Zorbax ODS Column Research Articles

Rapid determination of abamectin in lettuce and cucumber by high-performance liquid chromatography

A rapid, sensitive and reliable method is presented for the determination of trace amounts of abamectin in lettuce and cucumber. Abamectin consists of ⩾80% avermectin B1a and ⩽20% avermectin B1b. Vegetables were extracted with ethyl acetate and the extract was purified by solid-phase extraction using Sep-Pak silica cartridges. The purified extracts were analysed by high-performance liquid chromatography with a 5-μm Zorbax ODS column and UV detection under isocratic conditions. The method yields recoveries for avermectin B1a and B1b of 76—109% in the 0.054—0.54 mg/kg range. The limit of detection of the method is 40 μ/kg each of avermectin B1a and B1b in vegetables.

An HPLC Method for the Determination of Theophylline Concentrations in Plasma from Both Healthy Rats and in Rats with Carbon Tetrachloride Induced Cirrhosis

Abstract An accurate and reproducible high performance liquid chromatographic assay for theophylline in rat plasma has been developed. After addition of ammonium sulfate to the samples, theophylline is extracted from the plasma into chloroform. Beta-hydroxyethyl theophylline is used as the internal standard and separation is achieved using a Zorbax ODS (C18) column with a gradient mobile phase system, consisting of 0.05 M sodium acetate and acetonitrile. Samples are quantitated by UV detection of the column effluent at 275 nm. The assay can accurately and precisely measure theophyliine concentrations in rat plasma ranging from 0.25 to 7.5 μ/ml. This procedure has been successfully applied to the analysis of rat plasma samples obtained from both healthy rats and rats with carbon tetrachloride induced cirrhosis.

Provitamin A activity of specialty fruit marketed in the United States

The provitamin A content (α-carotene, β-carotene and β-cryptoxanthin) was determined in 33 different specialty fruits that are primarily of tropical origin and common to the U.S. market. After saponification, carotenoids were extracted with hexane and quantitated by reverse-phase HPLC using a Zorbax ODS column and an isocratic solvent system of acetonitrile, methylene chloride + 0.001% triethylamine, and methanol (350:150:1). Recoveries, determined by standard addition before saponification, were in the range of 70–100% for the three carotenoids. Proximate analysis was also conducted. Variation in carotenoid content was noted at different sampling times and for different cultivars of some of the specialty fruit. Provitamin A activity reported as retinol equivalents varied from nondetectable levels in fruit such as atemoya to relatively high levels in tamarillo (208 ± 63 RE/ 100 g) and purple passion fruit (196 ± 13 RE/ 100 g). Since the fruits included in the study represent an expanding market, they could provide significant levels of vitamin A activity to U.S. consumers. This study provides information necessary to judge their contribution of vitamin A activity to the U.S. diet.

Automatic sugar analysis in the beet industry. Part I: Parameter optimization of a reversed phase HPLC carbohydrate determination

AbstractAn evaluation of the most commonly used HPLC system (reversed phase octadecyl sillica gel) was undertaken in order to determine the level of certain carbohydrates in molasses produced in the refining of sugar beet. Chromatographic parameters and purification operations prior to analysis are discussed in order to develop an analytical method permitting automation of sugar determination. A Zorbax ODS column (250 × 9.4 mm), water elution, and light scattering detection allow easy determination of glucose + fructose, sucrose, and raffinose in molasses using an internal standard (maltose).

PROVITAMIN A (ALPHA‐CAROTENE, BETA‐CAROTENE AND BETA‐CRYPTOXANTHIN) AND ASCORBIC ACID CONTENT OF JAPANESE AND AMERICAN PERSIMMONS

ABSTRACTProvitamin A (alpha‐carotene, beta‐carotene and beta‐cryptoxanthin) and ascorbic acid were determined in 14 cultivars of Japanese persimmons (Diospyros kaki) and one American persimmon (D. virginiana). The study included 14 varieties grown at the USDA Persimmon test plot at the Southeastern Fruit and Nut Tree Laboratory, Byron, GA and one commercial variety, Hachiya, produced in California and purchased locally. After saponification, carotenoids were extracted with hexane and quantitated by a reverse‐phase HPLC system using a Zorbax ODS column and an isocratic solvent system of acetonitrile, methylene chloride and methanol (350:150:1). The total and reduced ascorbic acid were determined using AOAC methods. Variation in carotenoid content and carotenoid profiles was noted among the cultivars. The provitamin A activity ranged from 17 RE/100 g in Aizumi Shiraza to 120 RE/100 g in Hana Gosho. Beta‐carotene was the predominate provitamin A isomer in 11 of the 15 cultivars with beta‐cryptoxanthin predominate in the rest. Total ascorbic acid ranged from 35 mg/100 g in Hachiya to 218 mg/100 g in Fuyu. Mean provitamin A (RE/100 g) and ascorbic acid (mg/100 g) levels were higher in nonastringent persimmons compared to astringent persimmons (p < 0.0001). The data indicate that the Japanese persimmon is a good source of provitamin A activity and an excellent source of ascorbic acid.

Leaf Xanthophyll content and composition in sun and shade determined by HPLC

As a part of our investigations to test the hypothesis that zeaxanthin formed by reversible de-epoxidation of violaxanthin serves to dissipate any excessive and potentially harmful excitation energy we determined the influence of light climate on the size of the xanthophyll cycle pool (violaxanthin + antheraxanthin + zeaxanthin) in leaves of a number of species of higher plants. The maximum amount of zeaxanthin that can be formed by de-epoxidation of violaxanthin and antheraxanthin is determined by the pool size of the xanthophyll cycle. To quantitate the individual leaf carotenoids a rapid, sensitive and accurate HPLC method was developed using a non-endcapped Zorbax ODS column, giving baseline separation of lutein and zeaxanthin as well as of other carotenoids and Chl a and b.The size of the xanthophyll cycle pool, both on a basis of light-intercepting leaf area and of light-harvesting chlorophyll, was ca. four times greater in sun-grown leaves of a group of ten sun tolerant species than in shade-grown leaves in a group of nine shade tolerant species. In contrast there were no marked or consistent differences between the two groups in the content of the other major leaf xanthophylls, lutein and neoxanthin. Also, in each of four species examined the xanthophyll pool size increased with an increase in the amount of light available during leaf development whereas there was little change in the content of the other xanthophylls. However, the α-carotene/β-carotene ratio decreased and little or no α-carotene was detected in sun-grown leaves. Among shade-grown leaves the α-carotene/β-carotene ratio was considerably higher in species deemed to be umbrophilic than in species deemed to be heliophilic.The percentage of the xanthophyll cycle pool present as violaxanthin (di-epoxy-zeaxanthin) at solar noon was 96-100% for shade-grown plants and 4-53% for sun-grown plants with zeaxanthin accounting for most of the balance. The percentage of zeaxanthin in leaves exposed to midday solar radiation was higher in those with low than in those with high photosynthetic capacity.The results are consistent with the hypothesis that the xanthophyll cycle is involved in the regulation of energy dissipation in the pigment bed, thereby preventing a buildup of excessive excitation energy at the reaction centers.

Liquid Chromatographic-Atomic Absorption Spectrophotometric Method for Determination of Methyl Mercury in Seafood: Collaborative Study

A previously developed method that uses a simplified sample preparation procedure and atomic absorption detection of liquid chromatographic eluates for the determination of methyl mercury in seafood has been collaboratively studied. The unique feature of the method involves the use of a specially designed interface for the generation of mercury vapor. Methyl mercury is isolated from the blended sample by chloroform elution from a diatomaceous earth-hydrochloric acid column. The organomercury compound is then extracted into a small volume of 0.01M sodium thiosulfate solution. An aliquot of this solution is injected onto a Zorbax ODS column and eluted with methanol-ammonium acetate solution (3 + 2), pH 5.7, containing 0.01% mercaptoethanol. Mercury is detected by flameless atomic absorption spectrophotometry using the interface. The samples analyzed in the study were unspiked swordfish, unspiked and spiked lobster, and unspiked and spiked tuna. The spiked samples contained methyl mercury both above and below the U.S. Food and Drug Administration guideline level of 1 microgram Hg/g. Reproducibility relative standard deviations ranged from 10.5% at 1 microgram Hg/g to 18.2% at about 0.1 microgram Hg/g. Accuracy, measured by comparison to reference values obtained by the Associate Referee, ranged from 94.4 to 99.6%. The method has been adopted official first action.

Modification of sturgeon protamines with succinic anhydride

The modification of the α- and e-NH2 groups of sturins A and B — protamines fromAcipenser guldenstadti — with succinic anhydride has been studied. Succinylation was carried out at 20°C, pH from 7.5 to 10.5, using a 50 to 180-fold excess of succinic anhydride. The modification products were separated by reversed-phase HPLC on a Zorbax ODS column. A linear relationship has been established between the retention times of the succinyl derivatives and the number of succinyl groups introduced. The influence of the pH and of the excess of succinic anhydride on the modification of the sturins has been studied by the method of experimental planning and mathematical modeling. The optimum conditions have been found for the succinylation of the proteins: a 150- to 180-fold excess of succinic anhydride and pH values of 9.8–10.2 for sturin A and of 8.4–9.4 for sturin B.

Separation and quantitation of free cholesterol and cholesteryl esters in a macrophage cell line by high-performance liquid chromatography

A method for the direct high-performance liquid chromatographic (HPLC) determination of free cholesterol and the individual cholesteryl esters in cell culture experiments is described. The murine macrophage-like J774 cell line was loaded with cholesterol by incubation with low-density lipoproteins. After extraction of the cellular lipids with hexane-isopropanol (3:2, v/v), the cholesteryl esters were identified and quantified by isocratic HPLC. Unesterified cholesterol and its esters were eluted with acetonitrile-isopropanol (50:50, v/v) on a Zorbax ODS column within 25 min and detected at 210 nm. Cholesteryl heptadecanoate was used as an internal standard. The detection response is linear in the analytical range of interest; the overall coefficients of variation are less than 8% and the detection limit is between 50 and 150 ng. The results demonstrate that HPLC is suitable for the determination of cellular cholesteryl ester profiles and could usefully contribute to the understanding of the mechanism of foam cell formation during the development of atherosclerosis. This method can also be applied to all experimental systems involving the study of cholesteryl esters.

Separation of amino- and acetylamino-polycyclic aromatic hydrocarbons by reversed- and normal-phase high-performance liquid chromatography

In the field of chemical carcinogenesis, amino- and acetylamino-polycyclic aromatic hydrocarbons (PAHs) are among the most studied compounds. Many of these compounds have recently been detected in the environment. Presently, knowledge permitting predictions of the high-performance liquid chromatographic (HPLC) retention order of amino- and acetylamino-PAHs, particularly among their geometric isomers is lacking. In order to obtain a better understanding of the separation of these types of compounds, we have studied the separation of a series of structurally related amino- and acetylamino-PAHs derived from naphthalene, phenanthrene, anthracene, pyrene, benz[ a]anthracene, benzo[ a]pyrene, and benzo[ e]pyrene by using reversed-phase and normal-phase HPLC columns of different types (monomeric, polymeric, and chiral stationary phase). The results indicate: (i) Pirkle-type chiral stationary phase columns and the Zorbax SIL column can efficiently separate both the amino-PAHs and acetylamino-PAHs; (ii) in general, there was no correlation between retention time and molecular size; (iii) when acetylamino-PAHs were separated on the monomeric Zorbax ODS column, the isomer with the acetylamino group located at the carbon position of higher electron density has a shorter retention time; and (iv) separation of the parent PAHs was better than that of the amino-PAHs and acetylamino-PAHs. Our results thus may provide useful information for the analysis of amino-PAHs, particularly for distinguishing the geometric isomers of environmental samples.

Simultaneous determination of metoprolol and alpha-hydroxymetoprolol in human plasma and urine by liquid chromatography with a preliminary observation on metoprolol oxidation in Japanese subjects.

A simple, sensitive, and highly reliable liquid chromatographic method using fluorescence detection is described for the simultaneous determination of metoprolol and alpha-hydroxymetoprolol in plasma and urine. This method involves a single extraction of the compounds with the internal standard pindolol from alkalinized plasma or urine into dichloromethane. A reconstituted aliquot with a mobile phase is injected onto a reversed-phase, Zorbax ODS column, and the detection is achieved by the excitation and emission wavelengths at 230 and 300 nm, respectively. The assay is reproducible and precise for metoprolol and alpha-hydroxymetoprolol in both plasma and urine samples, as judged by a coefficient of variation of less than 9.2% at all concentrations examined. The standard curves for metoprolol and alpha-hydroxymetoprolol are linear over 10-200 ng/ml in plasma and over 0.5-10 micrograms/ml in urine. The lower detection limit is 2 ng/ml for each of the compounds in plasma using a 0.5-ml sample. Preliminary data on the oxidation polymorphism of metoprolol in Japanese subjects are reported using the current assay method. In 183 Japanese subjects no poor metabolizer of metoprolol has been identified so far.

High Performance Liquid Chromatographic Separation and Spectral Characterization of the Pigments in Turmeric and Annatto

ABSTRACTHigh performance liquid chromatographic separations for the determination of the pigments in the food colorants, annatto and turmeric, were developed. Chromatographic analysis time for the isocratic system was 10 min and 22 min for the gradient system. The isocratic separation employed a 25 cm × 4.6 mm i.d. Zorbax ODS column with a 58/42 (v/v) mixture of water/tetrahydrofuran, THF, at 1 mL/min. A gradient water/THF system using the same components was also developed to improve the resolution between the curcuminoids. Sample preparation consists of dilution and filtration of the extracts. The three major pigments in turmeric, identified from visible and fluorescence stop flow spectra, were curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Absorbance, excitation and emission maxima for the three curcuminoids plus the carotenoids, bixin and norbixin, were determined.

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 μm) and detected at 269 nm with a sensitivity limit of 1–5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1–13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.

Simultaneous measurement of eight corticosteroids by liquid chromatography, and application of the procedure to diagnosis of congenital adrenal hyperplasia.

We describe a liquid-chromatographic procedure for simultaneously determining eight steroids in serum. We used a Zorbax ODS column and a mobile phase of methanol/isopropanol/water (44/10/46, by vol), which well resolves the steroids cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, androstenedione, 17-hydroxyprogesterone, and progesterone, but not 11-deoxycorticosterone and androstenedione. Analytical recoveries of the steroids ranged from 89.27% to 99.58%. CVs were less than 10%. Prednisone and dexamethasone do not interfere. Using this method, we studied the serum steroid profiles of six patients with congenital adrenal hyperplasia (CAH) due to deficiency of 21-hydroxylase or 17-hydroxylase. Not only could we make a clearcut diagnosis and distinguish the subtle types of CAH, but also we could investigate clinical and biochemical variants of CAH. For example, we confirmed 17-hydroxylase deficiency in a patient who was normotensive with primary amenorrhea and streak gonads.

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.

Application of 3 alpha-hydroxysteroid dehydrogenase column to the determination of bile acids fractionated by high-performance liquid chromatography: advantage of pretreating human bile acids with Seppak C18 and piperidinohydroxypropyl Sephadex LH-20.

The analysis of bile acids in human bile was tried by high-performance liquid chromatography (HPLC). Bile acids in human bile were first prefractionated into free, glycine- and taurine-conjugated bile acids using a Seppak C18 cartridge and a piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20) column. Each fraction was then processed through a HPLC system consisting of a Zorbax ODS column and a 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) column. By these procedures the major 15 bile acids were clearly separated, and each bile acid of 10-125 ng was accurately analyzed. More than 400 times of analysis could be repeated on one 3 alpha-HSD column without loss of sensitivity. Thus the pretreatment through Seppak C18 and PHP-LH-20 made the HPLC analysis of human bile acids accurate and applicable to clinically obtained materials.

Reversed-phase high-performance liquid chromatographic separation of phenolic derivatives of benzo[ a]pyrene, benz[ a]anthracene, and chrysene with monomeric and polymeric C 18 columns

The separation of monohydroxylated derivatives (phenols) of benzo[ s]pyrene, benz[ a]anthracene, and chrysene was studied by reversed-phase high-performance liquid chromatography using a monomeric Zorbax ODS column and a polymeric Vydac C 18 column. The Vydac C 18 column resolved the phenols of each hydrocarbon with a wide range of retention times that the Zorbax ODS column. Four K-region phenols of benzo[ a]pyrene are either not separated or marginally separated on both monomeric and polymeric columns. Other K-region and non-K-region phenols of all three hydrocarbons can be separated by using the monomeric and polymeric columns in combination.

High Performance Liquid Chromatography of 2,5‐Dimethyl‐4‐Hydroxy‐3 (2H)‐Furanone in Pineapple and Grapefruit Juices

ABSTRACTA reversed‐phase HPLC method was developed for rapid analysis of 2,5‐dimethyl‐4‐hydroxy‐3(2H)‐furanone (DMHF), the compound responsible for the “burnt‐pineapple” flavor in pineapple juices. A Zorbax ODS column was used with a mobile phase of 0.05M sodium acetate (pH 4.0)/methanol (70:30); detection was by UV at 290 nm. Sample clean up was accomplished by solid‐phase extraction with C‐18 Sep‐Pak cartridges. DMHF contents ranged from 1.6 to 27.3 ppm from 10 fresh pineapple juices. Using this system, the production of DMHF was monitored in canned grapefruit juices during storage at varying temperatures.

Structures of Subsidiary Colors Isolated from Food Green No. 3 and Their Separation and Determination by High Performance Liquid Chromatography

Food Green No. 3 (Fast Green FCF, Colour Index No. 42053, FD & C Green No. 3) is prepeared by condensation of 1mol of p-hydroxy-o-sulfobenzaldehyde (HSBA) with 2mol of N-ethyl-N-benzylaniline sulfonic acid (EBASA). In this work, the isolation of two major lower-sulfonated subsidiary colors in Food Green No. 3, the structures of the subsidiary colors, and the separation and identification of the subsidiary colors by high performance liquid chromatography were investigated.Two subsidiary colors were isolated from Food Green No. 3 by thin layer chromatography. One of the subsidiary colors was identified as 3-[N-ethyl-N-[4-[(4-ethylaminophenyl) (4-hydroxy-2-sulfophenyl) methylene]-2, 5-cyclohexadienylidene] ammoniomethyl] benzenesulfonate (EA-subsidiary color) from the infrared (IR), nuclear magnetic resonance and FAB mass spectra (Figs. 2, 5 and 6). The other appear to be the color (EBA-subsidiary color) obtained by condensation of 1mol of N-ethyl-N-benzylaniline (EBA), 1mol of HSBA and 1mol of EBASA as judged from the IR and absorption spectra. The absorption maxima of EA- and EBA-subsidiary colors in 30v/v% methanol were at 610nm and 624nm, respectively (Table 1 and Fig. 7).Food Green No. 3 and EA- and EBA-subsidiary colors could be simultaneously separated by reversed phase high performance liquid chromatography on a Zorbax ODS column with 0.5% ammonium carbonate solution-methanol as the eluant (J. Food Hyg. Soc. Japan 27, 30 (1986)), with detection at 624nm for Food Green No. 3 and EBA-subsidiary color and 610nm for EA-subsidiary color. The retention times of Food Green No. 3, and EA- and EBA-subsidiary colors were about 6min, 8min and 10min, respectively (Fig. 8). Linear calibration curves were obtained in the range of 10-100ppm (50-500ng) for Food Green No. 3, and EA- and EBA-subsidiary colors (Figs. 9 and 10). The minimum detectable amount was 5ng (1ppm, 5μl) for Food Green No. 3 and EBA-subsidiary color, and 25ng (5ppm, 5μl) for EA-subsidiary color. The recoveries of EA- and EBA-subsidiary colors added to pure Fast Green FCF (free of subsidiary colors) at levels of 1-10% were 98.4-101. 8% and 98.4-102.1%, respectively (Table 2).EA- and EBA-subsidiary colors in six commercial samples of Food Green No. 3 were analyzed by this method. EA-subsidiary color content was determined to be 0.4-5.7%, and EBA-subsidiary color was detected at levels of 0.2-3.5% (Table 3 and Fig. 11). This method is considered to be suitable for the analysis of EA- and EBA-subsidiary colors in Food Green No. 3.

Rapid and Simultaneous Analysis of Hippuric and Benzoic Acids in Fermented Milk or Raw Milk by High Performance Liquid Chromatography

Benzoic acid (BA) of natural origin in fermented milk is known to be derived from hippuric acid (HA) in raw milk. Since it is forbidden to fortify fermented milk with BA, BA in fermented and HA in raw milk should be precisely determined in order to assess the source of BA.Our method of simultaneous analysis for both organic acids consists of extracting with acetone, making the extract alkaline with sodium bicarbonate solution, concentrating the mixture, and injecting a sample into a high performance liquid chromatography (HPLC) apparatus. HPLC was performed on a Zorbax ODS column, with 2% acetic acid-acetonitrile (78:22) as the mobile phase and o-methylhippuric acid as an internal standard. The detection limits of BA and HA were each 1ng, and the average recoveries of BA and HA added to milk at levels of 5-40ppm were not less than 98%.