Synchronized separation of seven medications representing most commonly prescribed antihypertensive classes by using reversed‐phase liquid chromatography: Application for analysis in their combined formulations

A reversed‐phase high‐performance liquid chromatography method was developed for the simultaneous determination of the diuretic, hydrochlorothiazide, along with six drugs representing the most commonly prescribed antihypertensive pharmacological classes such as atenolol, a selective β₁ blocker, amlodipine besylate, a calcium channel blocker, moexipril hydrochloride, an angiotensin‐converting‐enzyme inhibitor, valsartan and candesartan cilexetil, which are angiotensin II receptor blockers, and aliskiren hemifumarate, a renin inhibitor, using irbesartan as an internal standard. The chromatographic separation was achieved using acetonitrile/sodium phosphate dibasic buffer (0.02 M, pH 5.5) at a flow rate of 1 mL/min in gradient elution mode at ambient temperature on a stationary phase composed of an Eclipse XDB‐C₁₈ (4.6 × 150 mm, 5 μm) column. UV detection was carried out at 220 nm. The method was validated according to ICH guidelines. Linearity, accuracy, and precision were satisfactory over the concentration ranges of 2–40 μg/mL for hydrochlorothiazide and candesartan cilexetil, 20–120, 10–160, 5–40, 20–250, and 5–50 μg/mL for atenolol, valsartan, moexipril hydrochloride, aliskiren hemifumarate, and amlodipine besylate, respectively. The method was successfully applied for the determination of each of the studied drugs in their combined formulations with hydrochlorothiazide. The developed method is suitable for the quality control and routine analysis of the cited drugs in their pharmaceutical dosage forms.     

Chromatographic behavior of doxylamine succinate, phenylpropanolamine hydrochloride, chlorpheniramine maleate, dextromethorphan hydrobromide, paracetamol, and guaifenesin in ion pair reverse-phase high performance liquid chromatography

A study of the chromatographic behavior of some cough syrup ingredients has led to the optimum chromatographic separation of four ingredients (doxylamine succinate, phenylpropanolamine hydrochloride, chlorpheniramine maleate, and dextromethorphan hydrobromide). The paracetamol and guaifenesin were overlapping under all chromatographic conditions. The application of 1% chlorotrimethylsilane in methanol to the column (Partisil 5 CCS/C₈) was found to improve the column efficiency significantly. This separation can be applied for the analysis of cough syrup for these ingredients after a study of the interferences due to normal excipients.²     

Quantitative analysis of ripasudil hydrochloride hydrate and its impurities by reversed‐phase high‐performance liquid chromatography after precolumn derivatization: Identification of four impurities

We report the development and validation of a stability‐indicating reversed‐phase high‐performance liquid chromatography method with precolumn derivatization for the separation and identification of the impurities of ripasudil hydrochloride hydrate, a novel protein kinase inhibitor. 2,3,4,6‐Tetra‐O‐acetyl‐β‐d ‐glucopyranosyl isothiocyanate was chosen as the derivatizing reagent and triethylamine was added as catalyst. 200 μL sample solution (1 mg/mL), 600 μL derivatizing reagent (1 mg/mL), and 200 μL triethylamine solution (1%, v/v) were mixed and reacted at 40°C for 30 min. The separation was achieved on an Inertsil C₁₈ ODS‐3 (250 mm × 4.6 mm, 5 μm) column using mobile phases including 10 mmol monopotassium phosphate buffer (pH 3.0) and methanol in gradient mode. The column temperature was adjusted at 25°C and the flow rate at 1 mL/min. The detection was carried out at 220 nm. Different precolumn derivatization conditions as well as the high‐performance liquid chromatography conditions were optimized. Ripasudil hydrochloride hydrate and its four impurities were detected and quantitated, among which two new compounds were characterized. The proposed method was validated and proven to be selective, accurate, and precise and suitable for the quantitative analysis of ripasudil hydrochloride hydrate.     

The Simultaneous Determination of Phenylephrine Hydrochloride,Paracetamol, Chlorpheniramine Maleate and Dextromethorphan Hydrobromide in Pharmaceutical Preparations

A new rapid and sensitive method has been developed and validated for the simultaneous determination of phenylephrine hydrochloride, paracetamol, chlorpheniramine maleate and dextromethorphan hydrobromide in pharmaceutical preparations. The separation was achieved on a C18 column using a gradient mobile phase of acetonitrile–sodium perchlorate (pH 3, 0.01 M) at a flow rate of 1.4 mL min⁻¹. Detection was at 204 nm. Pseudoephedrine hydrochloride was selected as internal standard. The recovery of the drugs ranged from 97.8 to 100.9%. Central composite design was used during validation to calculate method robustness and the percentage of sodium perchlorate, temperature and flow rate were investigated as factors. The method was found to be applicable for the determination of the four compounds in sugar-coated tablets.

     

Analysis of duloxetine hydrochloride and its related compounds in pharmaceutical dosage forms and in vitro dissolution studies by stability indicating UPLC

A reproducible gradient reversed-phase ultra-performance liquid chromatographic method is developed for quantitative determination of duloxetine hydrochloride in pharmaceutical dosage forms. The method is also applicable for analysis of related substances and for study of in vitro dissolution profiles. Chromatographic separation is achieved on a 50 mm × 4.6 mm, 1.8 μm C-18 column. Mobile phase A contains a mixture of 0.01 M KH(2)PO(4) (pH 4.0) buffer, tetrahydro furan, and methanol in the ratio 67:23:10 (v/v/v), respectively, and mobile phase B contains a mixture of 0.01 M KH(2)PO(4), (pH 4.0) buffer, and acetonitrile in the ratio 60:40 (v/v), respectively. The flow rate is 0.6 mL/min, and the detection wavelength is monitored at 236 nm. Resolution of duloxetine hydrochloride and three potential impurities is greater than 2.0 for all pairs of components. The drug was subjected to ICH prescribed hydrolytic, oxidative, photolytic, and thermal stress conditions. Method is validated for linearity, specificity, accuracy, precision, ruggedness, and robustness.     

Rapid separation and determination of process-related substances of paracetamol using reversed-phase HPLC with photo diode array as a detector.

A simple and rapid gradient reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of paracetamol and its related compounds in bulk drugs and pharmaceutical formulations has been developed. As many as nine process impurities and one degradation product of paracetamol have been separated on a Symmetry C18 column (4.6 x 250 mm i.d., particle size 5 microm) with gradient elution using 0.01 M potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile as mobile phase and photo diode array detection at 215 nm. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH values. The correlation coefficients for calibration curves for paracetamol as well as impurities were in the range of 0.9951 - 0.9994. The proposed RP-LC method was successfully applied to the analysis of commercial formulations; the recoveries of paracetamol were in the range of 99-101%. The method could be of use not only for rapid and routine evaluation of the quality of paracetamol in bulk drug manufacturing units but also for detection of its impurities in pharmaceutical formulations.     

Comparison of C18 silica and multi‐walled carbon nanotubes as the adsorbents for the solid‐phase extraction of Chlorpyrifos and Phosalone in water samples using HPLC

A comparison between C₁₈ silica and multi‐walled carbon nanotubes (MWCNTs) in the extraction of Chlorpyrifos and Phosalone in environmental water samples was carried out using HPLC. Parameters affecting the extraction were type and volume of elution solvent, pH and flow rate of sample through the adsorbent. The optimum conditions obtained by C₁₈ cartridge for adsorption of these pesticides were 4 mL dichloromethane as elution solvent, sample pH of 5, flow rate of 1 mL/min, and those for MWCNT cartridge were 3 mL dichloromethane, pH of 5 and flow rate of 10 mL/min, respectively. Optimized mobile phase for separation and determination of these compounds by HPLC was methanol/water (80:20 v/v) with pH=5 (adjusted with phosphate buffer). Under optimal chromatographic and SPE conditions, LOD, linear range and precision (RSD n=8) were 3.03×10^?3, 0.01–5.00 μg/mL and 2.7% for Chlorpyrifos and 4.03×10^?4, 0.01–5.00 μg/mL and 2.3% for Phosalone, in C₁₈ cartridge, respectively. These values for MWCNT were 4.02×10^?6, 0.001–0.500 μg/mL and 1.8% for Chlorpyrifos and 1.02×10^?6, 0.001–0.500 μg/mL and 1.5% for Phosalone, respectively.     

A Stability-Indicating LC Method for Analysis of Balsalazide Disodium in the Bulk Drug and in Pharmaceutical Dosage Forms

A novel, sensitive, stability-indicating gradient RP-LC method has been developed for quantitative analysis of balsalazide disodium and its related impurities both in the bulk drug and in pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C₁₈ stationary phase with a simple mobile-phase gradient prepared from methanol and phosphate buffer (10 mm potassium dihydrogen orthophosphate monohydrate, adjusted to pH 2.5 by addition of orthophosphoric acid). The mobile-phase flow rate was 1.0 mL min^?1. Quantification was achieved by use of ultraviolet detection at 240 nm. Under these conditions resolution of balsalazide disodium from its three potential impurities was greater than 2.0. Regression analysis resulted in a correlation coefficient greater than 0.99 for balsalazide disodium and all three impurities. This method was capable of detecting the three impurities at 0.003% of the test concentration of 0.3 mg mL^?1, using an injection volume of 10 μL. Inter-day and intra-day precision for all three impurities and for balsalazide disodium was within 2.0% RSD. Recovery of balsalazide disodium from the bulk drug (99.2–101.5%) and from pharmaceutical dosage forms (99.8–101.3%), and recovery of the three impurities (99.1–102.1%) was consistently good. The test solution was found to be stable in 70:30 (v/v) methanol–water for 48 h. When the drug was subjected to hydrolytic, oxidative, photolytic, and thermal stress, acidic and alkaline hydrolysis and oxidizing conditions led to substantial degradation. The RP-LC method was validated for linearity, accuracy, precision, and robustness.     

Stability‐indicating chromatographic methods for determination of flecainide acetate in the presence of its degradation products; isolation and identification of two of its impurities

In this work, two stability‐indicating chromatographic methods have been developed and validated for determination of flecainide acetate (an antiarrhythmic drug) in the presence of its degradation products (flecainide impurities; B and D). Flecainide acetate was subjected to a stress stability study including acid, alkali, oxidative, photolytic and thermal degradation. The suggested chromatographic methods included the use of thin layer chromatography (TLC‐densitometry) and high‐performance liquid chromatography (HPLC). The TLC method employed aluminum TLC plates precoated with silica gel G.F₂₅₄ as the stationary phase and methanol–ethyl acetate–33% ammonia (3:7:0.3, by volume) as the mobile phase. The chromatograms were scanned at 290 nm and visualized in daylight by the aid of iodine vapor. The developed HPLC method used a RP‐C₁₈ column with isocratic elution. Separation was achieved using a mobile phase composed of phosphate buffer pH 3.3–acetonitrile–triethylamine (53:47:0.03, by volume) at a flow rate of 1.0 mL/min and UV detection at 292 nm. Factors affecting the efficiency of HPLC method have been studied carefully to reach the optimum conditions for separation. The developed methods were validated according to the International Conference on Harmonization guidelines and were applied for bulk powder and dosage form. Copyright © 2016 John Wiley & Sons, Ltd.     

Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate

The current work entails development of rapid, sensitive, and inexpensive high-performance liquid chromatographic method of quercetin dihydrate using the quality by design approach. Quality target method profile was defined and critical analytical attributes (CAAs) were earmarked. Chromatographic separation was accomplished on a C₁₈ column using acetonitrile and ammonium acetate buffer (35:65) %v/v (containing 0.1% acetic acid, pH 3.5) as mobile phase at 0.7?mL/min flow rate with UV detector at 237?nm. Screening studies using fractional factorial design revealed that organic modifier, injection volume, column temperature, and buffer strength have significant influence on method CAAs, namely, peak area, retention time, and peak tailing. The critical method parameters were systematically optimized using Box–Behnken design. Response surface mapping was used along with numerical optimization and desirability function for identifying the optimal chromatographic conditions. Linearity was observed in the drug concentration ranging between 2 and 50?µg/mL. Accuracy analysis revealed mean % recovery between 93.6 and 96.2%, while precision study revealed mean % recovery between 93.7 and 96.5%. Limits of detection and quantification of the developed method were found to be 12.1 and 36.6?ng/mL. Overall, the studies construed successful development of chromatographic method of quercetin with enhanced method performance.     

Simultaneous determination of aceclofenac, paracetamol, and chlorzoxazone by RP-HPLC in pharmaceutical dosage form

A simple, rapid, and precise reversed-phase liquid chromatographic method is developed for simultaneous determination of paracetamol, aceclofenac, and chlorzoxazone in their ternary mixtures of commercial pharmaceutical preparation. This method uses a Zorbax SB C18, 250 x 4.6 mm, 5 microm analytical column. Mobile phase is acetonitrile and buffer (40:60, v/v), buffer containing 50 mM orthophosporic acid; pH of the buffer is adjusted to 6 with 10% w/v sodium hydroxide solution. The instrumental settings are at a flow rate of 1 mL/min; the column temperature is 25 degrees C, and detector wavelength is 270 nm. The sample concentrations are measured on weight basis to avoid the internal standard. The method is validated and shown to be linear. The correlation coefficients for paracetamol, aceclofenac, and chlorzoxazone are 0.9981, 0.9990, and 0.9986, respectively. The recovery values for paracetamol, aceclofenac, and chlorzoxazone ranged from 100.7-101.4%, 100.4-101.0%, and 100.5-101.3%, respectively. The relative standard deviation for six replicates is always less than 2%. This HPLC method is successfully applied to the simultaneous quantitative analysis of the title drugs in tablets and can be applied for assay and dissolution test of tablets for the estimation of paracetamol, aceclofenac, and chlorozoxazone in their commercial samples.     

Simultaneous high-performance liquid chromatographic determination of paracetamol, phenylephrine HCl, and chlorpheniramine maleate in pharmaceutical dosage forms

A rapid, precise, and specific high-performance liquid chromatographic method is described for the simultaneous determination of paracetamol, phenylephrine HCI, and chlorpheniramine maleate in combined pharmaceutical dosage forms. The method involves the use of a microBondapak CN RP analytical column (125 A, 10 microm, 3.9 x 150 mm) at 22 degrees C as the stationary phase with the mixture of acetonitrile and phosphate buffer (pH 6.22, 78:22) as the mobile phase. Derivatization of the drugs is not required. The method is applied to commercial pediatric cough-cold syrups, tablets, and capsules marketed in Turkey. The relative standard deviation for 10 replicate measurements of each drug in the medicaments is always less than 2%.     

Ultra high-performance liquid chromatography of porphyrins

An ultra high‐performance liquid chromatographic (UHPLC) system was developed and optimized for the separation of porphyrins of clinical interest. Optimum conditions for the simultaneous separation of uroporphyrin, hepta‐, hexa‐, penta‐carboxylic acid porphyrins and coproporphyrin and their type I and III isomers on a Thermo Hypersil BDS C₁₈ column (2.4 µm particle size, 100 × 2.1 mm i.d.) using a gradient elution with 10% (v/v) acetonitrile in 1.0 m ammonium acetate buffer (pH 5.16) and 10% (v/v) acetonitrile in methanol at a flow‐rate of 0.4 mL/min. The effect of mobile phase buffer molarity on the sensitivity of fluorescence detection and resolution of porphyrin isomers was investigated. The method was successfully applied to the analysis of porphyrins extracted from the urine and faeces of patients with various human porphyrias. Copyright © 2011 John Wiley & Sons, Ltd.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

High‐performance liquid chromatographic analysis: applications to nutraceutical content and urinary disposition of oxyresveratrol in rats

A high‐performance liquid chromatographic (HPLC) method was developed for the analysis of the stilbene, oxyresveratrol. This method involves the use of a Luna® C₁₈ column with ultraviolet detection at 320 nm. The mobile phase consisted of acetonitrile, water and formic acid (30 : 70 : 0.04 v/v) with a flow rate of 0.6 mL/min. The calibration curves were linear over the range of 0.5–100.0 μg/mL. The mean extraction efficiency was between 98.9 and 109%. The precision of the assay was 0.069–18.4% (RSD%), and within 20% at the limit of quantitation (0.5 μg/mL). The bias of the assay was <15% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and to nutraceutical product analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

New approach for determination of the degradation products of fenspiride hydrochloride found in oral liquid formulations

Fenspiride hydrochloride (FNS) is used in treating chronic inflammatory diseases, most commonly as a liquid oral solution. FNS produces degradation products along with fenspiride N‐oxide (FNO) and 1‐phenylethyl‐4‐hydroxy‐4‐aminomethyl piperidine hydrochloride (PHAP). We aimed to develop and validate a chromatographic method in order to identify the main degradation products in the presence of other compounds from a liquid preparation. The method used a dual gradient using two buffer solutions: the first with pH 4.5 (buffer 1, pH 4.5–MeOH 90:10%, v/v) and the second with pH 2.9 (buffer 2, pH 2.9–acetronitrile–methanol, 65:15:10%, v/v/v). As mentioned, there was a modification of the organic mixture, starting with 10% methanol and ending with a mixture of acetonitrile–methanol (15:10%, v/v). The flow‐rate was 1.5 mL/min. According to the elution program, experimental conditions started with 100% solution S1, which decreased to 0% and, simultaneously, solution S2 increased to 100% during the first 10 min and was maintained for a further 5 min. After 15 min, initial conditions were re‐established. The linearity interval was 0.5–2 μg/mL and the minimum correlation coefficient was 0.999. The recovery factor was 100.47–103.17% and the limit of quantification was 0.19–0.332 μg/mL. Intra‐day maximum precision was 4.08% for FNS and 2.65% for PHAP. This double‐gradient mobile phase produced good specificity in relation to the degradation products of FNS and other constituents of the oral liquid formulation. Forced degradation studies revealed other related substances that were confirmed in mass balance analyses. Degradation products were confirmed in acidic, basic and oxidative media.     

A Selective LC Method for the Determination of Reboxetine in Human Plasma with Fluorescence Detection

A rapid, simple, accurate, sensitive and reproducible high performance liquid chromatographic method for the quantitation of reboxetine (REB) in human plasma using fluvoxamine as an internal standard (IS) has been developed and validated. The method is based on derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The NBD-derivatives in plasma were extracted by liquid–liquid extraction and chromatographed on a reversed phase C₁₈ column with isocratic elution using acetonitrile and aqueous nitric acid (pH 3) solution. Calibration curve was linear over the range 2.0–200.0 ng mL⁻¹ with inter- and intra-assay precision (RSD%) of less than 4%. The mean recovery was about 94% for REB. The applicability of the method to the plasma was also studied.

     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Stress Degradation Behavior of Desipramine Hydrochloride and Development of Suitable Stability-Indicating LC Method

A simple reverse phase liquid chromatographic method was developed for the quantitative determination of desipramine hydrochloride and its related impurities in bulk drugs which is also stability-indicating. During the forced degradation at hydrolysis, oxidative, photolytic and thermal stressed conditions, the degradation results were only observed in the oxidative stress condition. The blend of the degradation product and potential impurities were used to optimize the method by an YMC Pack Pro C₁₈ stationary phase. The LC method employs a linear gradient elution with the water–acetonitrile–trifluoroacetic acid as mobile phase. The flow rate was 1.0 mL min^?1 and the detection wavelength 215 nm. The stressed samples were quantified against a qualified reference standard and the mass balance was found close to 99.0% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. desipramine). The developed RP-LC method was validated in agreement with ICH requirements.     

High-Performance Liquid Chromatographic Determination of Cefepime and Cefazolin in Human Plasma and Dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of cefepime and cefazolin in human plasma and dialysate. For component separation, the method utilized a C₁₈ column with an aqueous mobile phase of dibasic potassium hydrogen phosphate (pH 7.0) and methanol gradient at a flow rate of 1 mL min⁻¹. The method demonstrated linearity from 2.0 to 100.0 μg mL⁻¹ (r > 0.999) with detection limit of 1 μg mL⁻¹ for both cefepime and cefazolin. The method was utilized for evaluation of plasma and dialysate samples in a clinical study evaluating the dialyzer clearance of cefepime and cefazolin using high-flux hemodialysis with varying blood flow rates in chronic kidney failure patients undergoing hemodialysis and peritoneal dialysis treatment.