Development and Validation of High‐Performance Liquid Chromatographic Method for Estimation of Lercanidipine in Rabbit Serum

Abstract

A new, simple, and sensitive reverse‐phase liquid chromatographic method was developed and validated for the estimation of Lercanidipine hydrochloride in rabbit serum using UV detector under isocratic conditions. After subjecting serum to simple and efficient one‐step extraction procedure, 100 µl of sample was injected onto high‐performance liquid chromatography system. The detector response was linear in the concentration range of 25–1000 ng/ml. The developed method was validated as per standard guidelines. Validation demonstrated accuracy, precision, and selectivity of the proposed method. The drug was found to be stable under various processing and storage conditions.     

Development and Validation of a RP-HPLC–PDA Method for Determination of Curcuminoids in Microemulsions

A sensitive, specific and rapid high-performance liquid chromatography method was developed in an effort to quantify extremely low curcuminoid levels for the future transdermal experiments where the curcuminoids are incorporated with excipients such as microemulsion, liposomes, and micelles. The chromatographic separation was performed using a Symmetry^® C₁₈, 250 × 4.6 mm, 5-μm column, with a mobile phase composed of 5 mM acetonitrile:phosphoric acid (45:55, v/v) at a flow rate of 1.0 mL min^?1, it was sensitive with a low limit of quantitation for curcuminoids (0.626 ng mL^?1 for curcumin) and good linearity (r ² ≥ 0.999) over the range 1–100 ng mL^?1. All the validation data, such as accuracy and precision, were within the required limits from the ICH guideline. The assay method was successfully applied during forced degradation of curcuminoid solutions. The method retained its accuracy and precision when the standard addition technique was applied.     

Development and Validation of a RP-HPLC–PDA Method for Determination of Curcuminoids in Microemulsions

A sensitive, specific and rapid high-performance liquid chromatography method was developed in an effort to quantify extremely low curcuminoid levels for the future transdermal experiments where the curcuminoids are incorporated with excipients such as microemulsion, liposomes, and micelles. The chromatographic separation was performed using a Symmetry^® C₁₈, 250 × 4.6 mm, 5-μm column, with a mobile phase composed of 5 mM acetonitrile:phosphoric acid (45:55, v/v) at a flow rate of 1.0 mL min⁻¹, it was sensitive with a low limit of quantitation for curcuminoids (0.626 ng mL⁻¹ for curcumin) and good linearity (r ² ≥ 0.999) over the range 1–100 ng mL⁻¹. All the validation data, such as accuracy and precision, were within the required limits from the ICH guideline. The assay method was successfully applied during forced degradation of curcuminoid solutions. The method retained its accuracy and precision when the standard addition technique was applied.

     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear in the concentration range of 1.00–400 ng mL^?1 in rat plasma, with a 1.00 ng mL^?1 lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from ?2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.     

Development and Validation of a Reverse-phase High Performance Liquid Chromatography Method for Determination of Exenatide in Poly（lactic-co-glycolic acid） Microspheres

Exenatide(synthetic exendin-4), which has been approved by the Food and Drug Administration(FDA) for the adjunctive treatment of patients with type 2 diabetes, is an incretin mimetic agent. The development and validation of a RP-HPLC method for the quantification of the exenatide in poly(lactic-co-glycolic acid)(PLGA) microspheres is described. Separation was performed on a C4 column via a mobile phase consisting of ACN:KH2PO4(0.02 mol/L, pH=2.5) gradient elution from 30:70 to 45:55(volume ratio) in 30 min. Multi-diode array detection(DAD) appears to be most appropriate to evaluate the spectral purity of exenatide. The limits of detection and quantification of exenatide were 0.4 and 1.2 μg/mL, respectively. The calibration curve of exenatide was linear in a range of 0.025―0.2 mg/mL with a correlation coefficient of 0.9995. The results of validation study show that this method is specific, accurate(recovery95%), precise(RSD2.0%) and robust.     

Development and Validation of a GC–MS Method for the Determination of Methyl and Ethyl Camphorsulfonates in Esomeprazole Magnesium

A rapid gas chromatography–mass spectrometry method for the identification and determination of two carcinogenic impurities viz. methyl camphorsulfonate (MCS) and ethyl camphorsulfonate (ECS) in esomeprazole magnesium (EOM) is developed for the first time. The factors affecting method development and the fragmentation patterns of MCS and ECS based on the mass spectral analysis are described. The limit of detection and limit of quantitation values for both compounds were established as 3 and 10 ppm, respectively with respect to 50 mg mL^?1 of EOM. The method is linear within the range of 10–120 ppm and found to be precise, accurate, specific and robust.     

Liquid Chromatographic Method for Determination of Free and Niosome‐Entrapped Nimodipine in Mouse Plasma and Different Tissues

Abstract

A rapid HPLC method for the quantification of nimodipine in mouse plasma and tissues has been developed in this study, with simple procedure of sample preparation by one‐step protein precipitation. The results of HPLC analysis indicated that linear calibration curves were obtained over the concentration range 0.10–10.00 µg/ml for plasma, 0.10–20.00 µg/g for heart, liver, spleen, kidneys, and brain, and 1.00–200.00 µg/g for lung, respectively. The desirable precision and accuracy were achieved, both intraday and interday for plasma and tissue homogenates. Thus, this newly developed procedure was successfully applicable for determination of nimodipine in mouse plasma and tissues following intravenous administration of free and novel niosome‐entrapped nimodipine.     

Development and Validation of an HPTLC–Densitometric Method for Determination of ACE Inhibitors

A high-performance thin layer chromatographic method coupled with densitometric analysis has been developed for measurement of benazepril and cilazapril, both pure and in their commercial dosage forms. The active substances were extracted from tablets with methanol (mean recovery 102%) and chromatographed on silica gel 60 F₂₅₄ HPTLC plates in horizontal chambers with ethyl acetate–acetone–acetic acid–water, 8:2:0.5:0.5 (v/v), as mobile phase. Chromatographic separation of these ACE inhibitors was followed by UV densitometric quantitation at 215 nm. Calibration plots were constructed in the range 0.4 to 2.0 g L^–1 for benazepril (2.0–10.0 g spot^–1) and from 0.5 to 1.5 g L^–1 for cilazapril (4.0–12.0 g spot^–1) with good correlation coefficients (r 0.990). The method was used to determine benazepril and cilazapril in pharmaceutical preparations with satisfactory precision (1.4% < RSD < 5.6%) and accuracy (1.7 < RE < 5.1).     

Development and Validation of a HPLC Method for the Determination of Five Bioactive Compounds in the “Xuebijing” Injection

A reversed phase high performance liquid chromatographic (RP-HPLC) method with ultraviolet (UV) detector was developed for simultaneously determining five bioactive components (i.e., hydroxysafflor yellow A: HSYA; paeoniflorin, ferulic acid, benzoic acid, and danshensu) in “Xuebijing” (XBJ) injection, a widely used traditional Chinese medicine (TCM) for treating sepsis and multiple organ dysfunction syndrome. A Zorbox SB C₁₈ column was used with 0.2% phosphoric acid (V/V)-acetonitrile as the mobile phase under the condition of gradient elution. The five components were analyzed by using a timed wavelength measure according to their maximum absorption wavelength. The intraday and interday precisions of the five investigated compounds were less than 1.17% and the average recoveries ranged from 97.3% to 103.2%. There were good linear correlations between the concentrations of the five components and their chromatographic peak areas (R² ≥ 0.9998), the proposed method was successfully applied to determine the five components in different batches of XBJ injection products, the results indicated that the proposed method is simple, stable, and accurate and could be readily utilized as a quality control method for manufacturing process of XBJ injection.     

Development of Polar Organic Mode Chromatographic Method by Polysaccharide-Based Immobilized Chiral Selector and Validation for the Determination of the Enantiopurity of Novel Mineralocorticoid Receptor Antagonist Atropisomer–Esaxerenone

Esaxerenone is a new nonsteroidal mineralocorticoid receptor antagonist utilized to treat high blood pressure. Chemically, esaxerenone is a pyrrole derivative consisting of hindered rotation, which results in stereoisomers named atropisomers. Currently, no methods exist for the separation and quantification of these atropisomers. A new and accurate chiral liquid chromatographic technique was developed and validated to estimate the enantiomeric purity of esaxerenone. Polar organic chiral separation was carried out on an immobilized amylose-based chiral stationary phase (Chiralpak IG) with methanol:acetonitrile:diethylamine (9:1:0.1, v/v/v) mixture as a mobile phase. The total runtime was 15 min, and the resolution (R_s) between the atropisomers was more than 3.0. The detection and quantification thresholds for the R-atropisomer were found to be 0.03 and 0.1 µg mL^?1, respectively, for a test concentration of esaxerenone (1000 µg mL^?1). Over the range from the limit of quantification to 0.3 percent, the method's linearity for the R-atropisomer was excellent (R²?>?0.999). The R-atropisomer recovery varied from 95 to 102%, confirming the method’s good accuracy. For a 48-h research period, the chemical was shown to be stable.

     

Development and Validation of a High-Performance Thin-Layer Chromatographic—Densitometric Method for the Quantification of Guggulsterones E and Z in a Polyherbal Formulation Containing Commiphora Mukul

JPC – Journal of Planar Chromatography – Modern TLC - The present article focuses on the quantitative analysis of a polyherbal formulation containing arjuna, guggul, and pushkarmoola in...     

Development and Validation of Liquid Chromatography Method for the Separation of Valdecoxib and its SC‐77852 Impurity

Abstract

A reversed‐phase liquid chromatography method has been developed for the separation of valdecoxib and impurity SC‐77852. The best results were achieved using a mobile phase—methanol: 1% water solution TEA (52∶48 v/v), pH 7.35 (adjusted with 85% orthophosphoric acid), column temperature 24°C. Separation was carried out on XTerra? RP₁₈ (150 mm×4,6 mm), particle size 5 µm, flow rate 1 ml/min, using detection on 220 nm. The method was statistically validated for its selectivity, linearity, precision (repeatability), and robustness. Quantitation and detection limits were determined for both valdecoxib and SC‐77852. Method robustness was further evaluated by performing full factorial design experiment. Validated method was used for assay of valdecoxib and SC‐77852 in Bextra^® film‐coated tablets.     

Development and Validation of a High-Performance Thin-Layer Chromatographic—Densitometric Method for the Quantification of Apigenin in Ocimum basilicum L. Seed (Takhmaria)

JPC – Journal of Planar Chromatography – Modern TLC - A simple, selective, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been developed for the...     

Development of an RPLC-based Method for Measurement of Indoprofen Stability and Validation of the Method

A method for quantitative measurement of the photochemical decomposition of the anti-inflammatory agent, indoprofen （INP） is descriped. An RPLC-based assay that could determine the extent of degradation of INP in a rapid, sensitive, and accurate manner was developed. The method was validated under photoirradiation. Quantitation was monitored with an Inertsil ODS-3V column using a mobile phase of acetonitril and 1% HOAc solution in deionized H2O. Statistics relevant to the system criteria, peak integrity and resolution among the parent drug and its degradation products were performed. From the intra- and inter-day tests, the coefficients of variation were found to range from 0.59% to 4.25% for the former and from 0.71% to 4.86% for the latter. The good selectivity and specificity of this RPLC-based procedure render it suitable for measurements of INP stability.     

Development and Validation of Spectrophotometric Methods for the Determination of Cefetamet in Pharmaceutical Dosage Forms

Two simple and sensitive spectrophotometric methods for the determination of cefetamet in either pure form or in its pharmaceutical formulations were described. The method Ⅰ is based on the interaction of 3-methylbenzo[d]- thiazolin-2-one hydrazone （MBTH） with cefetamet in the presence of freshly prepared ferric chloride in a neutral medium. The resulting blue colored product has λmax at 628 nm. The method Ⅱ describes the reduction of ferric ion by the drug to ferrous ion followed by a complex formation reaction with 1,10-phenanthroline （1,10-phen） to form an orange red colored chromogen exhibiting 2max at 510 nm. The products are stable for more than 5 and 8 h respectively. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods. Both methods are highly reproducible and have been applied to a wide variety of pharmaceutical preparations and the results are comparable with those of official methods.     

Development and Validation of a High-Performance Thin-Layer Chromatography—Densitometry Method for the Quantitative Estimation of Morphine in the Classical Ayurvedic Formulation Kamini Vidrawan Ras

JPC – Journal of Planar Chromatography – Modern TLC - Kamini Vidrawan Ras is a classical Ayurvedic medicine, referred to in Bhaisjya Ratnavali, a book recognized by the Drugs and...     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.