Reproducible Reversed-phase High-performance Liquid Chromatography Research Articles

Development and Validation of a RP‐HPLC Method for Determination of Nimodipine in Sustained Release Tablets

A rapid, sensitive, and reproducible reverse phase high performance liquid chromatographic (RP‐HPLC) method with UV detector for the determination of nimodipine in sustained release tablets was developed. The method involved using a SinoChoom ODS‐BP C18 reversed phase column (5 μm, 4.6 mm × 200 mm) and mobile phase consisting of methanol‐acetonitrile‐water (35 : 38 : 27, v/v). The flow rate is 1.0 mL/min, the UV detector was operated at 237 nm, and the column was maintained at 25°C. The method was validated according to official compendia guidelines. The calibration curve of nimodipine for RP‐HPLC method was linear over the range of 10–100 μg/mL. The retention time was found at 7.50 min for nimodipine. The variation for interday and intraday assay was found to be less than 0.72%. The proposed RP‐HPLC was proved to be suitable for the determination of nimodipine in sustained release tablets.

RP-HPLC determination of vildagliptin in pure and in tablet formulation

AimThe present work aims to a simple, rapid and reproducible reverse phase high performance liquid chromatographic (RP-HPLC) method for the estimation of vildagliptin in pure form and in tablet dosage form using Agilent XDB C18, 150 × 4.6 mm, 5 μm, column. MethodsThe mobile phase consists of 0.1 M Phosphate buffer and acetonitrile in the ratio of 85:15% v/v, and was pumped at rate of 1.0 mL/min. The detection was carried out at 210 nm and the calibration curve was linear in the concentration range of 10–150 μg/mL. ResultsThe method was statistically validated for its linearity, precision, accuracy, stability, specificity, LOD and LOQ. Due to its simplicity, rapidness, high precision and accuracy, the proposed RP-HPLC method may be used for determining vildagliptin in pure form and in tablet formulation. ConclusionThe proposed method was found to be simple, precise, accurate and rapid for determination of vildagliptin from pure form and tablet dosage form.

DETERMINATION OF TELMISARTAN AND FORCED DEGRADATION BEHAVIOR BY RP-HPLC IN TABLET DOSAGE FORM

A simple, rapid, precise, sensitive and reproducible reverse phase high performance liquid chromatographic (RP-HPLC) method for determination of Telmisartan (TELM) in tablet dosage form was developed and validated. Chromatographic separation was achieved on a 250 4.6 mm, 5?, Waters symmetry column in gradient mode, with mobile phase consisting of a mixture of solution (10 mM potassium dihydrogen phosphate, pH 3.5 0.01): acetonitrile (64:40) was used. The quantitation performed at flow rate of 1.0 mL/min at 230 nm and run time was 12 min. The analytical method was validated as per ICH guideline for linearity, accuracy, precision, specificity, limit of detection, limit of quantification, robustness and stability and method can be extended to the analysis of TELM in tablet formulations. The relative standard deviation values for precision was less than 2%, and % recovery was greater than 98% for TELM. The drug undergoes oxidative degradation, thermal degradation and in alkali medium.

DETERMINATION OF ATORVASTATINE IN PHARMACEUTICAL FORMULATIONS BY REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, sensitive and reproducible reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitative estimation of Atorvastatin calcium (ATOR-C) in the pharmaceutical formulations. Chromatographic separation was achieved on a 250 4.6 mm, 5?, Waters symmetry column. The flow rate was 1mL/min and eluent was monitored by absorbance at 246.0 nm using a mixture of Methanol and Acetonitrile (pH 3.00.01) in the ratio of 25:75 (v/v). The retention times of ATOR-C was found to be 5.5 min. Calibration plots were linear in the concentration range of 5-25 ?g/mL for ATOR-C calcium. The total run time was 12 min. The proposed method was validated by testing its linearity, recovery, specificity, system suitability, precision (Interday, intraday, analyst and instrument precision), robustness and LOD/LOQ values and it was successfully employed for the determination of ATOR-C in pharmaceutical tablet formulations.

DETERMINATION OF TELMISARTAN IN PHARMACEUTICAL FORMULATIONS BY REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, sensitive and reproducible reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitative estimation of Telmisartan (TELM) in the pharmaceutical formulations. Chromatographic separation was achieved on a 250 × 4.6 mm, 5μ, Waters symmetry column. The flow rate was 1 ml/min and eluent was monitored by absorbance at 230 nm using a mixture of Methanol and Acetonitrile (pH 3.0±0.01) in the ratio of 30:70 (v/v). The retention time of Telmisartan was found to be 7.9 min. Calibration plots were linear in the concentration range of 10-50 μg/ml for Telmisartan with correlation coefficient (R 2 ) 0.999. The proposed method was validated by testing its linearity, recovery, specificity, system suitability, precision (Interday, intraday, analyst and instrument precision), robustness and LOD/LOQ values and it was successfully employed for the determination of Telmisartan in pharmaceutical tablet formulations.

SIMULTANEOUS DETERMINATION OF ATORVASTATIN CALCIUM AND TELMISARTAN IN PHARMACEUTICAL FORMULATIONS BY REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, sensitive and reproducible reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for simultaneous quantitative estimation of Atorvastatin calcium (ATOR) and Telmisartan (TELM) in the pharmaceutical formulations. Chromatographic separation was achieved on a 250 × 4.6 mm, 5μ, Waters symmetry column. The flow rate was 1ml/min and eluent was monitored by absorbance at 267 nm using a mixture of Methanol and Acetonitrile (pH 3.0

STABILITY-INDICATING RP-HPLC METHOD FOR ANALYSIS OF TELMISARTAN IN THE DOSAGE FORM

A simple, rapid, precise, rapid, sensitive and reproducible reverse phase high performance liquid chromatographic (RP-HPLC) method has been developed for quantitative analysis of Telmisartan (TELM) in pharmaceutical dosage forms. Chromatographic separation of TELM and its degradation products was achieved on a C18, 250 × 4.6 mm, 5μ, Waters symmetry column.

Development of a Stability-Indicating RP-HPLC Method for Determination of Rupatadine and its Degradation Products in Solid Oral Dosage Form

A simple, sensitive, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method, coupled with a photodiode array detector, was developed for the determination of rupatadine (RUPA) and its related substances in pharmaceutical dosage forms. Chromatographic separation was achieved on the Hypersil BDS (150 x 4.6 mm, 5 μm) column with a mobile phase containing a gradient mixture of a buffer (acetate buffer pH-6.0) and solvent (methanol). The eluted compounds were monitored at 264 nm for the related substances and assay, the flow rate was 1.0 mL/min, and the column oven temperature was maintained at 50°C. The developed method separated RUPA from its four known and three unknown impurities within 15.0 min. Rupatadine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Rupatadine was found to degrade significantly under oxidative stress conditions, and degrade slightly under acid, base, hydrolytic, thermal, and photolytic stress conditions. All impurities were well-resolved from each other and from the main peak, showing the stability-indicating power of the method. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. The developed and validated RP-HPLC method is LC-MS compatible and can be explored for the identification of eluted unknown impurities of RUPA.

Development and validation of a dissolution test with reversed-phase high performance liquid chromatographic analysis for Candesartan cilexetil in tablet dosage forms

A simple, rapid, selective and reproducible reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the estimation of release of Candesartan cilexetil (CC) in tablets. Analysis was performed on an Agilent, Zorbax C8 column (150mm×4.6mm, 5μm) with the mobile phase consisting of phosphate buffer (pH2.5)–acetonitrile (15:85, v/v) at a flow rate of 1.0mL/min. UV detection was performed at 215nm and the retention time for CC was 2.2. The calibration curve was linear (correlation coefficient=1.000) in the selected range of analyte. The optimized dissolution conditions include the USP apparatus 2 at a paddle rotation rate of 50rpm and 900mL of phosphate buffer (pH7.2) with 0.03% of polysorbate 80 as dissolution medium, at 37.0±0.5°C. The method was validated for precision, linearity, specificity, accuracy, limit of quantitation and ruggedness. The system suitability parameters, such as theoretical plate, tailing factor and relative standard deviation (RSD) between six standard replicates were well within the limits. The stability result shows that the drug is stable in the prescribed dissolution medium. Three different batches (A, B and C) of the formulation containing 8mg of Candesartan cilexetil was performed with the developed method and the results showed no significant differences among the batches.

β-Glucosidase catalyzed synthesis of octyl-β- d-glucopyranoside using whole cells of Pichia etchellsii in micro aqueous media

Octyl-β- d-glucopyranoside was synthesized by transglucosylation between p-nitrophenyl β- d-glucopyranoside ( pNPG) and octanol as an acceptor using whole cells of thermo tolerant yeast Pichia etchellsii displaying cell wall bound β-glucosidase. Effect of several parameters such as glucosyl donor concentration, enzyme units and initial water activity was studied to optimize product yield. An initial water activity interval of 0.33–0.64 was favorable and increase in total enzyme units had marginal effect on conversion yield. An empirical model was developed to describe the relationship between various parameters and octyl glucoside yield. These factors were combined in a batch replacement strategy whereby octyl-β- d-glucopyranoside was synthesized in 4 h to a concentration of 30 mM (9.25 mg/ml) with a conversion yield of nearly 70% with pNPG as a glucosyl donor. Quantitative analysis was done by a highly reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method and detection was achieved using refractive index detector. The structure of the product was confirmed by 13C and 1H NMR spectroscopy. Additional products like octyl diglucoside were also formed, the structure of which was confirmed by mass spectrometry.

Development of RP-HPLC method for the analysis of levocetirizine. 2HCl and ambroxol. HCl in combination and its application

A simple, sensitive isocratic and reproducible reversed phase High Performance Liquid Chromatographic (RP-HPLC) method was developed for the estimation of ambroxol hydrochloride (ABH) and levocetirizine dihydrochloride (LCD) in combination using PDA detector. The system consisted of RP-C18 column and the detection was performed at 230nm. The mobile phase was a mixture of acetonitrile : phosphate buffer solution (60:40) (pH 7.0) pumped at room temperature and a flow rate of 1 ml/min. ABH and LCD were eluted at 2.75 and 5.01 sec respectively. The mean absolute recoveries of ABH and LCD were about 98 % and 99 % respectively and the limit of detection of LCD and ABH in the mixture of given proportion is observed to be 0.1 µg/ml and 1.5 µg/ml and the limit of quantitation is 0.3 µg/ml and 4.5 µg/ml respectively. The calibration was linear over a concentration range of 4.5 µg/ml to 15.0 µg/ml with r2 > 0.997 for ABH and 0.3 µg/ml to 1.0 µg/ml with r2 > 0.999 for LCD. The intra (n = 5) and inter (n = 5) day assay variations in the linear range are < 4 % for ABH and < 6 % for LCD. Three pharmaceutical products containing this combination are analyzed to test the applicability of the new method. The percentage of ABH and LCD in three marketed capsule dosage form studied range from 99 to 102 % and 100 to 103 % and respectively to the claimed value.

A simple and reliable preparative high-performance liquid chromatographic technique for isolation of a bioactive flavone diglycoside from and extract ofCuminum cyminumseeds

Summary In this paper we describe a sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) method with photodiode-array detection for isolation and quantification of the bioactive hydrophilic constituent 7-(1-O-β-d-galacturonide-4′-(1-O-β-d-glucopyranosyl)-3′,4′,5,7-tetrahydroxyflavone, 1, from the seeds of Cuminum cyminum. Compound 1 was separated isocratically on a C18 preparative column, in high purity, after removal of solvents. The purity and identity of the compound were established by use of LC-mass spectrometry and by spectroscopic techniques (1H and 13C NMR). The purity of 1 was also confirmed by HPTLC.

A Simple and Rapid Method for the Determination of Pennogenin Diglycoside in Rat Plasma by HPLC-MS: Application to the Pharmacokinetics of the Extract in Gongxuening Capsules

A sensitive, selective, and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) was developed for the simultaneous quantification of pennogenin diglycoside (PD) in a small volume (100 microL) of rat plasma. PD was extracted from rat plasma samples using liquid-liquid extraction with methanol, digoxin as internal standard. The chromatographic separation was performed on a reversed-phase Agilent C(18) (250 mm x 4.6 mm i.d., 5 mum particle size) analytical column using a mobile phase of 0.1% formic acid solution-acetonitrile (75:25, v/v) at a flow-rate of 1.0 mL/min. The mass spectrometer was operated in the negative ion mode at the deprotonated-molecular ions [M-H]- of parent drug. Calibration curve in spiked plasma was linear (correlation coefficient r = 0.999) from 0.5 to 50.0 mg/mL. For the different samples with concentration of 0.50, 5.00, and 50.0 microg/mL, recoveries of PD were (86.45 +/- 4.39)%, (91.40 +/- 4.40)%, and (93.79 +/- 3.29)%, respectively (n = 3). The intra-day assay relative standard deviation at 0.50, 5.00, and 50.0 mg/mL of PD were 4.29%, 5.66%, and 4.03% (n = 3), respectively. The inter-day assay %RSD at the previously mentioned concentrations were 5.53%, 4.99%, and 4.31% (n = 3), respectively. The method was successfully applied to the pharmacokinetic study of PD in rats following either intravenous administration of PD solution or oral administration of the extract in Gongxuening capsules, a famous patent Chinese botanic drug.

Simultaneous Estimation of Metformin Hydrochloride, Pioglitazone Hydrochloride, and Glimepiride by RP-HPLC in Tablet Formulation

A simple, precise, rapid, and reproducible reversed-phase high-performance liquid chromatography method is developed for the simultaneous estimation of metformin hydrochloride (MET), pioglitazone hydrochloride (PIO), and glimepiride (GLP) present in multicomponent dosage forms. Chromatography is carried out isocratically at 25 degrees C +/- 0.5 degrees C on an Inertsil-ODS-3 (C-18) Column (250 x 4.60 mm, 5 microm) with a mobile phase composed of methanol-phosphate buffer (pH 4.3) in the ratio of 75:25 v/v at a flow rate of 1 mL/min. Detection is carried out using a UV-PDA detector at 258 nm. Parameters such as linearity, precision, accuracy, recovery, specificity, and ruggedness are studied as reported in the International Conference on Harmonization guidelines. The retention times for MET, PIO, and GLP are 2.66 + 0.5 min, 7.12 + 0.5 min, and 10.17 + 0.5 min, respectively. The linearity range and percentage recoveries for MET, PIO, and GLP are 10-5000, 10-150, and 1-10 microg/mL and 100.4%, 100.06%, and 100.2%, respectively. The correlation coefficients for all components are close to 1. The relative standard deviations for three replicate measurements in three concentrations of samples in tablets are always less than 2%.

Quantitative Determination of Reserpine, Ajmaline, and Ajmalicine in Rauvolfia serpentina by Reversed- Phase High-Performance Liquid Chromatography

A sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) method using photodiode array detection is established for the simultaneous quantitation of important root alkaloids of Rauvolfia serpentina, namely, reserpine, ajmaline, and ajmalicine. A Chromolith Performance RP-18e column (100 x 4.6-mm i.d.) and a binary gradient mobile phase composed of 0.01 M (pH 3.5) phosphate buffer (NaH(2)PO(4)) containing 0.5% glacial acetic acid and acetonitrile are used. Analysis is run at a flow rate of 1.0 mL/min with the detector operated at a wavelength of 254 nm. The calibration curves are linear over a concentration range of 1-20 microg/mL (r = 1.000) for all the alkaloids. The various other aspects of analysis (i.e., peak purity, similarity, recovery, and repeatability) are also validated. For the three components, the recoveries are found to be 98.27%, 97.03%, and 98.38%, respectively. The limits of detection are 6, 4, and 8 microg/mL for ajmaline, ajmalicine, and reserpine, respectively, and the limits of quantitation are 19, 12, and 23 microg/mL for ajmaline, ajmalicine, and reserpine, respectively. The developed method is simple, reproducible, and easy to operate. It is useful for the evaluation of R. serpentina.

Determination of midazolam and its major metabolite 1′-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1′-hydroxymidazolam (1′-OHM) in a small volume (200 μl) of human plasma. Midazolam, 1′-OHM and 1′-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid–liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY™ Elite C18 (5 μm particle size; 100 mm × 2.1 mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water–acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 μl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M + l] + of parent drug and metabolite. Calibration curves in spiked plasma were linear ( r 2 ≥ 0.99) from 15 to 600 ng/ml (MDZ) and 5–200 ng/ml (1′-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1′-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4 ± 3.1% ( n = 6) and 84.2 ± 4.7% ( n = 8), respectively; for 1′-OHM at 30 and 200 ng/ml the values were 89.9 ± 7.2% ( n = 6) and 86.9 ± 5.6% ( n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1′-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1′-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).

Determination of alkylphenols and alkylphenol polyethoxylates by reversed-phase high-performance liquid chromatography and solid-phase extraction.

A simple, accurate and reproducible reversed-phase high-performance liquid chromatography (HPLC) method was developed for the separation and characterisation of alkylphenols (APs) and alkylphenol polyethoxylates (APEOs), using a C18 octadecyl silica (ODS) column. APs and each APEO oligomer were separated successfully within a reasonable time without gradient elution. An excellent resolution was obtained, even for mixtures of APs and low EO number APEOs, which are otherwise difficult to separate using conventional normal-phase HPLC methods. This method, combined with solid-phase extraction, was highly applicable for the simultaneous determination of alkylphenols and alkylphenol ethoxylates in real samples.

Rapid and simultaneous high-performance liquid chromatography assay of polyamines and monoacetylpolyamines in biological specimens

A rapid, resolutive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for polyamines and acetylpolyamines by adopting pre-column derivatization with benzoyl chloride. In a single run lasting less than 15 min ten polyamines were separated as well as traces of benzoic acid, methylbenzoate and benzoic anhydride. These contaminants, produced during the derivatization reaction, were almost all eliminated by washing steps envisaged in the same procedure. This simple and sensitive method can be applied to routine determination of polyamines in biological samples. A fine application of this procedure to the determination of endogenous content of polyamines in chick embryo retina was reported.

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min −1. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml −1 in mouse plasma. The inter-day relative standard deviations (RSDs) ( n=5) ranged from 18.4% and 12.1% at 0.05 μg ml −1 to 2.9% and 3.3% at 10.0 μg ml −1 for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma ( n=6) ranged from 8.2% and 14.2% at 0.05 μg ml −1 to 7.6% and 11.5% at 10.0 μg ml −1 for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml −1 with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.

Effect of Various Salts on the Stability of Lansoprazole, Omeprazole, and Pantoprazole as Determined by High-Performance Liquid Chromatography

A fast and reproducible reverse-phase high-performance liquid chromatography (HPLC) assay method has been developed for the simultaneous quantitation of omeprazole, lansoprazole, and pantoprazole. The three compounds were monitored at 280 nm using Zorbax Eclipse XDB C8 (5 μm, 150 cm × 4.6 mm i.d.) and a mobile phase consisting of 700:300 phosphate buffer:acetonitrile with the pH adjusted to 7.0 with phosphoric acid. The method was used to study the effect of pH and various salts on the stability of the three compounds. The pH rate profile curve showed that pantoprazole was the most stable compound and lansoprazole the least stable. The stabilities of the compounds in salt solutions were found to be in the following order: phosphate buffer < trisodium citrate < citrate buffer ≤ acetate buffer < citric acid ≤ monosodium citrate ≤ calcium carbonate < sodium bicarbonate < sodium chloride < water. The rate of degradation had a direct relationship with the H+ and salt concentration.