LC determination of glimepiride and its related impurities

Five impurities in glimepiride drug substance were detected and quantified using a simple isocratic reverse phase HPLC method. For the identification and characterization purpose these impurities were isolated from a crude reaction mixture of glimepiride using a normal phase HPLC system. Based on the spectroscopic data like NMR, FTIR, UV and MS these impurities were characterized and used as impurity standards for determining the relative response factor during the validation of the proposed isocratic reverse phase HPLC method. The chromatographic separation was achieved on a Phenomenex Luna C8 (2) 100 Å, 5 μm, 250 mm × 4.6 mm using a mobile phase consisting of phosphate buffer (pH 7.0)–acetonitrile–tetrahydrofuran (73:18:09, v/v/v) with UV detection at 228 nm and a flow rate of 1 ml/min. The column temperature was maintained at 35 °C through out the analysis. The method has been validated as per international guidelines on method validation and can be used for the routine quality control analysis of glimepiride as active pharmaceutical ingredient (API).     

Development of a HPLC method for the determination of cyclosporin-A in rat blood and plasma using naproxen as an internal standard

An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 205 nm has been developed for the determination of cyclosporin-A (CyA) in rat blood and plasma. Naproxen was successfully used as an internal standard. Blood or plasma samples were pretreated by liquid–liquid extraction with diethyl ether. The ether extract was evaporated and the residue was reconstituted in acetonitrile–0.04 M monobasic potassium phosphate buffer (pH 2.5) solvent mixture. After washing with n-hexane, 30 μl of the reconstituted solution was injected into HPLC system. Good chromatographic separation between CyA and internal standard peaks was achieved by using a stainless steel analytical column packed with 4 μm Nova-Pak Phenyl material. The system was operated at 75 °C using a mobile phase consisting of acetonitrile–0.04 M monobasic potassium phosphate (pH 2.5) (65:35 v/v) at a flow rate of 1 ml/min. The calibration curve for CyA in rat blood was linear over the tested concentration range of 0.0033–0.0166 M with a correlation coefficient of 0.989. For rat plasma, the range of the concentrations tested were between 0.002 and 0.0166 M and showed linearity with a correlation coefficient of 0.953. The intra- and inter-run precision and accuracy results were 1.24–21.87 and 3.1–12.23%, respectively. The low volume of blood or plasma needed (200 μl), simplicity of the extraction process, short run time (5 min) and low injection volume (30 μl) make this method suitable for quick and routine analysis.     

Determination of ethylenediamine tetraacetic acid in injection forms by ion-pair chromatography

A high performance liquid chromatographic method was developed for the determination of ethylenediamine tetraacetic acid (EDTA) in injection forms. The method consists of direct extraction of the samples with ethyl acetate; the organic layers were evaporated to dryness and further diluted to a 0.025% (w/v) copper nitrate in order to achive the formation of the EDTA–copper solution complex. The chromatographic separation was performed on a C8 Hypersil column. The mobile phase consisted of a mixture of acetonitrile–0.015 M tetrabutylammonium hydroxide (10:90, v/v), (pH* 7.0) pumped at a flow rate of 1.5 ml min⁻¹. The UV detector was operated at 300 nm. Correlation coefficients of the calibration graphs were better than 0.9995, relative standard deviation was less than 2.5%. Detection limit of EDTA was found to be 1.97 μg ml⁻¹.     

Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples

Liquid chromatographic–tandem mass spectrometric (LC–MS–MS) methods for the determination of metformin in plasma from different species are presented. The first method employed a YMC cyano 2 mm×50 mm, 3 μm analytical column. For minimum sample preparation direct injection of samples after protein precipitation was performed. The polar column used with highly organic mobile phases provided a normal phase retention mechanism. The elution conditions were optimized to obtain reproducible peak areas and good peak shape. A step gradient from 100% acetonitrile to acetonitrile–water 80:20 (v/v) containing 10 mM ammonium acetate and 1% acetic acid was applied, leading to a sample-to-sample cycle time of 2 min. In a second method, a column-switching LC–MS–MS assay for on-line trapping was developed. The analyte and internal standard were trapped on a YMC cyano 2 mm×10 mm, 5 μm column using acetonitrile–methanol 95:5 (v/v). Elution was performed isocratically in back-flush mode on to the analytical column (YMC cyano 2 mm×50 mm, 3 μm) using 10 mM ammonium acetate in acetonitrile–water 80:20 (v/v) with 1% formic acid. With this approach, the signal-to-noise ratio was improved and the run time could be shortened to 1 min. Calibration samples were prepared in the matrix to be assayed in the range of 10–10,000 ng/ml. Quality control (QC) samples were prepared at 40, 400 and 4000 ng/ml and interspersed with the unknown study samples in the assays. Deviations for precision and accuracy were less than 20% for the lower limit of quantification (LLOQ) and low QC sample and less than 15% for other calibrators and QCs.     

Development and validation of a bioanalytical method using automated solid-phase extraction and LC-UV for the simultaneous determination of lumefantrine and its desbutyl metabolite in plasma

A bioanalytical method for the determination of lumefantrine (LF) and its metabolite desbutyl-lumefantrine (DLF) in plasma by solid-phase extraction (SPE) and liquid chromatography has been developed. Plasma proteins were precipitated with acetonitrile:acetic acid (99:1, v/v) containing a DLF analogue internal standard before being loaded onto a octylsilica (3 M Empore) SPE column. Two different DLF analogues were evaluated as internal standards. The compounds were analysed by liquid chromatography UV detection on a SB-CN (250 mm × 4.6 mm) column with a mobile phase containing acetonitrile–sodium phosphate buffer pH (2.0; 0.1 M) (55:45, v/v) and sodium perchlorate 0.05 M. Different SPE columns were evaluated during method development to optimise reproducibility and recovery for LF, DLF and the two different DLF analogues. The within-day precisions for LF were 6.6 and 2.1% at 0.042 and 8.02 μg/mL, respectively, and for DLF 4.5 and 1.5% at 0.039 and 0.777 μg/mL, respectively. The between-day precisions for LF were 12.0 and 2.9% at 0.042 and 8.02 μg/mL, respectively, while for DLF 0.7 and 1.2% at 0.039 and 0.777 μg/mL, respectively. The limit of quantification was 0.024 and 0.021 μg/mL for LF and DLF, respectively. Different amounts of lipids in plasma did not affect the absolute recovery of LF or DLF.     

Determination of local anaesthetics and their impurities in pharmaceutical preparations using HPLC method with amperometric detection

A method for the determination of local anaesthetics and their impurities – 2,6-dimethylaniline and o-toluidine – by high-performance liquid chromatographic method with amperometric detection has been developed. The analysis was performed in an isocratic mode on a reversed phase Luna column 5 μm C-18 (100 mm × 4.6 mm). A mobile phase [0.01 mol l⁻¹ Tris buffer of pH 7.9:acetonitrile (45:55)] was selected for the separation and determination of studied anaesthetics and their impurities. Chromatograms were recorded for 500 s by means of an amperometric detector at a potential of +1.0 V of the glassy carbon electrode versus the reference electrode Ag/AgCl. The proposed liquid chromatographic method was successfully applied to the analysis of commercially available pharmaceutical preparations. The limit of the detection for 2,6-dimethylaniline and o-toluidine was 0.8 ng ml⁻¹. The limit of qantitation, considering a signal to noise ratio was 1.5 ng ml⁻¹. The method developed in this study is sensitive and selective and can be applied to routine studies of pharmaceuticals in the form of cream and injection.     

Analysis of flurbiprofen, ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug-protein binding in human plasma

A stereoselective reversed-phase high-performance liquid chromatography (HPLC) assay to determine the enantiomers of flurbiprofen, ketoprofen and etodolac in human plasma was developed. Chiral drug enantiomers were extracted from human plasma with liquid–liquid extraction. Then flurbiprofen and ketoprofen enantiomers reacted with the acylation reagent thionyl chloride and pre-column chiral derivatization reagent (S)-(−)--(1-naphthyl)ethylamine (S-NEA), and etodolac enantiomers reacted with S-NEA using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C₁₈ (4.6 mm × 250 mm, 5 μm) column with a mixture of acetonitrile–0.01 mol·L⁻¹ phosphate buffer (pH 4.5) (70:30, v/v) for flurbiprofen enantiomers, acetonitrile–0.01 mol·L⁻¹ phosphate buffer (pH 4.5) (60:40, v/v) for ketoprofen enantiomers and methonal–0.01 mol·L⁻¹ potassium dihydrogen phosphate buffer (pH 4.5) (88:12, v/v) for etodolac enantiomers as mobile phase. The flow of mobile phase was set at 0.8 mL·min⁻¹ and the detection wavelength of UV detector was set at 250 nm for flurbiprofen and ketoprofen enantiomers and 278 nm for etodolac enantiomers. The assay was linear from 0.5 to 50 μg·mL⁻¹ for each enantiomer. The inter- and intra-day precision (R.S.D.) was less than 10% and the average extraction recovery was more than 87% for each enantiomer. The limit of quantification for the method was 0.5 μg·mL⁻¹ (R.S.D. < 10%, n = 5). The method developed was used to study the drug–protein binding of flurbiprofen, ketoprofen and etodolac enantiomers in human plasma. The results showed that the stereoselective binding of etodolac enantiomer was observed and flurbiprofen and ketoprofen enantiomers were not.     

HPLC-DAD and LC-ESI-MS analysis of doxycycline and related impurities in doxipan mix, a medicated premix for incorporation in medicated feedstuff

HPLC-DAD and LC-ESI-MS methods have been developed for the analysis of doxycycline (DOX), including the identification of the related impurities metacycline (MTC) and 6-epidoxycycline (EDOX) and its determination in a medicated premix. The chromatographic separations have been performed on Luna C₁₈ stationary phase and on Synergi (4 μm) Polar-RP 80A, using both acidic (pH 2.5) and basic (pH 8.0) mobile phases. The Synergi Polar-RP column, in combination with a mobile phase of oxalic acid (0.02 M; pH 2.5)–acetonitrile 82:18 (v/v), allowed the complete separation of MTC, EDOX and DOX. The same separation was also obtained using Luna C₁₈ stationary phase with a pH 8 mobile phase. Application of a LC-ESI-MS system and MS/MS analysis, using both positive and negative polarity, allowed the peak identity to be confirmed. A method based on Luna C₁₈ column and UV detection at 346 nm was validated for the determination of DOX in a medicated premix for incorporation in medicated feedstuff.     

Determination of imperatorin in rat plasma by reversed-phase high-performance liquid chromatography after oral administration of Radix Angelicae dahuricae extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of imperatorin in rat plasma and applied to a pharmacokinetic study in rats after administration of Radix Angelicae dahuricae extract. After addition of fluocinonide as an internal standard (IS), plasma samples were extracted with diethyl ether. HPLC analysis of the extracts was performed on a Diamonsil C18 analytical column using methanol–water (70:30, v/v) as the mobile phase. The UV detector was set at 254 nm. The standard curve was linear over the range 0.04–4.0 μg/mL. The lower limit of quantification was 0.04 μg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of imperatorin in rat plasma after giving the animals Radix Angelicae dahuricae extract.     

An HPLC method for the determination of atorvastatin and its impurities in bulk drug and tablets

A simple high-performance liquid chromatographic (HPLC) method was developed for the analysis of atorvastatin (AT) and its impurities in bulk drug and tablets. This method has shown good resolution for AT, desfluoro-atorvastatin (DFAT), diastereomer-atorvastatin (DSAT), unknown impurities and formulation excipients of tablets. A gradient reverse-phase HPLC assay was used with UV detection. Some solvent systems prepared using methanol or acetonitrile and water or buffer systems with different pH values were tested. Capacity factors of related substances were calculated at all tested systems. Best resolution has been determined using a Luna C₁₈ column with acetonitrile–ammonium acetate buffer pH 4-tetrahydrofuran (THF) as mobile phase. Samples were eluted gradiently with the mobile phase at flowrate 1.0 ml min⁻¹ and detected at 248 nm. The proposed method was applied to the determination of impurities and were found to contain 0.057–0.081, 0.072–0.097, 0.608–0.664% of the DFAT, DSAT and total impurity, respectively.     

Simultaneous determination of 10 active components in traditional Chinese medicine "YIGONG" capsule by RP-HPLC-DAD

A simple, reliable and accurate method for the simultaneous separation and determination of 10 active components (psoralen, isopsoralen, emodin, oleanolic acid, stachydrine hydrochloride, ammonium glycyrrhizinate, glycyrrhizinate, schizandrol, imperatorin and isoimperatorin) in traditional Chinese medicine “YIGONG” capsule was developed using reverse phase high-performance liquid chromatography (RP-HPLC) coupled with diode array detection. The chromatographic separation was performed on a Eurospher C₁₈ column (250 mm × 4.6 mm i.d. with 5.0 μm particle size) with a acetonitrile–water gradient containing 0.5% (v/v) aqueous phosphoric acid as mobile phase. Two detection wavelengths (210 and 250 nm) were utilized for the quantitative analysis due to the different UV spectra of these components. Good linear behaviors over the investigated concentration ranges were observed with the values of R² higher than 0.999 for all the analytes. The recoveries, measured at three concentration levels, varied from 95.0 to 105.3%. The validated method was successfully applied to the simultaneously determination of these active components in “YIGONG” capsule from different production batches.     

Simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation by liquid chromatography

A rapid and accurate liquid chromatographic method is described for the simultaneous determination of roxithromycin and ambroxol hydrochloride in a new tablet formulation. Chromatographic separation of the two drugs was achieved on a Diamonsil™ C₁₈ column (200 mm×4.6 mm, 5 μm). The mobile phase consisting of a mixture of acetonitrile, methanol and 0.5% ammonium acetate (39:11:50 (v/v), pH 5.5) was delivered at a flow rate of 1.0 ml/min. Detection was performed at 220 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 201.2–2012.0 μg/ml for roxithromycin and 42.7–427.0 μg/ml for ambroxol hydrochloride, respectively. Separation was complete in less than 10 min. The proposed method can be used for the quality control of formulation products.     

Simultaneous separation and determination of coenzyme Q(10) and its process related impurities by NARP-HPLC and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS)

A non-aqueous reversed phase high performance liquid chromatographic (NARP-HPLC) method for determination of coenzyme Q₁₀ in pharmaceutical preparations has been developed using Kromosil C₈ column with acetonitrile and isopropyl alcohol (84:16, v/v) as a mobile phase. Photodiode array (PDA) detector set at 210 nm was used for monitoring of the eluents. The method is simple, rapid, selective and capable of separating all process impurities at trace level with detection limits <0.1 μg/ml. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 50–300 μg/ml. The percentage recoveries ranged from 95.10 to 101.02. The method was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of coenzyme Q₁₀. For identification of related substances atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS) was used.     

A validated stability indicating LC method for oxcarbazepine

The present paper describes the development of a stability indicating reversed phase liquid chromatographic (RPLC) method for oxcarbazepine in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of oxcarbazepine was observed under base hydrolysis. The drug was found to be stable to other stress conditions attempted. Successful separation of the drug from the synthetic impurities and degradation product formed under stress conditions was achieved on a C18 column using mixture of aqueous 0.02 M potassium dihydrogen phosphate–acetonitrile–methanol (45:35:20, v/v/v) as mobile phase. The developed HPLC method was validated with respect to linearity, accuracy, precision, specificity and robustness. The developed HPLC method to determine the related substances and assay determination of oxcarbazepine can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of oxcarbazepine.     

Simultaneous determination of triamcinolone acetonide and oxymetazoline hydrochloride in nasal spray formulations by HPLC

A high-performance liquid chromatography (HPLC) method with UV detection at 232 nm was developed and validated for the simultaneous determination of triamcinolone acetonide (TAA) and oxymetazoline hydrochloride (OXY) in nasal spray formulations. The chromatographic system consisted of a μBondapak™ CN column (150 mm × 3.9 mm), 5 μm particle size with a mobile phase composition of acetonitrile:ammonium acetate (pH 5.0, 20 mM) (10:90, v/v) at a flow rate of 1.0 mL/min. Calibration curves were linear for both TAA and OXY in the concentration range of 2.5–25.0 μg/mL. The limit of detection and quantitation were 0.29 and 0.88 μg/mL for OXY and 0.24 and 0.73 μg/mL for TAA. The described method was further applied to the analysis and stability studies of two nasal spray formulations I and II prepared from TAA and OXY commercial nasal spray products. The stability of OXY and TAA in the commercial products and the nasal formulations I and II were analyzed after 30 days at room temperature and 30 days at 40 °C/60% relative humidity. The results of the stability study showed that OXY and TAA in the commercial nasal spray products and the nasal formulations I and II were stable at 20–25 °C (room temperature) but TAA was unstable at 40 °C/60% relative humidity. TAA exhibited more than 10% loss at 14 days in both the nasal formulations and in the commercial products. OXY showed increased degradation at 40 °C/60% relative humidity but <10%.     

Method development and validation of repaglinide in human plasma by HPLC and its application in pharmacokinetic studies

In this study, the development and validation of a high-performance liquid chromatography (HPLC) assay for determination of repaglinide concentration in human plasma for pharmacokinetic studies is described. Plasma samples containing repaglinide and an internal standard, indomethacin were extracted with ethylacetate at pH 7.4. The recovery of repaglinide was 92% ± 55.31. Chromatographic separations were performed on Purospher^® STAR C-18 analytical column (4.8 mm × 150 mm; 5 μm particle size). The mobile phase composed of acetonitrile–ammonium formate (pH 2.7; 0.01 M) (60:40, v/v). The flow rate was 1 ml/min. The retention time for repaglinide and indomethacin were approximately 6.2 and 5.3 min, respectively. Calibration curves of repaglinide were linear in the concentration range of 20–200 ng/ml in plasma. The limits of detection and quantification were 10 ng/ml and 20 ng/ml, respectively. The inter-day precision was from 5.21 to 11.84% and the intra-day precision ranged from 3.90 to 6.67%. The inter-day accuracy ranged 89.95 to 105.75% and intra-day accuracy ranged from 92.37 to 104.66%. This method was applied to determine repaglinide concentration in human plasma samples for a pharmacokinetic study.     

Pharmacokinetics and tissue distribution study of orientin in rat by liquid chromatography

A simple HPLC–UV method was established for the determination of orientin in plasma and different tissues of rat (heart, liver, spleen, lung, kidney, brain, stomach and small intestine). The separation was achieved by HPLC on a C₁₈ column with a mobile phase composed of acetonitrile–0.1% acetic acid (20:80, v/v), UV detection was used at 348 nm. Good linearity was found between 0.250–50.0 μg/ml (r² = 0.9966) for plasma samples and 0.050–50.0 μg/ml (r² ≥ 0.9937) for the tissue samples, respectively. Within- and between-day precisions expressed as the relative standard deviation (R.S.D.) for the method were 2.3–9.6% and 3.0–7.4%, respectively. The relative recoveries of orientin ranged from 95.4 to 100.6% for plasma and 93.1 to 107.9% for tissue homogenates. The developed method was successfully applied to the pharmacokinetics and tissue distribution research after intravenous administration of a 20 mg/kg dose of orientin to healthy Sprague–Dawley rats. The main pharmacokinetics parameters obtained presented that orientin was quickly distributed and eliminated within 90 min after intravenous administration. The tissue distribution results showed that liver, lung and kidney were the major distribution tissues of orientin in rats, and that orientin had difficulty in crossing the blood–brain barrier. It was also found that there was no long-term accumulation of orientin in rat tissues.     

Simultaneous determination of cinnarizine and domepiridone maleate from tablet dosage form by reverse phase ion pair high performance liquid chromatography

A new simple, precise, rapid and selective reverse phase ion pair high performance liquid chromatography (HPLC-RP) method has been developed for the simultaneous determination of cinnarizine (CINN) and domepiridone maleate (DOME) from tablets using acetonitrile–methanol–water–0.1 N sulfuric acid (37:10:48:5 v/v/v/v) containing sodium lauryl sulfate (0.01 M), as a mobile phase and a Machery Nagel nitrile column (10 μ, 25 cm×4.0 mm i.d.) as the stationary phase. The flow of mobile phase through the column was kept at 1.0 ml min⁻¹ through out the analysis. Detection was carried out using a UV detector at 225 nm. The retention times for CINN and DOME were 4.73 and 9.41 min, respectively. The linearity range and percentage recoveries for CINN and DOME were 4–1000 and 60–750 μg ml⁻¹ and 99.90 and 99.60%, respectively.     

HPLC method for enantioselective analysis of cloprostenol

A new HPLC method for the separation and quantification of cloprostenol enantiomers was developed. The optimized separation system consisted of Chiralcel OD-RH column and acetonitrile–sodium dihydrogenphosphate (pH 3.0; 20 mM) (33:67, v/v) as the mobile phase. Baseline resolution of (±)-cloprostenol (R = 2.16) was achieved and the analysis time did not exceed 10 min. Limits of detection and quantification were units of μmol/l at 274 nm. The respective values decreased an order of magnitude at 210 nm. The R.S.D. values obtained for the retention factor, peak area and peak height of each enantiomer were less than 2%. Conditions for semipreparative separation of the enantiomers can be achieved easily just by a small adaptation of the mobile phase composition.     

Simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column

A simple, rapid and convenient high performance liquid chromatographic method, which permits the simultaneous determination of paracetamol, 4-aminophenol and 4-chloracetanilide in pharmaceutical preparation has been developed. The chromatographic separation was achieved on porous graphitized carbon (PGC) column using an isocratic mixture of 80/20 (v/v) acetonitrile/0.05 M potassium phosphate buffer (pH 5.5) and ultraviolet detection at 244 nm. Correlation coefficient for calibration curves in the ranges 1–50 μg ml⁻¹ for paracetamol and 5–40 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide were >0.99. The sensitivity of detection is 0.1 μg ml⁻¹ for paracetamol and 0.5 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide. The proposed liquid chromatographic method was successfully applied to the analysis of commercially available paracetamol dosage forms with recoveries of 98–103%. It is suggested that the proposed method should be used for routine quality control and dosage form assay of paracetamol in pharmaceutical preparations. The chromatographic behaviour of the three compounds was examined under variable mobile phase compositions and pH, the results revealed that selectivity was dependent on the organic solvent and pH used. The retention selectivity of these compounds on PGC was compared with those of octadecylsilica (ODS) packing materials in reversed phase liquid chromatography. The ODS column gave little separation for the degradation product (4-aminophenol) from paracetamol, whereas PGC column provides better separation in much shorter time.