. Known for their hydrogen-donating antioxidant activity as well as their ability to complex divalent transition metal cations, flavonoids are propitious to human health. Computer-controlled high-performance liquid chromatography (HPLC) has become the analytical method of choice. Many systems have been developed for the detection and quantification of flavonoids across one, two, or three subclasses. A summary of the various HPLC and sample preparation methods that have been employed to quantify individual flavonoids within a subclass or across several subclasses are tabulated in this review.     

Determination of dexamethasone in bovine liver by chemiluminescence high-performance liquid chromatography.

A new method for the determination of dexamethasone (9alpha-fluoro-11beta,17alpha,21-trihydroxy-16alpha -methylpregna-1, 4-diene-3,20-dione) in bovine liver was developed. This new liquid-liquid extraction method comprises the addition of sodium hydroxide to the tissue sample followed by extraction with ethyl acetate. After centrifugation, the extract is evaporated to dryness and the residue dissolved in acetonitrile. The cleaning of the fat is performed with n-hexane, and the acetonitrile layer is evaporated. Analysis of the extracts is performed using high-performance liquid chromatography with chemiluminescence detection employing luminol as CL reagent. A series of recovery curves performed at spiking levels of 50, 30, 10, 5, and 2.5 ppb show that at least 80% of DEX can be recovered from liver and that the chemiluminescence detection yields satisfactory results with respect to sensitivity (LOD 0.2 ppb), reproducibility (CV% 10.7) and repeatability (CV% 6.2-8.9).     

Determination of carotenoid stereoisomers in commercial dietary supplements by high-performance liquid chromatography

A method for the determination of beta-carotene, lutein, and zeaxanthin including their cis-isomers and alpha-carotene in commercial dietary supplements by HPLC has been developed. The study comprises 11 oral dosage forms, including 9 soft gelatin capsules, 1 dragée, and 1 effervescent tablet formulation. The capsule content was extracted with an acetone-hexane mixture, and the gelatin shell was digested with papain to release carotenoids that had migrated into the coat. Sample preparation for tablets and dragées was carried out as described for the capsule content. Extraction recoveries exemplified for all-trans-beta-carotene and all-trans-lutein were 95 +/- 5% and 93 +/- 2%, and 95 +/- 2% and 79 +/- 5% after enzymatic treatment, respectively. Apart from all-trans-beta-carotene, its 9-cis- and 13-cis-isomers were detected in all samples, whereas no evidence for cis-isomerization of lutein and zeaxanthin could be obtained. Migration of carotenoids into the shells was only observed in the case of beta-carotene. With the exception of one preparation, the beta-carotene contents determined exceeded the dosage specified on the label by up to 48%, which results from stability overages necessary to compensate for losses during storage.     

Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of anthocyanidins from berries and red wine is described. Delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin contents of bilberry (Vaccinium myrtillus), black currant (Ribes nigrum), strawberry (Fragaria ananassa cv. Jonsok), and a Cabernet sauvignon (Vitis vinifera) red wine were determined. The aglycon forms of the anthocyanins present in the samples were revealed by acid hydrolysis. A reversed phase analytical column was employed to separate the anthocyanidins before identification by diode array detection. The suitability of the method was tested by determining the recovery (95-102% as aglycons and 69-104% from glycosides) for each anthocyanidin. Method repeatability was tested by charting the total aglycon content of two samples over a period of 14 analyses and determining the coefficients of variation (1.41% for bilberry and 2.56% for in-house reference material). The method developed proved thus to be effective for reliable determination of anthocyanidins from freeze-dried berry samples and red wine. The total anthocyanidin content of the tested samples was as follows: in-house reference material, 447 +/- 8 mg/100 g; strawberry, 23.8 +/- 0.4 mg/100 g; black currant, 135 +/- 3 mg/100 g; bilberry, 360 +/- 3 mg/100 g; and Cabernet sauvignon red wine, 26.1 +/- 0.1 mg/100 mL.     

Separation and identification of nine penicillins by reverse phase liquid chromatography

A simple and rapid high performance liquid chromatographic method was developed for the separation and identification of amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, nafcillin, penicillin G potassium, and penicillin V potassium. The antibiotics were separated at ambient temperature on a Chromegabond 10 microns C18 column with acetonitrile-methanol-0. 01M potassium dihydrogen phosphate buffer, pH 4.7 (19 + 11 + 70), at 1 mL/min. A variable wavelength detector set at 225 nm, 0.16 AUFS , and a recorder set at 0.25 cm/min were used for the detection. Individual antibiotics and their mixtures were dissolved in the mobile phase and injected into the chromatograph through a 20 microL injection loop. Baseline separation was observed for virtually all 9 antibiotics. The entire mixture was resolved in less than 30 min. The method was sensitive, reproducible, and applicable to the qualitative analysis of commercial dosage forms.     

高效液相色谱法测定发酵醪中的γ-氨基丁酸

建立了高效液相色谱测定发酵醪中γ-氨基丁酸(γ-aminobutyric acid,GABA)的方法。采用7%(V/V)乙酸水溶液对发酵醪进行预处理,以异硫氰酸苯酯为衍生剂,用反相C18柱为分离柱,柱温27°C,阶段洗脱,在254 nm下进行检测。结果表明,发酵醪中的GABA获得了很好的分离,GABA在0~1.5 mmol/L内线性相关性好,其线性方程为y=14106.5713x-258.2493(r=0.9993)。最小检测浓度为0.5μmol/L(RSD≤10%),组间样品测定相对误差为3.571%,加样平均回收率达到了99.038%。所建立的方法稳定、灵敏、重现性好,可用于测定发酵醪中的GABA。     

Rapid analysis of cholesterol-elevating compounds in coffee brews by off-line high-performance liquid chromatography/high-resolution gas chromatography

A new method is proposed to analyze the cholesterol-elevating cafestol and kahweol which allows their rapid and reliable determination in different coffee brews. The method involves the preseparation of the sample by high-performance liquid chromatography, the collection of the selected fraction, and its subsequent analysis by high-resolution gas chromatography using a programmed temperature vaporizer operated in the split mode as sampling system. Under the experimental conditions investigated, recoveries as high as 87% (cafestol) and 94% (kahweol) were achieved while detection limits equal to 0.06 and 0.04 ppm for cafestol and kahweol, respectively, were obtained. Examples are given comparing levels of cafestol and kahweol resulting from the same ground roasted coffee by different brewing methods, which show the lowest values for brews prepared from coffee bags.