A fast and green reversed‐phase HPLC method with fluorescence detection for simultaneous determination of amlodipine and celecoxib in their newly approved fixed‐dose combination tablets

A fast, green, sensitive, and accurate analytical method using high‐performance liquid chromatography couple with fluorescence detection was established and validated for the simultaneous determination of amlodipine besylate and celecoxib in their recently approved fixed‐dose combination tablets (1:20). Separation of the two drugs was achieved on C₁₈ reversed‐phase column (Thermo ODS Hypersil, 4.6 × 250 mm, particle size 5 µm) using acetonitrile:potassium phosphate buffer (50 mM; pH 5.5, 60:40 v/v) as a mobile phase at 40°C, which eluted at a rate of 1 mL/min. Detection was carried out with excitation and emission wavelengths of 360 and 446 nm for amlodipine and 265 and 359 nm for celecoxib, respectively. The method was linear over a concentration range of 0.05‐2 and 0.05‐10 µg/mL and limit of detection reached to 0.017 and 0.0167 µg/mL for amlodipine and celecoxib, respectively. The developed method was successfully applied to assess the cited drugs in their newly FDA approved fixed‐dose combination tablet dosage form. Furthermore, the method was found to be sensitive and eco‐friendly green alternative to the reported methods as it was evaluated according to the green analytical procedure index tool guidelines and analytical Eco‐Scale.     

Green micellar HPLC analysis of three angiotensin‐converting enzyme inhibitors in their mixtures with hydrochlorothiazide and modeling of their retention behavior by fitting to Foley's model

We present an environmentally friendly method for the analysis of three angiotensin‐converting enzyme inhibitors and hydrochlorothiazide simultaneously using a green micellar eluent for the first time. The chromatographic separation of enalapril maleate, lisinopril dihydrate, benazepril hydrochloride, and hydrochlorothiazide was implemented on an octadecyl silica column with a solution containing sodium dodecyl sulfate (0.12 M), 1‐propyl alcohol (10% v/v), triethylamine (0.3% v/v), and H₃PO₄ (0.02 M) at pH 3.6 as the mobile phase and UV detection at 210 nm. Validity of the method was confirmed and it exhibited good linearity within the ranges of 5.0–50.0 μg/mL for hydrochlorothiazide and 10.0–60.0 μg/mL for the three angiotensin‐converting enzyme inhibitors with a limit of detection of 0.39 to 1.15 μg/mL for all the studied drugs. The developed micellar high‐performance liquid chromatography method enables the quantification of the targeted angiotensin‐converting enzyme inhibitors in combined tablets with hydrochlorothiazide by isocratic elution. There is no need for special precautions to prevent broadening and splitting of their chromatographic peaks. The method fulfills the society rights for safe and green analytical methods. The retention behavior of the four studied drugs was fitted to Foley's model and their association equilibria to the micelles (K _AM) and to the surface‐modified stationary phase (K _AS) were calculated.     

Simultaneous Determination of Losartan Potassium, Atenolol and Hydrochlorothiazide in Pharmaceutical Preparations by Stability-Indicating UPLC

A stability-indicating UPLC method was developed for the simultaneous quantitative determination of losartan potassium, atenolol, and hydrochlorothiazide in pharmaceutical dosage forms in the presence of degradation products. The separation was achieved on a simple isocratic method (water: acetonitrile: triethyl amine: ortho phosphoric acid (60:40:0.1:0.1, v/v) at 0.7 mL min^?1, a detection wavelength of 225 nm). The retention times of losartan potassium, atenolol, and hydrochlorothiazide were 2.3, 0.6 and 0.9 min. The total runtime was 3 min. Losartan potassium, atenolol, and hydrochlorothiazide were subjected to different ICH prescribed stress conditions. The method was validated with respect to linearity, accuracy, precision, robustness and ruggedness.     

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.     

Development of a method for the determination of vardenafil in human plasma by high performance liquid chromatography with UV detection

A simple high‐performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed‐phase Kromasil KR 100 C₁₈ (5 µm particle size) column using a mobile phase of acetonitrile–potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10–1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of chemometric approach for development and validation of high performance liquid chromatography method for estimation of ropinirole hydrochloride

A systematic Quality by Design approach was employed for developing an isocratic reversed‐phase liquid chromatographic technique for the estimation of ropinirole hydrochloride in bulk drug and pharmaceutical formulations. LiChrospher RP 18‐5 Endcapped column (25 cm × 4.6 mm id) at ambient temperature (25 ± 2°C) was used for the chromatographic separation of the drug. The screening of factors influencing chromatographic separation of the active pharmaceutical ingredient was performed employing fractional factorial design to identify the influential factors. Optimization of the selected factors was carried out using central composite design for selecting the optimum chomatographic conditions. The mobile phase employed was constituted of Solvent A/Solvent B (65:35 v/v) (Solvent A [methanol/0.05 M ammonium acetate buffer, pH 7, 80:20 v/v] and Solvent B [high performance liquid chromatography grade water]) and used at 0.6 mL/min flow rate, while UV detection was performed at 250 nm. Linearity was achieved in the drug concentration range 5–100 µg/mL (R² = 0.9998) with limits of detection and quantification of 1.02 and 3.09 µg/mL, respectively. Method validation was performed as per ICH guidelines followed by forced degradation studies, which indicated good specificity of the developed method for detecting ropinirole hydrochloride and its possible degradation products in the bulk drug and pharmaceutical formulations.     

Determination of tryptophan in bee pollen and royal jelly by high‐performance liquid chromatography with fluorescence detection

A method for tryptophan analysis in bee pollen and royal jelly was developed using HPLC with fluorescence detection. To determine the free tryptophan in bee pollen and royal jelly, ultrasonic extraction was performed using water (pH 6.3)–acetonitrile (10:1, v/v) as extraction solvent. While determining the total tryptophan in these bee products, the method involves alkaline hydrolysis of the proteins with 4 mol/L sodium hydroxide at 110°C for 20 h under anaerobic conditions. The operating conditions for the HPLC analysis were: Symmetry C₁₈ column (4.6 × 250 mm, 5 µm), 0.1% trifluoroacetic acid–methanol (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min at 30°C. The fluorescence detector was operated at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. A linear response (r² > 0.9998) was obtained in the range 0.0625–5.0 µg/mL. The method was successfully applied to the determination of the free and total tryptophan contents in bee pollens, which were 0.069 ± 0.003 and 2.693 ± 0.476 mg/g, respectively, while only the total tryptophan was detected in royal jelly, with a content of 1.743 ± 0.066 mg/g. Copyright © 2009 John Wiley & Sons, Ltd.     

RP-HPLC-UV validation method for levofloxacin hemihydrate estimation in the nano polymeric ocular preparation

The specific and accurate reversed-phase HPLC-UV method has been validated to determine levofloxacin hemihydrate (LEVH) level. The separation was conducted at C 18 analytical column by administering mobile phase acetonitrile, methanol, and phosphate buffer (pH 3) with the ratio of 17:3:80. The flow rate of the mobile phase was 1 mL/min with a UV detector at 295 nm wavelength. Analytical methods validation evaluated includes specificity, linearity, accuracy, precision, LOD, LOQ, and robustness. The implementation of the analytical method was employed to determine LEVH level in ocular polymeric nanoparticles preparations. The test was specific for LEVH with the retention time of 7.66 min. Linearity was obtained from the concentration range of 4.8 µg/mL to 29.04 µg/mL. All method validation criteria are within the acceptable range. The developed method can be applied for LEVH polymeric nano-formulation analysis.     

HPLC determination of irbesartan in human plasma: its application to pharmacokinetic studies

A simple and rapid HPLC method using fluorescence detection was developed for determination of irbesartan in human plasma. Sample preparation was accomplished through a simple deproteinization procedure with 0.4 mL of acetonitrile containing 800 ng/mL of losartan (internal standard), and to a 0.1 mL plasma sample. Chromatographic separation was performed on a Zorbax Xclipse XDB C₁₈ column (150 × 4.6 mm, i.d., 5 µm) at 40°C. An isocratic mobile phase, acetonitrile:0.1% formic acid (37:63, v/v), was run at a flow‐rate of 1.0 mL/min, and the column eluent was monitored using a fluorescence detector set at excitation and emission wavelengths of 250 and 370 nm, respectively. The retention times of irbesartan and losartan were 4.4 and 5.9 min, respectively. This assay was linear over a concentration range of 10–5000 ng/mL with a lower limit of quantification of 10 ng/mL. The coefficient of variation for this assay precision was less than 8.48%, and the accuracy exceeded 94.4%. The mean relative recoveries of irbesartan and losartan were 98.4 and 99.1%, respectively. This method was successfully applied for pharmacokinetic study after oral administration of irbesartan (300 mg) to 23 Korean healthy male volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

Hansen solubility parameters for assay method optimization of simvastatin,ramipril, atenolol,hydrochlorothiazide and aspirin in human plasma using liquid chromatography with tandem mass spectrometry

A simple, specific, sensitive, validated method was developed using liquid chromatography with tandem mass spectrometry with electrospray ionization of human plasma for the simultaneous estimation of drugs (simvastatin, ramipril, atenolol, hydrochlorothiazide, and aspirin) of Polycap^TM capsule used in cardiovascular therapy. The interaction of these actives including internal standards between the stationary and mobile phase were investigated using Hansen solubility parameters. Chromatographic separation was performed on Phenomenex Synergi Polar‐RP (30 × 2 mm, 4 μm) column with a gradient mobile phase composition of acetonitrile and 5 mM ammonium formate for positive mode and 0.1% formic acid in both water and acetonitrile for negative mode. The flow rate and runtime were 1.0 mL/min and 3.5 min, respectively. Sample extraction was done by protein precipitation using acetonitrile, enabling a fast analysis. The calibration ranges from 0.1 to 100, 0.1 to 100, and 1 to 1000 ng/mL for simvastatin, ramipril, and atenolol using internal standard carbamazepine in positive mode, respectively, whereas it was 0.3–300 and 2–2000 ng/mL for hydrochlorothiazide and aspirin using internal standard 7‐hydroxy coumarin in negative mode, respectively. Hansen solubility parameters can be used as a high‐throughput optimizing tool for column and mobile phase selection in bioanalysis. This validated bioanalytical method has the potential for future fixed dose combination based preclinical and clinical studies that can save analysis time.     

Quantitative separation of oxytocin,norfloxacin and diclofenac sodium in milk samples using capillary electrophoresis

A simple, sensitive and rapid method has been developed for simultaneous separation and quantification of three different drugs: oxytocin (OT), norfloxacin (NOR) and diclofenac (DIC) sodium in milk samples using capillary electrophoresis (CE) with UV detection at 220 nm. Factors affecting the separation were pH, concentration of buffer and applied voltage. Separation was obtained in less than 9 min with sodium tetraborate buffer of pH 10.0 and applied voltage 30 kV. The separation was carried out from uncoated fused silica capillary with effective length of 50 cm with 75 µm i.d. The carrier electrolyte gave reproducible separation with calibration plots linear over 0.15–4.0 µg/mL for OT, 5–1000 µg/mL for NOR and 3–125 µg/mL for DIC. The lower limits of detection (LOD) were found to be 50 ng/mL for OT, and 1 µg/mL for NOR and DIC. The method was validated for the analysis of drugs in milk samples and pharmaceutical preparations with recovery of drugs within the range 96–100% with RSD 0.9–2.8%. Copyright © 2009 John Wiley & Sons, Ltd.     

Applying green analytical chemistry for development and validation of RP-HPLC stability indicating assay method for estimation of fenoverine in bulk and dosage form using quality by design approach

A green and robust reverse-phase liquid chromatographic method has been developed for the determination of fenoverine (FEN), by applying combined principles of green analytical chemistry and quality by design approaches on a Spherisorb C18 column (150?×?4.6?mm, 3?µm) with UV detection at 262?nm. A two level fractional factorial design (2^^7-3) Res IV was used for screening of influential chromatographic factors. The critical method parameters actively affecting critical quality attributes (CQAs) were identified and further optimized using Box–Behnken design. The predicted optimum assay conditions comprised of methanol and ammonium acetate buffer 20?mM, in an extent of 81:19% v/v individually having a flow rate of 1.0?mL/min with a column oven temperature of 33°C. The drug was stressed in hydrolytic, oxidative, reductive, thermal, and photolytic conditions. The developed method was validated successfully. The detector response was linear in the concentration of 0.5–160?µg/mL with a limit of detection (LOD) and limit of quantitation (LOQ) as 0.1 and 0.3?µg/mL, respectively. The % recovery was found to be 99.7%. The analytical method volume intensity value for developed method was 45?mL and the environment assessment tool (EAT) score was 41.07. The method is simple, environmentally benign, rapid, and robust for the determination of FEN in bulk and in its dosage form.     

Determination of levetiracetam in human plasma by online heart‐cutting liquid chromatography: Application to therapeutic drug monitoring

Levetiracetam is an antiepileptic drug for the treatment of psychiatric patients. In this study, a selective, straightforward, and rapid online heart‐cutting liquid chromatography method was developed for the therapeutic drug monitoring of levetiracetam. This method allows for the determination of levetiracetam in human plasma without complex sample preparation. The mobile phases consisted of 30 mM aq. orthophosphoric acid solution/methanol (70:30) at a flow rate of 1 mL/min for the first system and 10 mM aq. orthophosphoric acid solution/methanol (55:45) at a flow rate of 1 mL/min for the second system. The first separation was carried out on a GL Sciences Intersil ODS‐3 column (4.6 mm × 150 mm, 3 µm) and the second separation was carried out on a Restek Ultra PFPP column (4.6 mm × 150 mm, 5 µm). The detection was carried out at 205 nm for both systems. The method was validated for selectivity and linearity, which were in the 6–60 µg/mL range. Intra‐ and interassay accuracies were <112.6%, and the intra‐ and interassay precisions were <6.4% for all quality control samples. The lower limit of quantitation was 6 µg/mL. The developed method was successfully applied for therapeutic drug monitoring of plasma samples from patients.     

Determination of ASP3258, a novel phosphodiesterase type 4 inhibitor,in rat plasma by high‐performance liquid chromatography with fluorescence detection and its application to pharmacokinetic study

The potent phosphodiesterase 4 inhibitor ASP3258 contains a carboxylic acid moiety and a naphthyridine ring and is a novel therapeutic agent for asthma and chronic obstructive pulmonary disease. To support the drug development of ASP3258, we developed and validated a simple method for its determination in rat plasma. Following the addition of the analog AS1406604‐00 as an internal standard, plasma samples were processed using C₁₈‐bonded solid‐phase extraction cartridges under acidic conditions and injected into a high‐performance liquid chromatography system with fluorescence detection. Chromatographic separation was achieved on a Shiseido Capcell Pak C₁₈ UG120 column (3.0 × 150 mm, 5 µm) with a mobile phase consisting of acetonitrile–0.5% acetic acid (50:50, v/v). HPLC eluent was monitored with a fluorescence detector set at a wavelength of 315 nm for excitation and 365 nm for emission. The calibration curve was linear over a range of 2.5–250 ng/mL. Validation data demonstrated that the method is selective, sensitive and accurate. In addition, the present method was successfully applied to rat plasma samples from a pharmacokinetic study. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Analysis of meloxicam by high-performance liquid chromatography with cloud-point extraction

A simple cloud-point extraction method for the determination of meloxicam in human serum was developed. Meloxicam was extracted from serum sample after adding 1 mL of 3% (v/v) Triton X-114 aqueous solution in the presence of 1M HCl and 60 mg NaCl. The meloxicam, present in the surfactant-rich phase, was enriched again with acetonitrile. Tenoxicam was used as the external standard. The separation was achieved on a C18 analytical column with a mobile phase consisting of aqueous acetic acid (1%, v/v) and acetonitrile (54:46, v/v). UV detection was performed at 360 nm. The response was linear over the range 45–2000 ng mL⁻¹ in human serum, and intra- and interday precisions of less than 15.0% were obtained. The relative error was within ±3.0%. The recoveries of meloxicam were larger than 92.0%. The method was compared with liquid–liquid extraction. The results showed that the new method has a considerable LOQ and higher recoveries but poorer precision than liquid–liquid extraction, which exhibited poor recoveries of less than 86.0%, precisions of less than 5.0% and relative errors of less than 7.0%. The method was used for the determination of meloxicam in healthy human volunteers.     

An HPLC method for the quantitation of 3‐pentylbenzo[c]thiophen‐1(3H)‐one in dog plasma and its application to a pharmacokinetic study

A simple, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method was developed for the estimation of 3‐pentylbenzo[c]thiophen‐1(3H)‐one (S₅), a potential anti‐ischemic stroke agent, in dog plasma. The analytical procedure involves protein precipitation of S₅ and nobiletin (internal standard) from dog plasma with acetonitrile. Chromatographic separation was achieved on Sapphire C18 analytical column with methanol–water (80:20, v/v) as mobile phase. The eluate was monitored using a UV detector set at 260 nm. The calibration curves were linear over the range of 0.2–20 µg/mL. Absolute recoveries of S₅ were 79.2–86.1% from dog plasma. The intra‐ and inter‐day relative standard deviation precisions were <7 and 5%, respectively. The method was successfully applied to the pharmacokinetic study of S₅ in beagle dogs. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of four hormonal compounds in oral contraceptive tablet formulations by high performance liquid chromatography

A rapid, accurate, and precise HPLC method has been developed for simultaneous determination of four contraceptive hormonal compounds namely ethinylestradiol (EE), drospirenone (DR), gestodene (GS), and levonorgestrel (LV) in oral contraceptive tablet dosage form. The chromatographic separation was achieved on a C18 (150 × 4.6 mm, 5μ) column; the mobile phase consists of acetonitrile: water (50:50, v/v) pumped at a flow rate of 1.0 mL/min; and UV detection was set at 200 nm. The limit of detection was 0.0086 µg/mL for (EE), 0.0397 µg/mL for (GS), 2.80 µg/mL for (DR), and 0.229 µg/mL for (LV), whereas the limit of quantitation (LOQ) was 0.028 µg/mL for (EE), 0.132 µg/mL for (GS), 9.500 µg/mL for (DR), and 0763 µg/mL for (LV), respectively. The correlation coefficient (r) values of the four compounds ranged from 0.99995 to 0.99999. The method was validated as per ICH guidelines and USP 34 for estimation of (EE), (DR), (GS), and (LV) in commercially available tablet dosage form. The validation results were found satisfactory. The proposed method can be useful in quality control of bulk manufacturing and pharmaceutical dosage forms.     

Separation and simultaneous determination of seven bioactive components in Tripterygium wilfordii Hook. F. and Tripterygium preparations by micellar electrokinetic capillary chromatography

A simple, comprehensive, and highly selective MEKC method has been developed for simultaneous analysis of seven bioactive components (triptolide, wilfortrine, wilfordine, wilforgine, wilforine, triptophenolide, and triptonide) in the root extracts of Tripterygium wilfordii Hook. F. (TWHF) and Tripterygium preparations (TPs). Optimal BGE consisted of 10 mM sodium tetraborate, 30 mM SDS, and 30% v/v methanol. The separation voltage was 20 kV and the temperature was 25°C. A DAD was used and the detection wavelength was at 218 nm. Under the optimum conditions, the baseline separation of seven components was achieved in less than 26 min. Excellent precision, good stability, and accuracy were obtained. For all analytes, linear calibrations were established within 10–100 μg/mL. The LOD and LOQ were within 1.2–4.2 μg/mL and 4.0–14 μg/mL, respectively. The developed method was suitable for the determination of key components in TWHF and TPs.     

Determination of metoprolol in human plasma and urine by high‐performance liquid chromatography with fluorescence detection

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol in human plasma and urine. Separation of metoprolol and atenolol (internal standard) was achieved on an Ace C₁₈ column (5 μm, 250 mm×4.6 mm id) using fluorescence detection with λ_ex=276 nm and λ_em=296 nm. The mobile phase consists of methanol–water (50:50, v/v) containing 0.1% TFA. The analysis was performed in less than 10 min with a flow rate of 1 mL/min. The assay was linear over the concentration range of 3 – 200 and 5 – 300 ng/mL for plasma and urine, respectively. The LOD were 1.0 and 1.5 ng/mL for plasma and urine, respectively. The LOQ were 3.0 and 5.0 ng/mL for plasma and urine, respectively. The extraction recoveries were found to be 95.6 ± 1.53 and 96.4 ± 1.75% for plasma and urine, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.