High Performance Liquid Chromatographic Method for Quantitation of a New Antidiabetic Agent in Plasma

Abstract

An HPLC procedure for the detection and quantitation of a new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), in dog plasma was developed. The drug and internal standard were extracted from plasma using a reversed phase C₁₈ extraction column (Sep-pak). Separation was accomplished on a ERC-ODS-1161 reversed-phase column with a mobile phase of acetonitrile/0.1M phosphate buffer, pH 6.6 (30/70). Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. A linear relationship between concentration and peak height ratio (A4166/internal standard) was obtained. The method has been successfully used for analysis of plasma samples from beagle dogs following oral administration of A4166.     

High Performance Liquid Chromatographic Determination of Pentamidine in Plasma

Abstract

A high performance liquid chromatographic method for quantitating pentamidine in plasma has been developed. Sample clean-up involved precipitating plasma with acetonitrile containing the internal standard, hexamidine. The supernatant was passed through a C₈ Bond Elut column and eluted with a methanolic solution of sodium 1-heptanesulfonate. The eluate was then analyzed on an Altex C₈ column with a mobile phase consisting of 45% CH₈CN, 0.02% detramethylammonium chloride and 0.1% H₃PO₄. Using fluorescence detection (EX: 275 nm and EM: 340 nm), the detection limit was 1.25 ng/ml for 0.5 ml of plasma. The coefficients of variation for interday and intraday were around 10%.     

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     

Determination of phenyltin compounds by reversed-phase liquid chromatography

An analytical procedure for the determination of phenyltin compounds in environmental sample waters was studied. Chromatography of mono-, di- tri-phenyltin (MPT, DPT and TPT) was performed on a reversed-phase C₁₈ column with the mobile phase comprising methanol/10⁻² M H₃PO₄ (80:20 v/v) at pH 3 and UV detection at 214 nm. To enhance the sensitivity of the detection system, the post-column reaction between morin or 3-hydroxyflavone and phenyltin compounds was formed before fluorescence detection. Several parameters affecting the fluorescence intensity were studied systematically, including the optimum condition for the post-column reagent that was also compatible with the eluent. The parameters concerned in this study were the pH, the percentage of Triton X-100, the ratio of fluorigenic reagent to phenyltin compounds and the amount of methanol in the eluent. Detection limits before the preconcentration process were in the region of 1.5 ppb for TPT and 150–250 ppb for MPT and DPT, respectively. Utilizing solid-phase extraction on a C₁₈ cartridge for sample clean-up as well as preconcentration successfully reduced the detection limit of TPT to the level of ng dm⁻³ and can be applied to seawater analysis. Recovery in the range 95.0–98.0% was obtained by developing the optimum elution profile in the preconcentration step. © 1998 John Wiley & Sons, Ltd.     

Separation of Twelve Catecholamine Metabolites and Determination of Some of the Metabolites in Human Urine by Liquid Chromatography with Electrochemical Detection

Abstract

Separation of twelve catecholamine metabolites on a reversed-phase column was achieved, when eluate from the column was amperometrically monitored at +800 mV vs. Ag/AgCl. The mobile phase was a 0.1 M acetate buffer (pH 5.0). The flow rate and column temperature were 1.5 ml/min and 25 ± 1°C, respectively. Ethyl acetate extraction of the catecholamine metabolites from acidified human urine was studied and optimized. We demonstrate the feasibility of the method showing some liquid chromatographic results.     

Determination of Fluoxetine and Norfluoxetine in Serum by Liquid Chromatography with Fluorescence Detection

Abstract

A reversed phase liquid chromatographic procedure with fluorescence detection for the simultaneous determination of fluoxetine and its active metabolite, norfluoxetine in human serum is described. A 0.5-mL aliquot of the sample after the addition of protriptyline as the internal standard is passed through a 1-mL BondElut C₁₈ silica extraction column. The column is selectively washed to remove polar, neutral, acidic and weakly basic compounds. The desired compounds are eluted with a 0.25-mL aliquot of a mixture of 0.1 N perchloric acid + acetonitrile (1:3). A 20-uL aliquot of the eluate is injected onto a 15 cm × 4.6 mm (i.d.) column packed with 5-um C₈-silica particles, which is eluted at ambient temperature with a mobile phase containing tetramethyl-ammonium perchlorate. The peaks are detected with a fluorescence detector (ex = 235 nm; em = 310 nm). In the resulting chromatogram, there are only few extraneous peaks, fluoxetines give sharp peaks which are well resolved from peaks for solvent and internal standard. The extraction recovery of fluoxetines and internal standard is in the range of 85%.     

An Accurate,Specific HPLC Method for the Analysis of a Decapeptide in a Lactose Matrix

Abstract

A high-performance liquid chromatographic (HPLC) method is described for the determination of an analog of the hormone LH-RH in lyophilized vials at the low parts-per-million level. The peptide (Schally analog) is quantitatively recovered from the glass lyophilization vials after reconstitution with mobile phase. The peptide solution is eluted on a reversed-phase, C₁₈ column and monitored with ultraviolet (UV) detection at 220 nm. The chromatography resolves Schally analog from a number of synthetic impurities and decomposition products.     

A new UPLC approach for the quantitation of ephedrine and guaifenesin in a syrup formulation using multivariate optimization strategy

A new ultraperformance liquid chromatography (UPLC) method with photodiode array detection was developed for the quantitative analysis of a commercial syrup formulation containing ephedrine (EPH) and guaifenesin (GUA). In the development of UPLC method, experimental chromatographic conditions, flow rate, column temperature, and percentage of 0.1?M H₃PO₄ in mobile phase, were optimized using chemometric multivariate strategy. From the application of a 3³ full factorial design, the optimal chromatographic conditions were obtained as the flow rate of 0.29?mL/min, column temperature of 36.4°C, and 56.9% of 0.1?M H₃PO₄ in the mobile phase. The optimal conditions gave us a good chromatographic separation of the analyzed drugs with short analysis runtime within 3?min. Calibration curves for EPH and GUA in the linear working range of 4–64 and 6–96?µg/mL, respectively, were obtained using peak areas detected at 215?nm. Performance and validity of the optimized UPLC method were estimated by analyzing independent binary mixtures, inter-day and intra-day samples, and standard addition solutions containing EPH and GUA substances. It was concluded that the proposed method was a promising approach for the quantitative determination and routine analysis of a commercial syrup formulation of the titled substances.     

Enantioselectivity of 6-O-alkyldimethylsilyl-2,3-di-O-methyl-β-cyclodextrins as chiral stationary phases in capillary GC

Summary Clenbuterol has been determined in urine by solidphase extraction on a C₁₈ cartridge, diazotization of the eluate with nitrite, coupling of the diazonium ion with 1-(naphthyl)ethylenediamine, and separation of the azo dye formed by HPLC with a C₁₈ column and a micellar mobile phase containing 0.1 M sodium dodecyl sulphate, 12%n-butanol and 0.05 M citrate buffer, pH 3. Recoveries higher than 90% were obtained by mixing the samples with a 20% 0.2 M NaOH before extraction. Limits of detection of 51 and 6.7 ng L⁻¹ were obtained with spectrophotometric and thermal lens spectrometric detection, respectively; respective repeatabilities were 3.1% (5 μg mL⁻¹) and 5.6% (0.16 μg mL⁻¹).     

Water-Phosphoric Acid Media: Electrochemical Properties

Abstract

Electroactivity ranges at polished Platinum, vitreous Carbon, and Mercury electrodes are determined in H₂O - H₃PO₄ mixtures (0.1 -14 M H₃PO₄). The Ferrocene system is considered as a comparison system. Ag⁺/Ag_s and AgCl/Ag_s systems are considered as reference systems. The R_o(H) acidity function is determined. It varies from -2.0 to -6.0 when H₃PO₄ varies from 5.5 to 11.5 M.     

High Performance Liquid Chromato-Graphic Method For The Determination Of Permethrin Deposits In A Forestry Spray Trial

Abstract

A convenient and sensitive reversed-phase high performance liquid Chromatographie method has been developed for the determination of perraethrin [3-phenoxybenzyl (±)- cis,trans -3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate] insecticide. Various isocratic and gradient mobile phases, consisting of acetonitrile:water (CH₃CN:H₂O) and methanol:water (CH₃OH:H₂O) solvent systems at two flow rates, were tested to separate and quantify the Isoners of permethrin using octadecylsilyl (ODS) (Regis 5μ m) and octylsilyl (OS) (RP-8, 10 μ m) bonded columns. The optimal mobile phase for perraethrin using the ODS column was 70:30 (v/v) CH₃CN:H₂O mixture at flow rates of either 1.0 or 1.5 mL/min. The measurement was done with a UV detector at 200 nm and 50°C. The OS column gave a less satisfactory separation than the ODS. Gradient elution systems examined did not improve the iso-meric separation of perraethrin. Using the method developed, deposit levels obtained on various sampling units during a perraethrin spray trial were analyzed after elution or extraction followed by necessary column cleanup. Minimum levels of detection for permethrin varied from 1 to 3 ng depending on the nature of the sampler used.     

Separation and quantification of basic hydroxycinnamic amides and hydroxycinnamic acids by reversed-phase high-performance liquid chromatography

A method for the separation of basic hydroxycinnamic amides and hydroxycinnamic acids by high-performance liquid chromatography is described. N-p-Coumaryl-, N-caffeyl- and N-ferulylpetrescine, N-p-coumryl-, N-caffeyl- and N-ferulylspermidine and p-coumaric, caffeic, ferulic and sinapic acids were chromatographed on a μBondapak C₁₈ reversed-phase column (particle size 9 μm) with different methanol—water gradients as the mobile phase. It is possible with this high-resolution and reproducible method to assay biological samples containing mre that 10⁻⁵ M of hydroxycinnamic amides, using either p-coumaric or ferulic acid as the internal standard: this is demonstrated for tobacco extracts.     

Quantitative Separation of Volatile Fatty Acids by High-Pressure Liquid Chromatography

Abstract

Volatile fatty acids (acetic, propionic, butyric, isovaleric, and valeric) are separated isocratically on a reverse phase C₁₈ μBONDAPAK column in less than 20 min. The eluent was 0.01 M NaH₂PO₄ buffer, pH 3.5, containing 10% methanol. Separations were monitored by UV absorption at 210 nm. Peak height measurements gave quantitative linear responses from 0.25 μmole to 2.50 μmole of each acid.     

Determination of Iodate in Iodized Salt by Reversed-Phase High-Performance Liquid Chromatography with UV Detection

A method for the determination of iodate was developed by reversed-phase high-performance liquid chromatography with UV detection. Iodate was converted to iodine, which was separated from the matrix using a reversed-phase Ultrasphere C₁₈ column (250 × 4.6 mm, 5 μm) with methanol-1 mmol L^?1 H₃PO₄ (20:80, v/v) as mobile phase at 1.00 mL min^?1 and UV detection at 224 nm. The calibration graph was linear from 0.05 μg mL^?1 to 5.00μg mL^?1 for iodine with a correlation coefficient of 0.9994 (n=7). The detection limit was 0.01 μg mL^?1. The method was successfully applied to the determination of iodate in iodized salt. The recovery was from 96% to 101% and the relative standard deviation was in the range of 1.5% to 2.9%.     

Reversed-phase liquid chromatographic separation of Fe(II) and Fe(III) as their respective 1,10-phenanthroline and 5-sulphosalicylate complexes

A method has been developed for the separation of Fe(II)-1,10-phenanthroline and Fe(III)-5-sulphosalicylate complexes on a reversed-phase C₁₈ column in the presence of an ion-pairing reagent. Samples were injected on the column in the pre-complexed form and separated using a mobile phase consisting of acetonitrile [0.1% (w/v) in 5-sulphosalicylic acid] ?0.02 M sodium acetate buffer (pH 6.9) [0.1% (w/v) in tetramethylammonium chloride] (1 + 1). Spectrophotometric detection of the complexes was carried out at 515 nm. Linear calibration graphs were obtained for 1–12 μg mol^?1 Fe(II) and Fe(III).     

Development and validation of a reversed-phase HPLC method with post-column iodine-azide reaction for the determination of thioguanine

A high-performance liquid chromatographic method of reversed-phase with a post-column iodineazide reaction has been developed and validated for the determination of thioguanine. Isocratic elution was performed on a column of C₁₈ using acetonitrile- water-sodium azide solution (1.5%; pH 6.5) 16: 34: 50 (v/v/v) as a mobile phase with flow-rate of 0.5 mL/min. Monitoring of unreacted iodine in post-column iodine-azide reaction induced by thioguanine resulted in its detection at 350 nm. The method applied to thioguanine was linear within the scope of values 8–100 nM (r ² > 0.9988). The relative standard deviation (RSD < 4.2%) and the recovery (>96%) prove that the intra-day precision and the accuracy were satisfactory. The lower limits of detection (LLD) and quantification (LLQ) of thioguanine were established at the levels of 6 and 8 nM, respectively. The elaborated method was validated and applied to thioguanine determination in tablets.     

Validated LC-MS/MS assay for the quantitative determination of clematichinenoside AR in rat plasma and its application to a pharmacokinetic study

This study aimed to develop and validate a liquid chromatography tandem mass spectrometry method for measuring clematichinenoside AR in rat plasma. Clematichinenoside AR was extracted by solid‐phase extraction and chromatographed on an XTerra MS C₈ column. Pulchinenoside B4 was used as the internal standard. Elution was achieved using an isocratic mobile phase of acetonitrile with 0.1% acetic acid (21:79, v/v) at a flow‐rate of 0.2 mL/min. The detection was performed by multiple reaction monitoring mode via a negative electrospray ionization interface. Standard curves were linear, ranging from 2.5 to 500 ng/mL. The intra‐ and inter‐day precision values were <14.0% and the accuracy was within ±13%. Extraction recovery ranged from 93.2 to 93.9%. This proposed method was successfully applied to a pharmacokinetic study on clematichinenoside AR in rats after oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Reversed-phase liquid chromatography and argentation chromatography of the minor capsaicinoids

An investigation of the liquid chromatography of the minor capsaicinoids in a commercial capsaicinoid mixture is reported. Twelve stationary phases including C₈, C₁₈, C₃₀, phenyl, and cation-exchange chemistries were examined in combination with isocratic aqueous methanol and aqueous acetonitrile mobile phases. A phenyl stationary phase and aqueous acetonitrile mobile phase baseline-resolved 7 of 11 capsaicinoids, and selected ion chromatograms (LC–ESI-MS) demonstrated this was the most effective reversed-phase separation. Argentation chromatography with an alkyl or phenyl column and aqueous silver nitrate–methanol mobile phase revealed the presence of the 6-ene-8-methyl and 6-ene-9-methyl homocapsaicin isomers and the absence of 7-ene-9-methyl homocapsaicin. A mixed phenyl–cation-exchange stationary phase (charged with silver ion) enabled unique and useful separations of the capsaicinoids.     

Determination Of Serum Cephalexin By High Performance Liquid Chromatography

Abstract

A simple and specific method for the assay of cephalexin in human serum by high performance liquid chromatography is described. Cephalexin was extracted from serum with 5 fold volume of methanol and chromatographed on a reversed-phase column using 30 % aqueous acetonitrile solution containing 0.005 M sodium 2-propanesulfonate (pH 3.0) as the mobile phase. Cephalexin was detected by the absorbance at 254 nm. Only 20 μl of serum was required and all of the operations for analysis were completed within 10 min. This method was proved to be effective in the rapid monitoring of serum cephalexin concentration in humans who received its ordinal and long-acting preparations.     

Determination of Ergotamine Tartarate and Cyclizine Hydrochloride in Pharmaceutical Tablets by Reverse Phase HPLC

Abstract

A high performance liquid chromatographic procedure is presented for the determination of ergotamine tartarate and cyclizine hydrochloride in pharmaceutical tablets. An aliquot of the sample is dissolved in methanol containing ethyl p-hydroxybenzoate as an internal standard and chromatographed on a 5 üm, C₁₈, Hibar pre-packed, Lichrospher (250 mm × 4.0 mm i.d.), column using a mobile phase of 0.01 M ammonium acetate in acetonitrile: water: triethylamine (35:64:1) solution, the pH was adjusted to 3.7 with glacial acetic acid. The method was tested for linearity, recovery, and specificity and was found to be fast, sensitive, accurate and reproducible with a total elution time of six min.