Validated LC Method for Simultaneous Analysis of Tramadol Hydrochloride and Aceclofenac in a Commercial Tablet

This paper describes development and validation of a high-performance liquid chromatographic method for simultaneous analysis of tramadol hydrochloride (TR) and aceclofenac (AC) in a tablet formulation. When the combination formulation was subjected to ICH-recommended stress conditions, adequate separation of TR, AC, and the degradation products formed was achieved on a C₁₈ column with 65:35 (v/v) 0.01 M ammonium acetate buffer, pH 6.5—acetonitrile as mobile phase at a flow rate of 1 mL min^?1. UV detection was performed at 270 nm. The method was validated for specificity, linearity, LOD and LOQ, precision, accuracy, and robustness. The method was specific against placebo interference and also during forced degradation. The linearity of the method was investigated in the concentration ranges 15–60 μg mL^?1 (r = 0.9999) for TR and 40–160 μg mL^?1 (r = 0.9999) for AC. Accuracy was between 98.87 and 99.32% for TR and between 98.81 and 99.49% for AC. Because degradation products were well separated from the parent compounds, the method was stability-indicating.     

Development of a Validated Stability-Indicating LC Assay and Stress Degradation Studies of Linezolid in Tablets

This paper describes the validation of an isocratic LC method for the assay of linezolid in tablets. Validation parameters such as linearity, precision, accuracy, specificity, limit of detection, limit of quantitation and robustness were determined. LC was carried out by reversed phase technique on an RP-18 column with a mobile phase composed of 1% acetic acid:methanol:acetonitrile (50:25:25, v/v/v). Linezolid and your combination drug product were exposed to acid, base, oxidation, dry heat and photolytic stress conditions. A linear response (r > 0.9999) was observed in the range of 8.0–20.0 μg mL⁻¹. The retention time of linezolid was 4.6 min. The method showed good recoveries and intra- and inter-day relative standard deviations were less than 1.0%. The LOD and LOQ were 0.21 and 0.63 μg mL⁻¹, respectively. The developed LC method for determination of related substances and assay determination of linezolid can be used to evaluate the quality of regular production samples. It can also be used to test the stability samples of linezolid.

     

Application of Multicriteria Methodology in the Development of Improved RP-LC-DAD for Determination of Rizatriptan and Its Degradation Products

A simple, rapid, and stability-indicating reversed-phase high-performance liquid chromatographic (LC) method for analysis for dutasteride has been successfully developed. Chromatography was performed on a 150 mm × 4.6 mm C₁₈ column with acetonitrile–water 60:40 (v/v) as isocratic mobile phase at 1.0 mL min⁻¹. Ultraviolet detection of dutasteride was at 210 nm. Its retention time was approximately 10 min and its peak was symmetrical. Response was a linear function of concentration over the range 0.2–1 μg mL⁻¹ (R ² = 0.997) and the limits of detection and quantitation were was 0.05 and 0.10 μg mL⁻¹, respectively. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting dutasteride stock solution to photolytic, acidic, basic, oxidative, and thermal degradation. The peaks from the degradation products did not interfere with that from dutasteride. The method was used to quantify dutasteride in pharmaceutical preparations.

     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min⁻¹ and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL⁻¹ (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL⁻¹, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.

     

Development and Validation of a Stability-Indicating LC Method for Determination of Ebastine in Tablet and Syrup

A simple stability-indicating reversed-phase liquid chromatographic method with diode-array detection was developed and validated for the quantitative determination of ebastine in tablets and syrup. The LC method was carried out on a C₁₈ column with acetonitrile:phosphoric acid 0.1% pH 3.0 (55:45, v/v) as mobile phase, at a flow rate of 1.2 mL min⁻¹. Ultraviolet detection of ebastine was at 254 nm. A linear response (r = 0.9999) was observed in the range of 10–80 μg mL⁻¹. The RSD values for intra- and inter-day precision studies showed good results (RSD < 2%) and accuracy was greater than 98%. Validation parameters such as specificity and robustness were also determined. The method was found to be stability-indicating and can be applied to quantitative determination of ebastine in tablets and syrup.

     

HPLC-ELSD Determination of Kryptofix 2.2.2 in the Preparations of 2-Deoxy-2-[18F]fluoro-d-glucose and other Radiopharmaceuticals

A new and simple high-performance liquid chromatography with evaporative light scattering detector method for the determination of Kryptofix 2.2.2 (K-222) in the radiopharmaceuticals of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) and 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) was developed. A C₁₈ column was used and the mobile phase was 10 % (v/v) methanol and 90 % (v/v) water (0.1 % trifluoroacetic acid, v/v) at a flow rate of 0.2 mL min⁻¹. The drift tube temperature was 40 °C. The pressure of nebulizing gas (N₂) was 3.0 bar. The gain was 10. Good separation of K-222 from main related substances could be achieved. Excellent linearity (r ² = 0.9995) was obtained over the range of 5–100 μg mL⁻¹. The precision ranged from 0.68 to 5.16 % (RSD) and the accuracy ranged from −3.05 to 2.62 % (RE). The limit of detection was 2 μg mL⁻¹. This method offers simple, rapid and quantitative detection of K-222, thus making it acceptable for routine determination.

     

Stability-Indicating LC Determination of a New Antihypertensive,Olmesartan Medoxomil in Tablets

A stability-indicating liquid chromatographic method was developed and validated for quantitative determination of olmesartan medoxomil (OLM) in coated tablets in the presence of degradation products generated under stress conditions. An isocratic LC separation was performed using a Phenomenex RP-18 column using a mobile phase consisting of water:triethylamine:acetonitrile (60:0.3:40 v/v/v, pH adjusted to 6.3 with phosphoric acid). The flow rate was 1.2 mL min⁻¹ and the detection was achieved with a photodiode array detector set at 257 nm. The response was linear over a range of 10.0 to 30.0 μg mL⁻¹ (r = 0.9999). The specificity and stability-indicating capability of the method was verified subjecting the reference substance and drug product to hydrolytic, oxidative, photolytic, and thermal stress conditions. The method showed a good and consistent recovery (100.2%) with low intra- and inter-day relative standard deviation (RSD) (≤1.0%). A considerable degradation occurred in all stress conditions and the degradation product was well resolved from the main peak. There was no interference of the excipients in the determination of the active pharmaceutical ingredient. Thus, the proposed method was found to be stability-indicating and can be used for routine analysis for quantitative determination of OLM in coated tablets without the interference of major degradation products.

     

Development of a Stability-Indicating LC Assay Method for Determination of Chloroquine

A simple and rapid development of a stability-indicating LC method for determination of chloroquine diphosphate in the presence of its hydrolysis, oxidative and photolysis degradation products is described. Stress testing showed that chloroquine diphosphate was degraded under basic conditions and by photolytic treatment but was stable under the other stress conditions investigated. Separation of the drug from its degradation products was achieved with a Nova Pack C18 column, 0.01 M PIC B7 and acetonitrile (40:60 v/v) pH 3.6, as mobile phase. Response was linear over the range 0.08–5.70 μg mL⁻¹ (r = 0.996), with limits of detection and quantification (LOD and LOQ) of 0.17 and 0.35 μg mL⁻¹, respectively.

     

Development and Validation of a Stability-Indicating HPLC Method for Determination of Ciprofloxacin Hydrochloride and its Related Compounds in Film-Coated Tablets

The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprofloxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250–750 μg mL⁻¹ for ciprofloxacin HCl and 0.5–1.5 μg mL⁻¹ for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 μg mL⁻¹, respectively. Correlation coefficients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL⁻¹ in plasma and 0.32–20 μg mL⁻¹ in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.

     

LC Method for Studies on the Stability of Sibutramine in Soft Gelatin Capsules

A sensitive and rapid liquid chromatographic method was successfully developed and validated for the determination of sibutramine hydrochloride in bulk and capsules. Sibutramine in the presence of its degradation products was analyzed using UV detection at 225 nm. Chromatography was performed on a reversed-phase C₈ (150 × 4.0 mm I.D., 5 μm) analytical column under isocratic conditions. The mobile phase was composed of acetonitrile:water (aqueous phase containing 0.3% triethylamine and pH adjusted to 7.0) (75:25, v/v) at a flow-rate of 1.1 mL min⁻¹. No chromatographic interference was found during the analysis. Light was the stress condition which most contributed to sibutramine degradation. The method showed a linear response (r > 0.999) from 30 to 90 μg mL⁻¹. The mean recovery for capsules was 101.2%. Inter-day assays showed relative standard deviations of 0.42 and 1.62% for bulk and capsules, respectively. The developed method is able to separate sibutramine from its major degradation products and it may be used in the quality control of this active pharmaceutical ingredient in both bulk and capsules.

     

LC Method for Quantification of ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Rabbit Plasma: Validation and Application to a Pharmacokinetic Study

ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic acid (5F), a diterpenoid isolated from the Chinese herb Pteris semipinnata L, has been suggested to show antitumor properties. A simple and sensitive LC method was developed for the determination of 5F in rabbit plasma. The method involved liquid–liquid extraction using ethyl acetate under acidic conditions using naproxen as an internal standard. Separations were performed on a reversed-phase column with a mixture of 1% (v/v) glacial acetic acid and methanol (45:55, v/v) as mobile phase and UV detection was utilized at 242 nm. The calibration plot was linear in the range 0.20–10.0 μg mL⁻¹ (correlation coefficients r ² > 0.998). The detection limit was 0.20 μg mL⁻¹, mean extraction recovery was above 82%, intra-day precision of the method was less than 6.4%, and inter-day precision was better than 8.7%, respectively. The validated assay was found to be suitable for the pharmacokinetic study of 5F in rabbits.

     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min⁻¹. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL⁻¹ for isomer I and 0.40–2.40 µg mL⁻¹ for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL⁻¹ for isomer I and 0.0356 and 0.1078 µg mL⁻¹ for isomer II, respectively.

     

LC Determination of Ketorolac in Human Plasma and Urine for Application to Pharmacokinetic Studies

A simple, specific and sensitive liquid chromatographic method has been developed for the assay of ketorolac in human plasma and urine. The clean-up of plasma and urine samples were carried out by protein precipitation procedure and liquid–liquid extraction, respectively. Separation was performed by a Waters sunfire C₁₈ reversed-phase column maintained at 35 °C. The mobile phase was a mixture of 0.02 M phosphate buffer (pH adjusted to 4.5 for plasma samples and to 3.5 for urine samples) and acetonitrile (70:30, v/v) at a flow rate of 1.0 mL min⁻¹. The UV detector was set at 315 nm. Nevirapine was used as an internal standard in the assay of urine sample. The method was validated over the concentration range of 0.05–8 and 0.1–10 μg mL⁻¹ for ketorolac in human plasma and urine, respectively. The limits of detection were 0.02 and 0.04 μg mL⁻¹ for plasma and urine estimation at a signal-to-noise ratio of 3. The limits of quantification were 0.05 and 0.1 μg mL⁻¹ for plasma and urine, respectively. The extraction recoveries were found to be 99.3 ± 4.2 and 80.3 ± 3.7% for plasma and urine, respectively. The intra-day and inter-day standard deviations were less than 0.5. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay demonstrated to be applicable for clinical pharmacokinetic studies.

     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

Development and Validation of a Stability-Indicating LC Method for Curcumin

A simple, isocratic, stability-indicating liquid chromatographic method for quantitative determination of curcumin was successfully developed. The chromatographic separations were achieved using a Hi-Q-Sil C18; 4.6 mm × 250 mm and 10 μm particle size column employing acetonitrile and acetate buffer (pH 3.0; 60: 40, v/v) as the mobile phase. The analyte was subjected to acidic, basic, oxidative, thermal and photo degradation. The method was validated with respect to linearity, precision, accuracy, limit of detection and limit of quantification. Curcumin was detected by UV-Vis detector at 425 nm whereas the degradation products were detected at 280 nm. The method was linear over the concentration range of 1–10 μg mL^?1. The limit of detection was found to be 0.06 μg mL^?1 and the quantification limit was 0.21 μg mL^?1. Considerable degradation of the analyte was observed when it was subjected to alkaline conditions. Accuracy, evaluated as recovery, was in the range of 97–103%. Intra-day precision and intermediate precision showed relative standard deviations <1% and <2% respectively.     

An LC Ion-Pairing Method for the Determination of Fe(II) in Ferrous Bisglycinate Pharmaceutical Formulation

A reversed-phase ion-pairing liquid chromatographic method was developed and validated for the assay of Fe(II) in ferrous bisglycinate (Fe-bis-gly) capsules using 4-(2-pyridylazo) resorcinol reagent. The analysis was carried out using a Gemini RP-18 (150 mm × 4.6 mm I.D., particle size 5 μm) analytical column; the mobile phase consisted of a mixture of acetonitrile–water (28:72 v/v) containing 1 mM tetrabutylammonium hydrogensulfate and 1% phosphate buffer (pH 8.0). The flow rate was 1.0 mL min⁻¹ and the detection was achieved with a photodiode array (PDA) detector at 706 nm. The specificity of the method was proved using stress conditions and evaluated using a PDA detector. The data validation showed that the method is specific, fast, accurate, and reproducible for the determination of Fe-bis-gly in dosage form. The response was linear over a range of 1.0–2.6 μg mL⁻¹ (r = 0.9999). The accuracy of the method ranged from 98.02 to 102.75%. The RSD values for intra- and inter-day precision studies were below 1.3 and 1.1%, respectively. There was no interference of the excipients on the determination of the active pharmaceutical ingredient.

     

Quantitative Analysis of Toosendanin in the Fruit of Melia toosendan Sieb. Et Zucc (Meliaceae) by High-Performance Liquid Chromatography Coupled with Charged Aerosol Detection

In this work, a simple and rapid high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) method was first developed for the quantitation of toosendanin, the major constituent of the dried fruit of Melia toosendan Sieb. Et Zucc. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 μm) by isocratic elution using 33 % acetonitrile and 67 % water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL min⁻¹. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 °C. The established method was well validated. Satisfactory linearity was achieved (r ² > 0.9997) in a relatively wide concentration range (5–500 μg mL⁻¹). The intra- and inter-day precisions, repeatability, and stability of the method were good with relative standard deviations (RSDs) of 1.05, 2.23, 2.39, and 2.03 %, respectively. The method also showed excellent accuracy with recovery rates of 97.42–101.87 %. Particularly, CAD showed much better sensitivity (LOQ 4 μg mL⁻¹) than evaporative light scattering detector (LOQ 100 μg mL⁻¹) for toosendanin’s determination. The established method was further applied in the quantitation of toosendanin in 39 batches of raw and stir-fried toosendan fructus. The HPLC-CAD method was rapid and accurate, and could be used for the routine analysis and quality control of toosendan fructus and its preparations.

     

Pharmacokinetics and Tissue Distribution Study of Tryptanthrin in Rats by RP-HPLC with DAD Detector

A simple RP-LC-UV method was established for the determination of tryptanthrin in plasma and different tissues of rats. The separation was achieved by HPLC on a C₁₈ column with a mobile phases composed of acetonitrile–water (47:53, v/v), UV detection was used at 251 nm. Good linearity was found between 0.0183–1.1712 μg mL⁻¹ (r ² = 0.999) for plasma and 0.0937–1.7568 μg mL⁻¹ for the tissue samples, respectively (r ² ≥ 0.9932). The intra- and inter-day precisions expressed as the relative standard deviation for the method were 0.92–6.01 and 1.06–9.11 %, respectively. The relative recoveries of tryptanthrin ranged from 95.26 to 97.89 % for plasma and 82.55 to 114.99 % for tissue homogenates (except heart). The developed method was successfully applied to the pharmacokinetics and tissue distribution research after orally administration of a 56-mg kg⁻¹ dose of tryptanthrin to healthy SD rats. The main pharmacokinetics distribution results showed that liver, lung, small intestine, and large intestine were the major distribution tissues of tryptanthrin in rats, and that tryptanthrin had difficulty in crossing the blood–brain barrier.