Optimization of dispersive liquid–liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC‐UV

A simple, rapid, and sensitive method based on dispersive liquid–liquid microextraction combined with HPLC‐UV detection applied for the quantification of chlordiazepoxide in some real samples. The effect of different extraction conditions on the extraction efficiency of the chlordiazepoxide drug was investigated and optimized using central composite design as a conventional efficient tool. Optimum extraction condition values of variables were set as 210 μL chloroform, 1.8 mL methanol, 1.0 min extraction time, 5.0 min centrifugation at 5000 rpm min^?1, neutral pH, 7.0% w/v NaCl. The separation was reached in less than 8.0 min using a C₁₈ column using isocratic binary mobile phase (acetonitrile/water (60:40, v/v)) with flow rate of 1.0 mL min^?1. The linear response (r² > 0.998) was achieved in the range of 0.005–10 μg mL^?1 with detection limit 0.0005 μg mL^?1. The applicability of this method for simultaneous extraction and determination of chlordiazepoxide in four different matrices (water, urine, plasma, and chlordiazepoxide tablet) were investigated using standard addition method. Average recoveries at two spiking levels were over the range of 91.3–102.5% with RSD < 5.0% (n = 3). The obtained results show that dispersive liquid–liquid microextraction combined with HPLC‐UV is a fast and simple method for the determination of chlordiazepoxide in real samples.     

Combined microwave-assisted isolation and solid-phase purification procedures prior to the chromatographic determination of phenolic compounds in plant materials

A fast, sensitive and selective procedure employing a combination of microwave-assisted extraction (MAE) and solid phase extraction (SPE) was applied prior to liquid chromatographic identification and quantification of phenolic compounds in plant materials. MAE has been tested and optimized for the isolation of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanilic, caffeic, syringic, p-coumaric, ferulic, sinapic, benzoic, m-coumaric, o-coumaric, rosmarinic, cinnamic acids) and 3,4-dihydroxybenzaldehyde, syringaldehyde, p-hydroxybenzaldehyde, and vanillin in various plants. The effects of experimental conditions on MAE efficiency, such as solvent composition, temperature, extraction time, have been studied. The extraction efficiencies were compared with those obtained by computer-controlled, two-step Soxhlet-like extractions. Plant extracts were purified and phenolic compounds were pre-concentrated using SPE on polymeric RP-105 SPE sorbent prior to HPLC analysis. Chromatographic separation was carried out on a Hypersil BDS C₁₈ column using a mobile phase consisted of 0.3% (v/v) acetic acid in water (solvent A) and methanol (solvent B) at flow rate 0.6 ml min⁻¹ and column temperature 30 °C with gradient elution.     

Chromatographic separation and analysis of chloropheniramine maleate, methscopolamine nitrate and phenylephrine hydrochloride in sustained release capsules

Summary A high performance liquid chromatographic method has been developed for the simultaneous determination of chlorpheniramine maleate (CPM), methscoplamine nitrate (MSN) and phenylephrine hydrochloride (PEH) in sustained release capsules. The separation was carried out on a reverse-phase CN-column with use of a mobile phase consisting of 70% (v/v) solution of acetonitrile in water containing 2% (v/v) acetic acid and 0.005M sodium 1-hepatane sulfonate at a flow rate of 2 mL min⁻¹. The eluted peaks were detected at 262 nm. The method is sensitive, accurate and rapid and can be used in the routine analysis of the mixture of the three compounds.     

A Simple and Sensitive LC Method for Quantification of Cefteram in Human Plasma

A simple and sensitive liquid chromatographic method was developed for quantification of cefteram in human plasma. Amoxicillin was used as an internal standard. The present method used protein precipitation for extraction of cefteram from human plasma. Separation was carried out on a reversed-phase C₁₈ column. The column effluent was monitored by UV detection at 262 nm. The mobile phase was a mixture of methanol and water containing 0.3% v/v triethylamine and 0.6% v/v glacial acetic acid (35:65:0.3:0.6 v/v) at a flow rate of 0.30 mL min^?1. The column temperature was 20 °C. This method was linear over the range of 47.5–4,750.0 ng mL^?1 with determination coefficient greater than 0.99. The mean extraction recovery of cefteram and IS was ≥76.82 and ≥76.49%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for a pharmacokinetic study of cefteram in human.     

Development and validation of a generic RP‐HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines

A novel generic reverse phase high performance liquid chromatography (RP‐HPLC) method is developed and validated for simultaneous determination of seven pharmaceutically active ingredients, namely, acetaminophen, dextromethorphan, doxylamine, phenylephrine, guaifenesin, caffeine and aspirin. All seven ingredients were quantified in soft gel, syrup and tablet formulations of the over‐the‐counter US‐marketed products, as per the guidelines of the International Conference on Harmonization. The separation was achieved in a 16 min run time on an Agilent Zorbax Phenyl column using a gradient method with two mobile phases. Mobile phase A was 0.15% trifluoro acetic acid in purified water and while mobile phase B was a mixture of acetonitrile and methanol (750:250 v/v) with 0.02% trifluoro acetic acid. The flow rate was 1.0 mL min^?1 and injection volume was 10 μL. Detection was performed at 280 nm using a photodiode array detector. As part of the method validation, specificity, linearity, precision and recovery parameters were verified. The concentration and area relationships were linear (R² > 0.999), over the concentration ranges 20–120 μg mL^?1 for acetaminophen, 75–450 μg mL^?1 for dextromethorphan, 31.25–187.5 μg mL^?1 for doxylamine, 25–150 μg mL^?1 for phenylephrine, 25–150 μg mL^?1 for aspirin, 6.5–39 μg mL^?1 for caffeine and 12–72 μg mL^?1 for guaifenesin. The relative standard deviations for precision and intermediate precision were <1.5%. The proposed RP‐HPLC generic method is applicable for routine analysis of cold and cough over‐the‐counter products.     

A simple and rapid HPLC‐UV method for the determination of retigabine in human plasma

A simple and rapid high‐performance liquid chromatographic method with ultraviolet detection was developed for the quantitative determination of retigabine, known also as ezogabine, in human plasma. The assay uses a simple solid‐phase extraction for sample preparation and direct injection of the extract into the chromatograph. Flupirtine is used as an internal standard. Chromatographic separation is achieved on a C₁₈ Chromolith column (Chromolith Performance, 100 × 4.6 mm i.d.), using as mobile phase water/acetonitrile/methanol (72:18:10 v/v/v) mixed with 0.1% of 85% phosphoric acid. Isocratic elution is conducted at a flow rate of 1.5 mL min⁻¹. The total duration of a chromatographic run is 7 min. Calibration curves are linear over the 25–2000 ng mL⁻¹ concentration range, with a limit of quantitation of 25 ng mL⁻¹. Other performance characteristics include high precision (intra‐ and inter‐day coefficients of variation ≤12.6%) and high accuracy (99.7%–108.7%). The method is suitable for the investigation of concentration–response relationships in patients receiving therapeutic doses of retigabine.     

Determination of N-methylcarbamate insecticides in water samples using dispersive liquid–liquid microextraction and HPLC with the aid of experimental design and desirability function

A rapid and simple method for the extraction and preconcentration of N-methylcarbamates (NMCs) (carbofuran, carbaryl and promecarb) in water samples using dispersive liquid–liquid microextraction (DLLME) using chemometrics was developed. Influence variables such as volume of extracting (CHCl₃) and dispersing solvents (ACN), pH and ionic strength, extraction time and centrifugation time and speed were screened in a 2^7–4 Plackett–Burman design was investigated. The significant variables were optimized by using a central composite design (CCD) combined with desirability function (DF). At optimum conditions values of variables set as 126 μL chloroform, 1.5 mL acetonitrile, 1 min extraction time, 10 min centrifugation at 4000 rpm min⁻¹, natural pH, 4.7% (w/v) NaCl, the separation was reached in less than 14 min using a C₁₈ column and an isocratic binary mobile phase (acetonitrile: water (50:50, v/v)) with flow rate of 1.0 mL min⁻¹. At optimum conditions method has linear response over 0.001–10 μg mL⁻¹ with detection limit between 0.0001 and 0.0005 μg mL⁻¹ with relative standard deviations (RSDs) in the range 2.18–5.06% (n = 6).     

Focused microwave-assisted solvent extraction and HPLC determination of effective constituents in Eucommia ulmodies Oliv. (E. ulmodies)

A new focused microwave-assisted solvent extraction method using water as solvent has been developed for leaching geniposidic and chlorogenic acids from Eucommia ulmodies Oliv. The extraction procedures were optimized using a two indexes orthogonal experimental design and graphical analysis, by varying irradiation time, solvent volume, solvent composition and microwave power. The optimum extraction conditions were obtained: for geniposidic acid, 50% micorwave power, 40 s irradiation, and 80% (v/v) aqueous methanol as extraction solvent (20 ml g⁻¹ sample); and for chlorogenic acid, 50% micorwave power, 30 s irradiation, and 20% aqueous methanol (20 ml g⁻¹ sample). The composition of the extraction solvent was optimized and can be directly used as the mobile phase in the HPLC separation. Quantification of organic acids was done by HPLC at room temperature using Spherigel C₁₈ chromatographic column (, i.d. 5 μm), the methanol:water:acetic acid (20:80:1.0, v/v) mobile phase and UV detection at 240 nm. The R.S.D. of the extraction process for geniposidic and chlorogenic acid were 3.8 and 4.1%, respectively.     

A new high-performance liquid chromatography assay for the determination of sesquiterpene lactone 15-deoxygoyazensolide in rat plasma

A simple, rapid and sensitive high-performance liquid chromatography method was developed for the analysis of the sesquiterpene lactone 15-deoxygoyazensolide (LAC15-D) in rat plasma samples. The chromatographic separation was achieved on a LiChrospher^® RP18 column using methanol:water (50:50, v/v) containing 0.6% acetic acid as mobile phase, at a flow rate of 0.7 mL min⁻¹. UV detection was carried out at 270 nm. Phenytoin was used as internal standard. Prior to the analysis, the rat plasma samples were submitted to liquid-liquid extraction with dichloromethane. The mean absolute recoveries were 73% with R.S.D. values lower than 3.5. The method was linear over the 6.0-2000 ng mL⁻¹ concentration range and the quantification limit was 6.0 ng mL⁻¹. Within-day and between-day assay precision and accuracy were studied at three concentration levels (15, 300 and 480 ng mL⁻¹) and were lower than 15%. The validated method was used to measure the plasmatic concentration of LAC15-D in rats that received a single intraperitoneal dose of 30 mg kg⁻¹.     

Development of Validated Chromatographic Methods for the Simultaneous Determination of Metronidazole and Spiramycin in Tablets

Two chromatographic methods have been described for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in their mixtures. The first method was based on a high performance thin layer chromatographic (HPTLC) separation of the two drugs followed by densitometric measurements of their spots at 240 nm. The separation was carried out on Merck TLC aluminum sheets of silica gel 60 F₂₅₄ using methanol: chloroform (9:1, v/v) as a mobile phase. Analysis data was used for the linear regression line in the range of 1.0–2.0 and 0.8–2.0 μg band⁻¹ for MET and SPY, respectively. The second method was based on a reversed-phase liquid chromatographic separation of the cited drugs on a C-18 column (5 μm, 250 × 4.6 mm, i.d.). The mobile phase consisted of a mixture of phosphate buffer of pH 2.4 and acetonitrile (70:30, v/v). The separation was carried out at ambient temperature with a flow rate of 1.0 mL min⁻¹. Quantitation was achieved with UV detection at 232 nm based on peak area with linear calibration curves at concentration ranges 0.4–50.0 and 0.5–50.0 μg mL⁻¹ for MET and SPY, respectively. The proposed chromatographic methods were successfully applied to the determination of the investigated drugs in pharmaceutical preparations. Both methods were validated in compliance with ICH guidelines; in terms of linearity, accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.

     

Thin-layer chromatographic fingerprinting of the nonvolatile fraction extracted from the medicinal herb Cistus incanus L.

ABSTRACT

The aim of this study was to provide the first from-start-to-end thin-layer chromatographic method of fingerprinting the Cistus incanus L. raw herbal material, with a purpose to further use it for rapid screening, authentication, and quality control of the traded C. incanus L. herbs. To this effect, 12 different C. incanus L. samples purchased as herbal teas from a local market were extracted by means of the accelerated solvent extraction (ASE) with chemometrically optimized solvent extraction mixture and temperature (methanol–water, 27:73, v/v; 130°C), to derive the polar fraction from the plant samples. Then, the extracts were developed in two thin-layer chromatographic systems, both using the commercially precoated silica gel 60 chromatographic plates, yet two different mobile phases (mobile phase 1, ethyl acetate–formic acid–acetic acid–water, 100:11:11:13, v/v/v/v, and mobile phase 2, ethyl acetate–dichloromethane–formic acid–acetic acid–water, 100:10:10:10:11, v/v/v/v/v). The chromatograms were densitometrically scanned in the reflectance mode at the wavelength λ?=?366?nm to obtain fingerprints of the extracts derived from individual C. incanus L. samples. Mobile phase 2 performed slightly better, because with its use, the maximum number of 11 peaks could be seen in the respective fingerprints, whereas with mobile phase 1, the maximum number of 10 peaks only. Then an antioxidant potential of the investigated herbal extracts was assessed, making use of mobile phase 2 and the 0.20% methanol solution of 2,2-diphenyl picrylhydrazyl as a visualizing reagent. The resulting chromatograms were densitometrically scanned in the extinction mode at the wavelength λ?=?550?nm to obtain biological fingerprints of the extracts. Finally, chromatographic and biological fingerprints underwent a semiquantitative evaluation in terms of the contents of the extracted polar fraction and an overall antioxidant potential of the individual plant species.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

LC and SPE Determination of Chlorophacinone in Corn Grains, Soil and Rat Plasma

A liquid chromatographic method was used for the analysis of chlorophacinone in corn, soil and rat plasma. Sample preparation differed from corn, soil and serum. Simple liquid extraction procedures were applied to soil and corn; solid phase extraction using Oasis HLB cartridges and easy protocol was used for plasma. Liquid chromatography was on X-terra C18 column. The mobile phase used did not contain ion-pairing reagent, it was constituted with methanol/ammonium bicarbonate (0.01 M, pH = 7), 70/30, v/v at 1 mL min⁻¹. UV detection was by using photodiode detector operating at 282 nm. Complete validation of the proposed procedure for corn, soil and serum was made.     

A comparative study of the separation of oleanolic acid and ursolic acid in Prunella vulgaris by high-performance liquid chromatography and cyclodextrin-modified micellar electrokinetic chromatography

A high-performance liquid chromatographic (HPLC) method and a cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method were developed to separate and determine oleanolic acid (OA) and ursolic acid (UA) in Prunella vulgaris. HPLC separations were carried out on a Hedera ODS C18 column with methanol -H₂O- acetic acid (85:15:0.3, v/v/v) as mobile phase at a flow-rate of 0.8 ml min^?1. CD-MEKC analysis was performed on a CL1030 capillary electrophoresis system with a 6% (v/v) methanol solution (pH = 9.0) containing 10 mM disodium tetraborate, 10 mM sodium dihydrogen phosphate, 50 mM sodium dodecylsulfate (SDS), 15 mM 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) as background electrolyte. The analytical results of HPLC and CD-MEKC were compared with each other. CD-MEKC has better analytical efficiency for two components, and the analytical time (15 min) was shorter than that of HPLC (35 min).     

Determination of Visnagin inAmmi visnagaHairy Root Cultures Using Solid-Phase Extraction and High-Performance Liquid Chromatography

A high-performance liquid chromatographic method was developed for separation of the furochromone fraction and for determination of visnagin inAmmi visnagahairy root cultures. Lyophilized samples were extracted with chloroform:methanol (1:1, v/v) and purified on solid-phase extraction cartridges. HPLC analyses were performed on a Eurospher 100-C₈Knauer column and the mobile phase was 29:28:526:417 (v/v/v/v) acetonitrile:tetrahydrofuran:30 mM citric acid (pH 3.0):methanol. Quercetin was used as internal standard. Peaks were identified by addition of authentic standards and/or by diode-array detection.     

Simultaneous quantification of the major bile acids in Artificial Calculus bovis by high‐performance liquid chromatography with precolumn derivatization and its application in quality control

An accurate and sensitive high‐performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis, including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2‐bromine‐4’‐nitroacetophenone and 18‐crown ether‐6 were used for derivatization. The chromatographic separation was performed on an Agilent SB‐C18 column (250 × 4.6 mm id, 5 μm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity (r² ≥ 0.9980), recovery (94.24–98.91%), limits of detection (0.25–0.31 ng) and limits of quantification (0.83–1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis, and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C₁₈ column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0–6.0 μg mL⁻¹ for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.

     

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C₁₈ column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0–6.0 μg mL⁻¹ for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.