Reversed‐Phase High‐Performance Liquid Chromatography with Electrochemical Detection of Anthocyanins

Abstract

Anthocyanins, flavonoid compounds present in grapes and wines, were determined by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) with electrochemical detection (RP‐HPLC‐ED). The method developed consists of RP‐HPLC gradient elution with voltammetric detection using a glassy carbon electrode after separation in an Inertsil ODS‐3V analytical column. Good peak resolution was obtained following direct injection of a 50 µL sample of anthocyanins in a mobile phase of pH 2.20. The results show that six different anthocyanins: cyanidin‐3‐O‐glucoside chloride (kuromanin chloride), cyanidin‐3,5‐di‐O‐glucoside chloride (cyanin chloride), malvidin‐3‐O‐glucoside chloride (oenin chloride), malvidin‐3,5‐di‐O‐glucoside chloride (malvin chloride), delphinidin‐3‐O‐glucoside chloride (myrtillin chloride), and peonidine‐3‐O‐glucoside chloride, all with antioxidant properties, can be separated in a single run by direct injection of solution. The limit of detection (LOD) for these compounds was lower than 0.3 µM. The method can also be applied to the analysis of these compounds in red wines and in skins and pulp extracts of red grapes, since all these antioxidants are electroactive.     

Reversed–Phase Liquid Chromatographic Determination of Zaleplon in Human Plasma and its Pharmacokinetic Application

Abstract

A simple, rapid, specific, and reliable high performance liquid chromatographic assay of zaleplon in human plasma has been developed. Reversed‐phase chromatography was conducted using a mobile phase of methanol∶ammonium acetate buffer (50∶50) v/v, pH 3.2 adjusted with orthophosphoric acid, UV detection at 232 nm. After extraction from plasma by precipitation the drug was chromatographed using a C₁₈ reversed‐phase analytical column. The average recoveries of zaleplon from spiked plasma in the concentration range from 0.005–0.2 µg/ml were 93.29%, and their respective CV% was 2.557%. Regression analysis for the calibration plot for plasma standards obtained on three different days for the drug concentrations between 0.005–0.2 µg/ml indicated excellent linearity (r>0.999) and the coefficient of variation of the slopes of the three lines was less than 2%. The limit of detection was 5 ng/ml. Analysis of variance of the data showed no detectable difference in the slopes of the three standard plots (F=3.1, P>0.01). The high correlation coefficients and the similarities in the slopes are good indications of the excellent reproducibility and linearity of the proposed method. The proposed method was applied to study the bioequivalence of a commercial product of zaleplon, using as reference standard the innovator drug product. The study was conducted by using one capsule (1×10 mg) of each of the commercial product and the reference standard in a two‐way open randomized crossover design involving 24 volunteers. The criteria used to assess bioequivalence of the products were AUC (0?∞), C_max, t_max, t_1/2, and K. The obtained values for these parameters were 0.246±0.03 µg h/ml, 0.150±0.013 µg/ml, 1 h, 1.26±0.36 h, and 0.5928±0.1732 h^?1 for product A whereas, for product B they were 0.256±0.044 µgh/ml, 0.142±0.014 µg/ml, 1 h, 1.18±0.33 h, and 0.63±0.1747 h^?1, respectively.     

A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and plasma     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

High Performance Liquid Chromatographic Separation of Bryostatins 1–12

Abstract

A reversed-phase high performance liquid chromatographic (RP-8; acetonitrile-water gradient) separation procedure was developed for detecting bryostatins 1–12, using a photodiode array detector system. While bryostatins 6 and 9 were found to co-elute they were easily separated using a silica gel column with 9:1 n-hexane-n-propanol as eluent.     

High－Performance Liquid Chromatographic Determination Of N－Nitrosoamines by Pre－Column Fluorescence Derivatization with 9－Phenanthrene Chloride

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Determination of Drugs in Blood Samples by High Performance Liquid Chromatography Using On-Line Sample Pretreatment Technique

Theflowdiagramofthison linesample pre treatmentsystemisshowninFig .1.Fig .1　Flowdiagramofon linesamplepretreatmentsystem1.degasunit;2 .pump ;3 .autosampler ;4.on linesampledi lutionbypass;5 .mixer;6.highpressureflowswitchingvalve;7.pretreatmentcolumn ;8.…     

Determination of Drugs in Blood Samples by High Performance Liquid Chromatography Using On-Line Sample Pretreatment Technique

The flow diagram of this on-line sample pretreatment system is shown in Fig. I.     

Integrated Micro Bio Systems and High Performance Liquid Chromatographic System on Chip

1　Microchemicalsystemonchip　　Microintegratedchemicalsystemsareexpect edaspromisingultrahighthroughputchemicalandbioprocessorswithextremelysmallvolume .Ourresearchgroupshaveproposedanddevelopedtheo riginalmethodologiesformicrointegrationofgen eralchemic…     

Integrated Micro Bio Systems and High Performance Liquid Chromatographic System on Chip

1 Micro chemical system on chip Micro integrated chemical systems are expected as promising ultra high throughput chemical and bio processors with extremely small volume. Our research groups have proposed and developed the original methodologies for micro integration of general chemical systems as well the electropho-     

Determination of the Flavonoids from Ginkgo Biloba Extract by High Performance Liquid Chromatography

Today Ginkgo biloba extract (GBE) is one of the most widely used food supplements and herbal medicines. The amounts of flavone glycosides, one of the key active components in GBE, vary according to the source of the ginkgo leaves and the extraction and enrichment procedures used to prepare the extract. A typical GBE contains from 22% to 27% of flavone glycosides. Ginkgo flavone glycosides are a group of small complex molecules that can be hydrolyzed to give kaempferol, quercetin and isor…     

β-Blocking Agents: Determination of Biological Levels Using High Performance Liquid Chromatography

Abstract

Nine β-blocking agents have been tested and dosed by high performance liquid chromatography. Six of them, acebutolol an acebutolol metabolite, atenolol, metoprolol, propranolol and sotalol are detected with a fluorometric detector. Oxprenolol, pindolol and timolol can be quantified by their UV absorption at variable wavelength. A method is developped to find the best conditions of extraction and detection for each blocking agent. Experimental trials have led to a simple procedure for all compounds. Only pindolol and timolol plasma levels are non suitable for high performance liquid chromatography and need mass fragmentography or gas chromatography with electron capture detection.

However, pharmacokinetic parameters can be reached, for timolol and pindolol, through urinary excretion since sensitivity of the procedure is within the range of urinary levels.

The method has been applied, as well, to pharmacokinetic studies on sotalol, acebutolol, acebutolol metabolite, atenolol, propranolol, pindolol and timolol.     

Development and Validation of High‐Performance Liquid Chromatographic Method for Estimation of Lercanidipine in Rabbit Serum

Abstract

A new, simple, and sensitive reverse‐phase liquid chromatographic method was developed and validated for the estimation of Lercanidipine hydrochloride in rabbit serum using UV detector under isocratic conditions. After subjecting serum to simple and efficient one‐step extraction procedure, 100 µl of sample was injected onto high‐performance liquid chromatography system. The detector response was linear in the concentration range of 25–1000 ng/ml. The developed method was validated as per standard guidelines. Validation demonstrated accuracy, precision, and selectivity of the proposed method. The drug was found to be stable under various processing and storage conditions.     

A Comparative Study of the Separation of the Tryptic Peptides of the β-Chain of Normal and Abnormal Hemoglobins by Reversed Phase High Performance Liquid Chromatography

Abstract

The separation of the tryptic peptides of the human hemoglobin A β-chain by reversed phase high performance liquid chromatography under different elution conditions on several microparticulate alkylsilica supports is described. Similar methods have been used to separate the tryptic peptides of β-chain hemoglobin variants including HbC, HbE, and Hb (Kempsey). Selectivity differences which can be achieved under the different chromatographic conditions have been exploited to permit the assignment of all the anticipated peptide fragments derived from the tryptic digestion of these β-chain Hb-variants.     

Determination of 2′,3′-Dideoxyinosine in Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatography analysis method has been developed for the quantitation of 2′,3′-dideoxyinosine (DDI) in plasma. Proteins were precipitated from plasma samples with acetonitrile containing the internal standard, 6-methylaminopurine riboside. The treated samples were evaporated to dryness and reconstituted in mobile phase for the analysis. Separation of the components was achieved on a 5 μm octadecylsilane column with ultraviolet detection at 254 nm. The method was validated at nine concentrations between 0.015 and 150 μg/mL. Using 500 μL of human plasma, the limit of quantitation was 120 ng/mL and the limit of detection was 60 ng/mL. The mean intra-day precision of the method was 1.6%. The mean accuracy of the method was within 2% of the actual values. This method is currently being used for pharmacokinetic studies in the rat.     

High‐Performance Liquid Chromatography Determination of Latanoprost in Pharmaceutical Formulations using UV Detection

Abstract

A simple, fast, and accurate high‐performance liquid chromatography (HPLC) method was developed to determine latanoprost in pharmaceutical formulations. The drug was chromatographed on a C₁₈ column. Eluents were monitored at a wavelength of 210 nm using a mixture of acetonitrile and 0.05 M potassium phosphate buffer pH 3.0 (70:30, (v/v). A linear response (r>0.9998) was observed in the range of 10.0–90.0 µg mL^?1. The method showed good recoveries (average 100.4%) and the relative standard deviations intra‐ and inter‐day were ≤1.0%. The method can be used for quality control assay of latanoprost in raw materials as well as in pharmaceutical formulations.     

Experimental Design Optimization of Solid‐Phase Microextraction Conditions for the Determination of Heterocyclic Aromatic Amines by High‐Performance Liquid Chromatography

Abstract

Solid‐phase microextraction, using Carbowax‐Templated Resin fiber, coupled with high performance liquid chromatography ultra violet/diode array detector (HPLC‐UV/DAD) has been optimized for the determination of the heterocyclic aromatic amines (HAs). Three variables (absorption time, soaking time, and desorption time) were considered as factors in the optimization process. Interactions between analytical factors and their optimal levels were investigated using two level factorial and Doehlert matrix designs. Absorption time and soaking time were significant variables, and 15 min for each one of the variables was chosen for the best response. The optimized procedure allowed the determination of HAs with detection limits that ranged from 1.58 to 16.8 ng/L (except PhIP: 23.8 ng/L). The reproducibility of the method (n=5), expressed as relative standard deviation was between 2.21% and 28.3%. The method was applied to the analysis of a meat extract sample and the range of recoveries for the amines was 64.14%–112.72%.     

Method Development and Validation for the Determination of Five Synthetic Food Colorants in Alcoholic Beverages by Reversed‐Phase High Performance Liquid Chromatography Coupled with Diode‐Array Detector

Abstract

An accurate method based on the use of reversed‐phase high performance liquid chromatography coupled with diode‐array detection was devised for the determination of five synthetic food colorants added to alcoholic beverages with natural colors. A C18 stationary phase was used and the mobile phase contained methanol and 40 mM ammonium acetate buffer solution. The synthetic food colorants were detected at their corresponding individual characteristic maxima of absorbance wavelength. Successful separation was achieved within 11 min for all the analytes using an optimized gradient elution, column temperature, buffer concentration and flow rate. Accurate sample quantification was feasible using matrix‐matched calibration curves. The method was successfully validated by determination of linearity ranges, the limits of quantification and detection, precision and recovery for all colorants tested. The proposed and validated method was used to analyze some alcoholic beverage samples, consisting of eight red wines, six coolers, four aromatized spirits, five bitters, three cocktails and four liquors from different Chinese manufacturers. The results showed the bitters and red wines did not have synthetic colorants, but colorants were found in all the samples of other kinds of alcoholic beverages. No analyzed sample exceeded the limit established by Chinese legislation.     

Determination by High Performance Liquid Chromatography of Stability of Tetrahydro-β-carbolines at Different Ambient Temperatures

Abstract

To determine the stability of tetrahydro-β-carboline compounds over time and at different temperatures, a reversed-phase high pressure liquid chromatography system with electrochemical detection was utilized. Noreleagnine (1,2,3,4-tetrahydro-β-carboline) and tetrahydroharman (1-methyl-1,2,3,4-tetrahydro-β-carboline) were dissolved in water or ascorbate (0.1 mg/ml) vehicle and stored at ?20°C, 22°C, or 37°C for one, seven or 12 days. After each solution was injected in the column in a concentration of 400–600 ng/10 μl, peak height values were obtained for the compound under each condition. Analysis of percent recovery showed that the two β-carbolines were relatively stable with a maximal degradation of 14% occurring only at the 12-day assay interval. These results suggest that this class of compound can be used in pharmacological studies in which they can be dispensed from a mini-pump implanted in tissue. Further, an HPLC system with electrochemical detector provides a valid and reliable procedure for quantification of indoleamine-aldehyde condensation products.     

Determination of Baicalin in Traditional Chinese Preparation by High Performance Liquid Chromatography with Chemiluminescence Detection

Herbal medicine, a form of complementary and alternative medicine, is becoming increasingly popular in the world1. Scutellariae radix is the root of Scutellariabaica -lensis georgi. The primary active constituent includes baicalin as follows: Clinical studies showed that baicalin exhibited therapeutic functions of antifever, moistening aridity, anti-inflammatory and detoxifying 2 and it is also an anti-abortion agent as well as can scavenge free radicals and against oxidation3. So it is…