Gradient Solvent System Research Articles

Microwave- and ultrasound-assisted extraction of vanillin and its quantification by high-performance liquid chromatography inVanilla planifolia

Microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and conventional extraction of vanillin and its quantification by HPLC in pods of Vanilla planifolia is described. A range of nonpolar to polar solvents were used for the extraction of vanillin employing MAE, UAE and conventional methods. Various extraction parameters such as nature of the solvent, solvent volume, time of irradiation, microwave and ultrasound energy inputs were optimized. HPLC was performed on RP ODS column (4.6 mm ID x 250 mm, 5 microm, Waters), a photodiode array detector (Waters 2996) using gradient solvent system of ACN and ortho-phosphoric acid in water (0.001:99.999 v/v) at 25 degrees C. Regression equation revealed a linear relationship (r2 > 0.9998) between the mass of vanillin injected and the peak areas. The detection limit (S/N = 3) and limit of quantification (S/N = 10) were 0.65 and 1.2 microg/g, respectively. Recovery was achieved in the range 98.5-99.6% for vanillin. Maximum yield of vanilla extract (29.81, 29.068 and 14.31% by conventional extraction, MAE and UAE, respectively) was found in a mixture of ethanol/water (40:60 v/v). Dehydrated ethanolic extract showed the highest amount of vanillin (1.8, 1.25 and 0.99% by MAE, conventional extraction and UAE, respectively).

Simultaneous analysis of N-nornuciferine, O-nornuciferine, nuciferine, and roemerine in leaves of Nelumbo nucifera Gaertn by high-performance liquid chromatography–photodiode array detection–electrospray mass spectrometry

A novel method has been developed for analysis of N-nornuciferine, O-nornuciferine, nuciferine, and roemerine in leaves of Nelumbo nucifera Gaertn by using high-performance liquid chromatography (HPLC)–photodiode array detection (DAD)–electrospray mass spectrometry (ESI-MS). The method was carried out by using a Shimadzu VP-ODS column with a gradient solvent system of 0.1% triethylamine aqueous solution–acetonitrile. N-Nornuciferine, O-nornuciferine, nuciferine, and roemerine were identified with authentic standard compounds and with MS-spectra. The contents of these compounds were measured by employing DAD. Linearity of around three orders in the magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 30–90 pg.

Simultaneous analysis of protoberberine, indolequinoline and quinolone alkaloids in coptis–evodia herb couple and the Chinese herbal preparations by high-performance liquid chromatography–electrospray mass spectrometry

A new and sensitive method has been developed for analysis of protoberberine, indolequinoline and quinolone alkaloids in coptis–evodia herb couple and the Chinese herbal preparations by using HPLC in combination with electrospray mass spectrometry (ESI-MS). Different LC and cone voltages have been optimized prior to this in order to obtain better results and sensitivity. The method was carried out by using a spherigel C 18 analytical column with a gradient solvent system of acetate buffer–acetonitrile–methanol. The contents of alkaloids were measured by pseudomolecule ion on the selective ion recording (SIR) mode. Linearity of around three orders in the magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 9–30 pg.

Determination of Fluquinconazole, Pyrimethanil, and Clofentezine Residues in Fruits by Liquid Chromatography with Ultraviolet Detection

A simple method was developed for the determination of fluquinconazole, pyrimethanil, and clofentezine in whole fruit; peel; and pulp of mango, apple, and papaya. These compounds were extracted from fruit samples with a mixture of ethyl acetate-n-hexane (1 + 1, v/v). An aliquot (2 mL) of the extract was evaporated to near dryness under a stream of nitrogen, and the residue was dissolved with 2 mL methanol. The analysis was performed by means of liquid chromatography with ultraviolet detection at 254 nm using a gradient solvent system. The method was validated with fortified fruit samples at concentration levels of 0.05, 0.10, 0.20, and 0.50 mg/kg. Average recoveries (4-8 replicates) ranged from 80 to 95% with relative standard deviations between 3.5 and 12.7%. Detection limits ranged from 0.03 to 0.05 mg/kg for fruit pulp and 0.03 mg/kg for whole fruit. The quantitation limits ranged from 0.05 to 0.10 mg/kg for fruit pulp and 0.05 mg/kg for whole fruit. The analytical method was applied to fruit samples obtained from local markets.

Isolation and Partial Characterization of Phytotoxic Mycotoxins Produced by Sclerotinia sp., a Potential Bioherbicide for the Control of White Clover(Trifoliorum repens)

Sclerotinia sp. (isolate BWC98-105) causes stem blight and root rot in Leghum sp., and is presently being evaluated as a potential mycoherbicide for the control of Trifoliorium repens. Bioassays have shown that Sclerotinia sp. produces phytotoxic substance which is biologically active against T. repens. Two biologically active compounds, designated as compoundsI and II, were produced in vitro from the culture filtrate of BWC98-105 isolate Sclerotium sp. Compounds I and II were purified by means of liquid-liquid extraction and <TEX>$C_{18}$</TEX> open column chromatography (300 <TEX>${\times}$</TEX> 30 mm, i.d). To determine the purity, the purified compounds were analyzed by RP-HPLC. The analytical RP-HPLC column was a TOSOH ODS-120T (150 <TEX>${\times}$</TEX> 4.6 mm i.d, Japan), of which the flow rate was set at 0.7 mL/min using the linear gradient solvent system initiated with 15 % methanol to 85 % methanol for 50 min with monitoring at 254 nm. Under these RP-HPLC conditions, compounds I and II eluted at 3.49 and 4.13 min, respectively. Compound II was found to be most potent and host specific. However, compound I had a unique antibiotic activity against phytopathogenic bacteria like bacterial leaf blight (Xanthomonas oryzae) on rice, where it played a less important role in producing toxicity on T. repens. No toxin activity was detected in the water fraction after partitioning with several organic solvents. However, toxin activity was detected in the ethyl acetate and butanol fractions. In the leaf bioassay using compound II, the disease first appeared within 4-5 h as water soaked rot, which subsequently developed into well-defined blight affecting the whole plant.

Multiple peak formation of avidin on an iminobiotin affinity support.

When pure avidin is chromatographed on a homemade iminobiotin column, two distinct peaks are observed using a gradient solvent system starting with a pH 9 buffer. Proteins in both peaks are shown to be identical by nondenatured polyacrylamide gel electrophoretic experiment. The weak interaction of the protein with the stationary phase at pH 9 could cause this unusual behavior.

Novel inhibition of cis/ trans retinoic acid interconversion in biological fluids—an accurate method for determination of trans and 13- cis retinoic acid in biological fluids

All- trans retinoic acid (tRA, or tretinoin) can be metabolized through stereoisomerzation to 13- cis retinoic acid (13-cRA) in vivo. We have developed a simple, sensitive and accurate method for analyzing tRA and 13-cRA in plasma with the addition of N-ethylmaleimide (NEM) and Vitamin C (Vit. C) to prevent the interconversion of cis/ trans retinoic acid. All- trans RA, 9-cRA, and 13-cRA were well separated from each other in plasma by using a C18 precolumn and a column with a gradient solvent system of mobile phases A and B at a flow rate of 1.0 ml/min. In addition, thermal stability of tRA and cRA in plasma during the sample preparation under the temperature of 0 and 25 °C were studied. Our results showed that (1) the interconversion ratios (%) (cRA/tRA and tRA/cRA) were decreased with the addition of NEM and Vit. C and the minimum concentrations of NEM and Vit. C to inhibit the interconversion were 50 and 150 μM, respectively, (2) higher concentrations of NEM and Vit. C were required to prevent the interconversion at higher temperature, (3) the precision and accuracy of calibration curve with various concentration of tRA (1–1000 ng/ml) and 13-cRA (5–800 ng/ml) in plasma showed good linearity ( r 2=0.9992 and 0.9994), and between-day errors expressed by coefficient of variation (CV, %) for tRA and 13-cRA which were both less than 5.6%, (4) the mean recovery of the analytes were 78–94% for tRA and 80–92% for 13-cRA at concentration range from 1 to 1000 ng/ml and 5 to 800 ng/ml, respectively, and (5) the limit of quantitation of tRA and 13-cRA were 1 and 5 ng/ml, respectively. In addition, the HPLC method had been successfully applied to the tRA pharmacokinetic study in two hepatoma patients after receiving 45 mg/m 2 per day orally. Thus, our results suggest that the HPLC method for analyzing tRA and 13-cRA in plasma with the addition of NEM and Vit. C is a simple, sensitive and accurate method.

Analysis of aristolochic acids in herbal medicines by LC/UV and LC/MS

AbstractTwo high‐performance liquid chromatographic methods, one combined with a photodiode array detector (LC/UV) and another with mass spectrometry (LC/MS), were developed for the analysis of aristolochic acid I and II in herbal medicines. The LC/UV method was carried out using a Cosmosil 5C18‐MS column with a gradient solvent system composed of phosphate buffer‐acetonitrile and a UV detector (390 nm). The LC/MS method was performed using an acetate buffer‐acetonitrile solvent system and positive‐ion electrospray ionization MS. The characteristic fragment ions for aristolochic acid I were selected at m/z 359, m/z 324, m/z 298, and m/z 296, and for aristolochic acid II at m/z 329, m/z 294, and m/z 268. In order to attain better resolution, solid phase extraction was used to prepare the test solution in this study.

Simultaneous determination of N-butyramide-tacrine and tacrine in mouse plasma and brain homogenate by high-performance liquid chromatography with a simple gradient solvent system

A novel reversed-phase HPLC method was developed for the simultaneous determination of tacrine (THA) and the newly synthesized prodrug ( N-butyramide-THA, BTHA) in mouse plasma and brain homogenate. The assay involves deproteinisation and subsequent detection at 240 nm with a gradient solvent system. Retention times were 18.5 and 9.3 min for BTHA and THA, respectively. Average recoveries for the analytes were 80.7% (BTHA) and 76.6% (THA) from plasma, and 75.0% (BTHA) and 68.4% (THA) from brain homogenate. Linear responses were observed over a wide range (0.25–20 μg/ml for BTHA in plasma and in brain homogenate, 0.025–20 μg/ml for THA in both matrices). Both BTHA and THA degraded from the prodrug can be detected even 12 h after intravenous administration of BTHA, indicating that BTHA is a promising prodrug for brain targeting.

Quantitative analysis of lycopene isomers in human plasma using high-performance liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of the concentrations of total lycopene and its cis and all-trans isomers in human plasma has been developed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method was based on the observation that, during negative ion atmospheric pressure chemical ionization with collision-induced dissociation, a unique fragment of m/z 467 was formed from the molecular ion of m/z 536 by elimination of a terminal isoprene group. The use of multiple reaction monitoring facilitated the selective detection of lycopene isomers and an internal standard without interference from the isobaric carotenoids a-carotene and beta-carotene, which are also abundant in human plasma. Measurement of total lycopene was carried out using a C18 high-performance liquid chromatography (HPLC) column and an isocratic mobile phase consisting of acetonitrile/methyl tert-butyl ether (95:5) so that all lycopene isomers eluted as a single chromatographic peak. all-trans-Lycopene was separated from its various cis isomers by using a C30 carotenoid column and a gradient solvent system from methanol to methyl tert-butyl ether. The effects of sample preparation and handling parameters on the stability of lycopene were evaluated such as the stability of lycopene in the HPLC autosampler and the effect of saponification upon lycopene isomerization. For example, the half-life of all-trans-lycopene in the HPLC mobile phase in the autosampler at 4 degrees C was determined to be approximately 16 h. Also, saponification of plasma samples was determined to cause lycopene degradation and isomerization so that lycopene recovery was reduced. The accuracy and interassay precision of this LC-MS-MS assay for lycopene showed a standard deviation of less than 10% over the range of 5-500 pmol injected on-column. The limit of detection was 11.2 fmol injected on-column, and the limit of quantitation was 22.8 fmol.

IDENTIFICATION AND SIMULTANEOUS ANALYSIS OF HARMANE, HARMINE, HARMOL, ISOVITEXIN, AND VITEXIN IN PASSIFLORA INCARNATA EXTRACTS WITH A NOVEL HPLC METHOD

A high performance liquid chromatographic method for the simultaneous analysis of two flavonoids (iso-vitexin and vitexin), and three indole alkaloids (harmane, harmine, and harmol) was developed. This method was then utilised to quantitate levels of these five constituents in methanolic extracts of Australian Passiflora incarnata. HPLC analysis was performed using a Waters™ Novapak C18 (150 × 4 mm, 4 μm) column, with a gradient solvent system of methanol-water-acetic acid. Detection was achieved by PDA UV (254 nm) and fluorescence (excitation 254 nm, emission 414 nm), utilising the external standard method to obtain quantification.

Determination of ephedra alkaloids by high-performance liquid chromatography

Two simple methods were developed for the simultaneous determination of six alkaloids (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine and methyl-pseudoephedrine) inEphedrae Herba by high-performance liquid chromatography. The first method was carried out by using a Cosmosil 5C18-MS column with a gradient solvent system consisting of a phosphate buffer and acetonitrile, and detection at 210 nm. The contents of alkaloids in non-pretreated ephedra herb extracts could be determined easily in 50 min. Alternatively, the alkaloids could be determined within 35 minutes by using a Cosmosil 5C18-MS column with an isocratic solvent system of a sodium dodecyl sulfate-acetonitrile solution. The two methods are compared and discussed.

A new high performance liquid chromatography multifactor methodology for systematic and simultaneous optimisation of the gradient solvent system and the instrumental/experimental variables

A simple HPLC methodology is proposed which takes into account changes in solvent composition and analysis time in order to speed up lengthy separations without affecting the chromatographic resolution. The main aspects discussed in the demonstration of the proposed approach are: routine analyses; long execution time separation processes; and an overall treatment of all the parameters. The combination of the new methodology and experimental design tools, such as a Doehlert matrix, has proved to be a reliable alternative to optimise simultaneously the HPLC solvent gradient system and the instrumental/experimental variables.

Reversed-phase high-performance liquid chromatography of catecholamines and indoleamines using a simple gradient solvent system and native fluorescence detection

A reversed-phase HPLC method using a C 18 column and a two-mobile-phase gradient elution system containing only volatile components has been developed for separation of norepinephrine, octopamine, epinephrine, dopamine, dihydroxyphenylalanine, tyramine, tyrosine, serotonin, 5-hydroxytryptophan, N-acetyl-serotonin and tryptophan. Mobile phase A contains 0.05% aqueous trifluoroacetic acid and methanol (97.5:2.5, v/v) and mobile phase B contains 0.05% aqueous trifluoracetic acid and methanol (40:60, v/v). This method has the advantage that the mobile phase can be removed completely, without salt residues, from the eluted fractions thus simplifying further analytical procedures on isolated fractions. The elution profile of standards is related to structural characteristics allowing prediction of retention times of known compounds and insight into possible structural characteristics of unknown components in a mixture. Detection is via native fluorescence using excitation at 220 nm and emission at 320 nm and under the conditions described has a sensitivity range from 2.5 to 25 pmol, although the sensitivity range can be extended depending on the emission wavelength used.

SIMULTANEOUS DETERMINATION OF ALDRIN, DIELDRIN, ENDRIN, HEPTACHLOR, AND p,p’-DDT IN MEDICINAL PLANT EXTRACTS USING A NOVEL HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD

A high performance liquid chromatographic method for the simultaneous determination of five organochlorine pesticides (aldrin, p,p’-DDT, dieldrin, endrin, and heptachlor) was developed. The method was used to determine the levels of these pesticides in medicinal plant samples. Analysis was carried out using a Merck LiChrospher 100 RP C18 (5 μm) column with a gradient solvent system of acetonitrile-water and PDA UV detection (224 nm). Quantification was carried out by the external standard method. The limit of detection for the utilized method was below the local legal limits (ANZFA) for similar plant materials for all 5 pesticides excepting endrin. Medicinal plant extracts were further analyzed by conventional GC-ECD and GC-NPD means using SPE and GPC cleanup as required.

Simultaneous determination of bile acids in rat liver tissue by high-performance liquid chromatography

A method for the simultaneous determination of bile acids in rat liver tissue by high-performance liquid chromatography was developed. Without prior fractionation and alkaline hydrolysis, 30 unconjugated, glycine- and taurine-conjugated bile acids were detected by post-column enzymatic reaction and fluorescence detection. They were separated on a reversed-phase column using a linear gradient solvent system of 10 m M tribasic ammonium phosphate–acetonitrile–methanol (44:12:5, v/v/v) and 20 m M dibasic ammonium phosphate–acetonitrile–methanol (2:1:2, v/v/v). The limits of detection were 1–5 pmol, and calibration curves were linear for concentrations ranging between 10 and 4000 pmol per 10 μl injection. This rapid and reliable method is effective for measuring bile acid levels in liver tissue not only of rats but also of patients with hepatobiliary and other diseases.

Separation of stereoisomers of C 27-bile acid CoA esters by liquid chromatography and its application to the study of the stereospecificities of D- and L-bifunctional proteins in bile acid biosynthesis

The separation and quantitation analysis of the stereoisomers of 3α,7α,12α,24-tetrahydroxy- and 3α,7α,24-trihydroxy-5β-cholestan-26-oyl CoAs (VA-CoA and DVA-CoA), known as one of the intermediates for bile acid biosynthesis, were achieved by liquid chromatography. The above stereoisomers of C 27-bile acids were effectively separated by a linear gradient solvent system of methanol/acetonitrile/sodium acetate buffer (pH 5) and acetonitrile/sodium acetate buffer (pH 5) using a reversed phase C18 column. Application for the study of the stereochemistry of the products by enzymatic reaction related to the β-oxidation of bile acids in rat liver was also demonstrated. The products (VA-CoA and DVA-CoA) from the corresponding 3α,7α,12α-trihydroxy- and 3α,7α-dihydroxy-5β-cholest-24-en-26-oyl CoAs (24 E-THCA-CoA and 24 E-DHCA-CoA) from a catalytic reaction with rat liver peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase or enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase were quantitatively determined. The results indicated different stereospecificities of these enzymes which might prove useful for the evaluation of their role and function.

Quantitative determination of constituents in various licorice roots by means of high performance liquid chromatography

During the course of our studies on the chemical constituents of botanically identified Chinese licorice roots of various origins, we have identified 15 kinds of saponins and 49 kinds of flavonoids (including flavonoid glycosides). With these chemical constituents at hand, we have carried out the chemical evaluation studies of licorice roots available in Japan by means of high performance liquid chromatography (HPLC). In the HPLC analysis using an ODS reversed-phase column, a combination of 1% aqueous acetic acid and acetonitrile was used as the mobile phase in a gradient solvent system, and the eluate was monitored with UV absorption at 254 nm (for saponins with a diene or a conjugated carbonyl moiety, liquiritin, and liquiritin apioside) or at 350 nm (for other flavonoid constituents). By our analytical method described here, quantitative analyses of saponin and flavonoid constituents were effected simultaneously. We have found an interesting piece of information concerning the origin of parent plants and their characteristic constituents such as saponins and flavonoids.

High Performance Liquid Chromatographic Determination of Pheophorbide-a and Its Related Chlorophyll Derivatives in Tea Leaves.

A simple method for the extraction of tea samples and conditions of HPLC analysis of pheophorbide-a (PB-a) and its related chlorophyll derivatives was developed. Tea samples were extracted with 85% acetone (v/v) with this solution injected directly into the HPLC column. The modified HPLC procedure developed included a gradient solvent system in which solvent A (95% ethanol (v/v) containing 0.005 M sodium chloride) and solvent B (80% ethanol (v/v) containing 0.005 M sodium chloride) were the mobile phase. PB-a, its derivatives and their C-10 epimers could be clearly separated and determined within 35 min. This analytical method could be routinely used to determine low levels of PB-a content (<10 mg/100g) and its related individual chlorophyll derivatives in green teas. Hence, it is applicable to the safety and quality control of green teas.

Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids

An improved straight-phase HPLC method for the separation and quantification of lipid classes is described. Two binary gradient solvent systems were used, one for polar and one for neutral lipids, and detection was performed with a light-scattering detector. The developed HPLC methods were highly reproducible and allowed base-line separation of all investigated polar lipid classes (phosphatidic acid, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine) and neutral lipid classes (triacylglycerol, free fatty acid, diacylglycerol, cholesterol and monoacylglycerol) except of cholesterol ester and wax ester. Application of the chromatographic systems demonstrated that the methods are suitable for quantitative analysis of the major lipid classes present in lipid extracts from livers and eggs of Atlantic salmon ( Salmo salar).