Mobile Phase Flow Rate Research Articles

Development and Validation of Fast and Robust Stability Indicating RP-HPLC Method for Simultaneous Estimation of Tolperisone Hydrochloride and Etoricoxib in Pharmaceutical Dosage Form

Objectives: A precise, accurate and selective stability-indicating reverse phase high performance liquid chromatographic assay method has been developed for the simultaneous quantitative determination of Tolperisone hydrochloride and Etoricoxib in tablets. Methods: The chromatographic separation of drugs was attained by using Eclipse plus C18 (150 mm × 4.6 mm, Agilent 5µm) column at room temperature. The composition of mobile phase was mixture of 0.035M triethylamine (pH 3.0 adjusted with orthophosphoric acid) and acetonitrile in ratio of 70:30 v/v and flow rate of mobile phase 1.0 ml/min with isocratic elution. The signal of eluents was observed at 290 nm by using diode array detector. Results: The retention time of Tolperisone hydrochloride and Etoricoxib were found to be 2.826 min and 7.566 min, respectively. The linearity ranges for both drugs were found to be 5-15 μg/ml and the percent recoveries were found to be 99.39% and 99.15% for Tolperisone hydrochloride and Etoricoxib respectively. Various forced degradation conditions, such as alkali hydrolysis, acid hydrolysis, thermal degradation, photolytic degradation, and oxidative degradation, were applied to the drug ingredients and drug formulation. The degradants were efficiently separated from the drugs by using optimized chromatographic conditions. The developed method was validated as per recommendation parameters of International council on harmonization guidelines Q2(R1). Conclusion: The validation parameters indicate that the drug substances were efficiently separated from its degradants and developed method can be routinely applied for the simultaneous quantitative determination of Tolperisone hydrochloride and Etoricoxib in combined tablet formulation in the quality control laboratory.

THE STUDY OF NETTLE HERB (URTICA DIOICA L.) GROWING IN SOME AREAS OF THE EUROPEAN RUSSIA AND SIBERIA

The report presents the results of the study of the chemical composition of nettle herb, 40%, 70% extracts, and extracts of polyphenolic complexes (PPC). Nettle herb were collected by the authors in June-July 2007–2020 in the territories of Tomsk, Kemerovo, Kursk, Novosibirsk, Moscow, Kirov, Tver regions, Altai and Krasnoyarsk Krai; Altai Republic. Extracts, polyphenolic and polysaccharide complexes were obtained from the herb. The studies was conducted using PC, TLC, UV-spectroscopy, HPLC, titrimetry, neutron activation. Reverse-phase HPLC were carried out on a chromatograph «Dionex Ultimate 3000» with UV-detector in the wave length range from 254 to 330 nm. Separation was performed by a column with reversed-phase sorbent «Restek Pinnacle IIC18» (150×4.6 mm, particle size 5 µm), flow rate of mobile phase is 1 ml/min; temperature is 20 °C, mobile phase (50 ml of acetonitrile, 50 ml of 0.1% trifluoroacetic acid) were carried out on. The study showed that nettle herb, 40%, 70% extracts and PPC contain phenolic compounds: phenolcarbonic acids, flavonoids, catechins, tannins; vitamin C and pigments. Baicalein, chrysoeriol, scutellarin identified for the first time. One of the dominant groups is hydroxycinnamic acids. The composition of flavonoids, phenolic carboxylic acids, catechins, hydroxycinnamic acids, including chlorogenic acid, chlorophyll, the sum of phenolic compounds, catechins are similar in 70% extract and its PPC. Additional information was obtained on the elements contained in nettle herb and its extracts.

Quantitative extraction of multiclass antibiotic residues in cow milk utilizing salting-out assisted liquid-liquid extraction followed by liquid chromatographic determination

ABSTRACT The salting-out assisted liquid–liquid extraction (SALLE) method with the liquid chromatography ultraviolet-visible detector (LC-UV) has been developed and validated for the simultaneous determination of seven antibiotic compounds of five different therapeutic classes (β-lactams), amoxycillin, ampicillin, penicillin G, (sulphonamides), sulfamethoxazole, (aminoglycosides) gentamicin, (fluoroquinolones) ciprofloxacin and (macrolide) erythromycin, in the cow milk samples. The LC conditions for separation of the selected antibiotic compounds were achieved with the reversed phase isocratic elution. The mobile phase consisted of 0.05 M Na2HPO4, acetonitrile and methanol (70:10:20 v/v/v). The mobile-phase flow rate was 1 mL min−1 with the injection volume of 20 μL and all the seven standard compounds were eluted within 14 min. The developed analytical method has been validated for the linearity, precision, sensitivity, accuracy, specificity, decision limit (CCα), and detection capability (CCβ). Experimental parameters influencing the extraction efficiency were evaluated and optimised. Under the optimum experimental conditions, good linearity over wide concentration ranges was obtained with the coefficient of determination R2 > 0.9989 for all the antibiotics. The limits of detection (LODs) and quantification (LOQs) ranged from 1.28 to 2.46 µg mL−1 and 3.87–7.46 µg mL−1, respectively. The relative standard deviations for the repeated analysis were below 8% indicating acceptable method precision. The accuracy study yielded satisfactory recoveries in the ranges 95.5–103% for the pure standard compounds and 83.8–112% for the spiked matrix-match milk samples. The results of the study revealed that the developed method is simple and fast and the SALLE method is appropriate for the simultaneous determination of targeted five multiclass antibiotic residues in the cow milk samples by LC-UV/Vis with a better efficiency.

Determination of atrazine and propazine metabolites deemed endocrine disruptors in human seminal plasma by LC\u2013ESI-MS/MS

BackgroundThe increasing prevalence of male infertility and the declining trend in sperm quality has been associated to compounds known as “endocrine-disruptors”. The proven endocrine-disrupting effects of atrazine and propazine herbicides led us to conduct long-term research based on highly accurate specific analytical methods with a view to confirming the suspected association. Among the proposed developments was a sensitive analytical method for the simultaneous determination of three metabolites of atrazine and propazine.ResultsIn this work, the method was for first time used for the chromatographic separation and determination of deethyl- and deisopropyl-atrazine (DEA and DIA, respectively) and propazine-2-hydroxy (PP-2OH) in human seminal plasma by LC–ESI-MS/MS using deuterated atrazine (d5-AT) as internal standard (IS). Chromatographic and mass spectrometric conditions such as the mobile phase composition and flow-rate, injected volume, dry gas source temperature and flow-rate, nebulizer pressure and capillary voltage were all carefully optimized. Analytes were identified and quantified by using the multiple reaction monitoring (MRM) mode as applied to positive ions ([M + H]+). Transitions at three different m/z values for each analyte were selected from precursor ions, and the 212.1 → [128]+, 188.1 → [146]+ and 174.1 → [68.1]+ transitions for PP-2OH, DEA and DIA, respectively, were found to be quantitative. The proposed method was validated in terms of precision (repeatability and reproducibility), linear range (10–240 ng mL–1), limit of detection (150–210 pg mL–1), and quantification (500–700 pg mL–1), recovery, accuracy and matrix effects on extracts from variably treated seminal plasma samples. The overall analytical method was successfully applied to human seminal plasma samples from volunteers. PP-2OH was found at concentrations from 1.10 to 11.3 ng mL–1 in four of the six samples, and so was DIA at 9.60 ng mL–1 in one.ConclusionsThese results are suggestive of bioaccumulation of the target analytes in humans. Untargeted analytes including suspected parent molecules (atrazine and propazine) and other ions [viz., deethyldeisopropyl-atrazine (DD) and diamino-s-chlorotriazine (DACT)] were also detected under the working conditions used. These results may open up new prospects for as yet very incipient research into the bioaccumulation of endocrine disruptors in seminal plasma.Graphical

Development and validation of liquid chromatography method for simultaneous determination of multiclass seven antibiotic residues in chicken tissues

BackgroundAntibiotics are routinely used on poultry for therapy and prevention of diseases and to enhance animal growth. The objective of this study was to develop and validate a sensitive and reliable liquid chromatography with UV detection (LC-UV) method for the simultaneous determination of seven multiclass antibiotic residues (amoxicillin, ampicillin, penicillin, sulfamethoxazole, gentamicin, ciprofloxacin, and erythromycin) in chicken tissues.MethodsThe liquid chromatography method with UV detection was optimized for complete separation of the seven selected antibiotic compounds with reversed phase and isocratic elution using Hypersil BDS-C18 (3 µm, 100 mm × 4 mm) column. The mobile phase consisted a ratio of 0.05 M Na2HPO4, acetonitrile and methanol (70:10:20), at UV absorption wavelength of 230 nm. The column thermostat was set at 40 °C, the mobile phase flow rate was 1 mL min−1, and the injection volume was 20 μL.ResultsAll the seven standard compounds were eluted within 14 min. The results for: linearity, precision, sensitivity, accuracy, specificity, decision limit (CCα), detection capability (CCβ), suitability and method robustness were validated according to the criteria of Commission Decision 2002/657/EC guidelines. Calibration plot correlation coefficients ranged from 0.9983 to 0.9998 and the percent relative standard deviations for repeated analysis were below 5% indicating acceptable method precision. The limits of detection (LODs) and quantification (LOQs) ranged from 0.098–0.255 μg kg−1 to 0.297–0.574 μg kg−1, respectively. The accuracy study yielded recoveries in the ranges 98.1–107% for the pure compounds and 94.0–102% for the spiked drug free chicken tissue samples.ConclusionsThe method was found to be appropriate for simultaneous determination of five different classes of seven antibiotic residues in chicken tissues. Furthermore, this is the first instance for the simultaneous determination of seven multiclass, multi-residues analysis using LC-UV from chicken tissue samples. This is a cost-effective and alternative method with simple instrumentation approach for laboratories that lack highly specialized state-of-the-art instrumentation.

The determination of monosaccharide in different years Qingzhuan Dark Tea polysaccharide by liquid chromatography-mass spectrometry.

To establish a fast, sensitive and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for determining the monosaccharide content of Qingzhuan Dark Tea polysaccharides in different years (2years, 5years and 11 years). The optimised chromatographic conditions were achieved on a C18 column (5.0μm, 250 mm × 4.6mm inner diameter). The mobile phase flow rate was 0.9mL/min and the column temperature was set to 27°C. The aqueous phase A (5 mM aqueous ammonium acetate) and organic phase B (acetonitrile) were used to elute the target analyses isocratically (0-60 min: 18% B). The mass spectrometer detector was equipped with an electron spray ionisation (ESI)source, and multiple reaction monitoring (MRM) mode was used for the determination of 1-phenyl-3-methyl-5-pyrazolone (PMP) derived monosaccharides. We carried out a comprehensive methodological validation of PMP derived monosaccharides, including linearity, precision, stability and repeatability. Nine monosaccharides (rhamnose, mannose, ribose, glucose, galacturonic acid, xylose, galactose, fucose and arabinose) of Qingzhuan Dark Tea polysaccharides were identified, in which ribose and fucose were reported for the first time. The results showed the contents of these nine monosaccharides differed significantly among different years. The validated method is reliable, accurate, repeatable and can be applied to quality assessment of these monosaccharides.

Chromatographic method development for the simultaneous assay of pseudoephedrine hydrochloride and chlorphenamine maleate in oral dosage formulations

Common cold medicines usually present as multi-component products and constitute one of the largest groups of ‘over-the-counter’ medicines marketed in Ghana. Due to their wide patronage and easy accessibility, their quality control is of utmost importance to medicine consumers. In this study, the Design of Experiment (DOE) concept is adopted to develop a chromatographic method for the simultaneous assay of pseudoephedrine hydrochloride and chlorphenamine maleate in commercially available products. The separation of the drugs was achieved on an Agilent Eclipse Plus C18 analytical column (4.6 × 150 mm; 5 µm) and detected at 252 nm, while maintaining column temperature at 30 oC. The method parameters, including acetonitrile concentration in the mobile phase, mobile phase pH and flow rate were optimized using a Central Composite Design study, while monitoring the method attributes, asymmetric factors, selectivity, retention times, and resolution. The outcome showed a good correlation between experimental and predictive values throughout the modeled Design Space, with a Desirability of 1.000. The conditions optimized included a mobile phase containing acetonitrile, 50 mM aqueous acetate buffer solution (pH = 3.19) and triethylamine (15.34:84.65:0.01 v/v/v), and a flow rate of 1.437 mL/min. The method was then validated following the International Council for Harmonization Q2(R) guidelines to establish linearity and range, accuracy, precision, robustness, and stability of the test solution. The developed method was successfully applied to estimate pseudoephedrine hydrochloride and chlorphenamine maleate contents in single and fixed-dose formulations commercially available (N = 38).

Optimization of chromatographic separation of aripiprazole and impurities: Quantitative structure-retention relationship approach

A new optimization strategy based on the mixed quantitative structure? retention relationship (QSRR) model is proposed for improving the RPHPLC separation of aripiprazole and its impurities (IMP A-E). Firstly, experimental parameters (EPs), namely mobile phase composition and flow rate, were varied according to Box?Behnken design and thereafter, an artificial neural network (ANN) as a QSRR model was built correlating EPs and selected molecular descriptors (ovality, torsion energy and non-1,4-van der Waals energy) with the log-transformed retention times of the analytes. Values of the root mean square error (RMSE) were used for an estimation of the quality of the ANNs (0.0227, 0.0191 and 0.0230 for the training, verification and test set, respectively). The separations of critical peak pairs on chromatogram (IMP AB and IMP D-C) were optimized using ANNs for which the EPs served as inputs and the log-transformed separation criteria s as the outputs. They were validated by application of leave-one-out cross-validation (RMSE values 0.065 and 0.056, respectively). The obtained ANNs were used for plotting response surfaces upon which the analyses chromatographic conditions resulting in optimal analytes retention behaviour and the optimal values of the separation criteria s were defined. The optimal conditions were 54 % of methanol at the beginning and 79 % of methanol at the end of gradient elution programme with a mobile phase flow rate of 460 ?L min-1.

AQbD Driven Development of an RP-HPLC Method for the Quantitation of Abiraterone Acetate for its Pharmaceutical Formulations in the Presence of Degradants

Abiraterone acetate is a well-known anticancer drug and a steroidal derivative of progesterone for treatment of patients with hormone-refractory prostate cancer. Chemometrics-assisted reverse phase high performance liquid chromatography (RP-HPLC) development of the drug abiraterone acetate has been employed in this study using an analytical quality by design (AQbD) approach. Drug separation was performed using a Princeton Merck-Hibar Purospher STAR (C18, 250 mm × 4.6 mm) i.d., 5 μm particle size) with ultraviolet detection at 235 nm. A Box-Behnken statistical experimental design with response surface methodology was executed for method optimization and desired chromatographic separation from its formulation with a few numbers of experimental trials. The impact of three independent variables, namely, composition of the mobile phase, pH, and flow rate, on response retention time and peak area was studied by constructing an arithmetic model from these variables. Optimized experimental conditions for the proposed work include the mobile phase acetonitrile and phosphate buffer (10 mM KH2PO4) (20:80 %v/v). At the concentration range of 2-100 μg/mL, a linear calibration curve was found. Recovery was performed at three concentrations and was foun to be between 98% and 102%. The 3D response surface curves revealed that mobile phase composition and flow rate were the most substantial critical factors affecting desired responses. An attempt has been made to develop and validate an economical, precise, robust, stability-indicating AQbD-based RP-HPLC method that can be employed successfully for the routine analysis of abiraterone acetate in quality control labs.

Rp-HPLC Method Development and Validation for Simultaneous Estimation of Atorvestatin Calcium and Fenofibrate in Bulk and Formulation

A reverse phase high performance liquid chromatographic method was developed for the simultaneous determination of atorvastatin and Fenofibrate in bulk and pharmaceutical dosage forms. The determination was performed by using Waters Symmetry C18 (250×4.6mm×5µ) as column stationary phase and Methanol: Acetonitrile: Water in the ratio of (70:10:20 %v/v) as mobile phase. The flow rate of mobile phase was optimized as 1mL/min and effluents were monitored at 274 nm. The retention time of atorvastatin and Fenofibrate were found as 4.15 min and 8.10 min respectively. The method shows linearity concentration ranges between 4-30 μg/mL and 80-400 μg/mL respectively. The developed method was validated for specificity, precision, linearity, accuracy, robustness, Ruggedness, LOD and LOQ. Recovery of atorvastatin and Fenofibrate in formulations was found to be 100.52% and 99.92% respectively which conforms the non-interferences of the excipients in the formulation. the proposed RP- HPLC method can be used for the simultaneous determination of these two drugs in bulk and formulation.

Development of a Liquid Chromatographic Method for Apomorphine Hydrochloride Quantitation in the Active Pharmaceutical Ingredients and in the Dosage Forms

Apomorphine is currently used as a dopamine agonist to treat advanced Parkinson Disease. In the present study, we developed a sensitive, simple, reliable, and robust HPLC method for quality control of apomorphine hydrochloride in bulk drug and pharmaceutical formulations. C18 column was used for separation. Isocratic elution was performed using mobile phase A: 50 mM potassium dihydrogen phosphate solution (pH:3 with ortho-phosphoric acid), B: acetonitrile in the ratio of 85:15 (A: B), and the mobile-phase flow rate was kept at 1 mL min-1. Analyses were carried out at 272 nm using a UV detector. A perfect linear relationship between peak-area versus drug concentration in the range of 10‑100 μg mL-1 was observed (r2, 0.9999). It has been found that the developed method is sensitive (Detection and quantification limits were determined as 1.3 μg mL-1 and 3.8 μg mL-1, respectively), precise (RSD

DEVELOPMENT OF A RAPID AND EFFICIENT METHOD FOR QUANTITATIVE DETERMINATION OF N-NITROSODIMETHYLAMINE IMPURITY BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN METFORMIN HYDROCHLORIDE, LOSARTAN POTASSIUM, VALSARTAN AND RANITIDINE MEDICINAL RAW MATERIALS AND ITS PRODUCTS

In this study, chromatographic conditions were developed for the separation and quantification of N-nitrosodimethylamine (NDMA) by HPLC. Our method would be useful for the rapid screening and quantification of NDMA impurity in Metformin hydrochloride, Losartan potassium, Valsartan and Ranitidine drugs substance and its products. The chromatographic separation was achieved on a Phenomenex C18 column (250 mm, 5 µm, 4.6 mm) by multistep gradient elution, using a water-acetonitrile mobile phase containing 0.1% formic acid, which provided better c hromatographic separation. Column temperature was maintained at 30℃; mobile phase flow rate: 1 mL/min; wavelength: 254 nm; and injection volume of 20 μL.

A simple HPLC-UV Method for Therapeutic Drug Monitoring of Linezolid in human Plasma in low-resourced settings

OBJECTIVE: A high-performance liquid chromatography method for the estimation of Linezolid in human plasma was developed and validated. METHODS: Samples (100µµL) were deproteinized with acetonitrile and analyzed using LiChrospher 100, RP18e column with PDA detection at 254 nm. The flow rate of the isocratic mobile phase comprising of 0.1% formic acid in 1000 ml of water and acetonitrile in the ratio of 60:40 (v/v) was set at 1.0 ml/min. RESULTS: The calibration curve ranged from 0.50 to 20.0 µg/ml and was linear. The recovery ranged from 96% to 101%. The accuracy ranged from 98 to 101% and intra- and inter-day relative standard deviation was <4.58%. The method reliably eliminated interfering materials from plasma and R2 was 0.9973. The method described was applied to the determination of plasma LZD concentration in multi-drug-resistant tuberculosis patients who are treated with a dose of 600 mg LZD once daily. CONCLUSIONS: The developed method is suitable for determination of plasma LZD in routine care and considered feasible in less-resourced settings

Green Stability Indicating Organic Solvent-Free HPLC Determination of Remdesivir in Substances and Pharmaceutical Dosage Forms

A green liquid chromatographic method is considered in this work to minimize the environmental impact of waste solvents. One important principle is to replace or eliminate the use of hazardous organic solvents. Organic impurities in any active pharmaceutical ingredient could arise either during the process of its synthesis, or as degradation products developed throughout the shelf-life. Remdesivir (RDS) is an antiviral drug, approved by the US Food and Drug Adminstration (-FDA), to treat SARS-Cov-2 virus during its pandemic crisis. We studied the stability of remdesivir against several degradation pathways using the organic solvent-free liquid chromatographic technique. Separation was performed on RP-C18 stationary phase using mixed-micellar mobile phase composed of a mixture of 0.025 M Brij-35, 0.1 M sodium lauryl sulfate (SLS), and 0.02 M disodium hydrogen phosphate, adjusted to pH 6.0. The mobile phase flow rate was 1 mL min−1, and detection was carried out at a wavelength of 244 nm. We profiled the impurities that originated in mild to drastic degradation conditions. The method was then validated according to International Conference of Harmonization (ICH) guidelines within a linearity range of 5–100 μg mL−1 and applied successfully for the determination of the drug in its marketed dosage form. A brief comparison was established with reported chromatographic methods, including a greenness assessment on two new metrics (GAPI and AGREE). This study is the first to be reported as eco-friendly, solvent-free, and stability indicating LC methodology for RDS determination and impurity profiling.

Determination, solvent extraction, and purification of artemisinin from Artemisia annua L.

Artemisinin, the main active compound in Artemisia annua L., has been used for antimalarial properties for centuries and currently attracting increasing interest for its antiviral activities. In addition, several recent publications indicated that this valuable compound can be effective on Sars-CoV-2 virus. In the study, a high-performance liquid chromatography (HPLC) method was optimized in terms of mobile phase compositions, column temperature and flow rate using response surface methodology for the determination of artemisinin from A. annua samples. The method was also validated for some parameters according to the Eurachem guideline. Validated method was applied on A. annua plant samples, cultivated in a controlled condition, and content of artemisinin was found in the range from 5825 to 7972 mg/kg (n = 20). Extraction conditions of artemisinin from the plant samples were also optimized. In the first step of the extraction, solvents with different polarities were applied to the samples for the evaluation of artemisinin solubility. Then, ethanol was chosen for extraction solvent due the high extraction yield and classification in safer chemical ingredients list by EPA. After the extraction, a purification step using various adsorbents was studied to remove remnant impurities such as chlorophyl. The results showed that powdered charcoal was found to be the most effective adsorbent. Amount of the adsorbent was also studied to evaluate for the reduction of chlorophyl without reducing the artemisinin concentration. Finally, purified solvent was dewaxed, evaporated, and dried under nitrogen to concentrate the artemisinin content. In conclusion, the optimized conditions could be regarded as a new alternative technique in pharmaceutical industry for the extraction of artemisinin from A. annua samples.

Response Surface Methodology Driven Systematic Development of a Stability- indicating RP-UPLC Method for the Quantification of Aliskiren: A Renin Inhibitor

Aims: The current study envisages experimental design enabled rapid, sensitive, and stability-indicating RP-UPLC method to quantify Aliskiren in its pharmaceutical formulations.
 Study Design: Box-Benkhen experimental Design using Response surface methodology.
 Place and Duration of Study: Department of Analytical Research and Development, Brawn laboratories ltd., Gurugram, India, and Department of Pharmacy KL College Pharmacy, KL Deemed to be University, Vaddesearam, Guntur, Andhra Pradesh, between May 2021 and September 2021.
 Methodology: The chromatographic partitioning was achieved on a Waters Acuity H class UPLC system, with BEH 130οA, C18 column (100 x 2.1 mm,1.7 μm) having isocratic elution containing (50:50 %v/v) of 0.2% Glacial acetic acid (GAA) : acetonitrile, at constant flow rate using PDA detection. The optimum conditions were delineated, selecting three influential factors (CMPs), i.e., mobile phase composition, flow rate, and injection volume. Systematic optimization was accomplished by 32 Box-Benkhen design using response surface methodology (RSM).
 Results: The selected variables are evaluated for obtained responses (CAAs), i.e., peak area, retention time (Rt), USP Plate count. The final optimized method employed, organic phase composition 0.2 % GAA (pH 3.0) and acetonitrile 50:50 (% v/v) with 0.3 mL min-1 flow rate. The injection volume was maintained as 2μL with 2 minutes run time and λmax 280 nm.
 Conclusion: The method was linear for 5-300 ppm, with regression co-efficient (R2) 0.9995. As per ICH guidelines, forced degradation studies were carried out to analyse the stability profile of drug. The short Rt 1.214, minute implies superior robustness, sensitivity, and cost-effectiveness for routine analysis. The results exhibited that RSM approach of QbD will be competently used to optimize the RP-UPLC method with fewer experimental trials and error-free investigation.

Fast analysis of derivatives of polycyclic aromatic hydrocarbons in soil by ultra-high performance supercritical fluid chromatography after supercritical fluid extraction enrichment

An efficient and environment-friendly method for simultaneous determination of 13 typical derivatives of polycyclic aromatic hydrocarbon (PAH) in petroleum-polluted soil with nitro-, oxy- and alkyl-functional group was developed using supercritical fluid extraction (SFE) followed by ultra-high performance supercritical fluid chromatography (UHPSFC). Parameters of UHPSFC, including type of stationary phase and mobile phase modifiers, gradient elution process, backpressure, column temperature, and the flow rate of mobile phase, were systematically optimized, achieving a fast separation within 4.2 min. Limits of detection (LOD) were 0.005–0.1 μg mL−1 or 0.1–2.0 ng g−1, respectively, with a good repeatability (RSD<5.0%). Before UHPSFC-PDA analysis, the PAH-derivatives in soil samples were effectively enriched in 15.0 min using SFE with an online carbon nanotubes (CNTs) collection trap. The soil samples were analyzed by the proposed method and the results were verified by GC-MS. Thus, SFE equipped with an online CNTs trap followed by UHPSFC-PDA analysis, which only consumed about 2.0 mL organic solvent for a whole run, has been demonstrated to be an efficient way for screening and quantitative analysis of trace-level PAH-derivatives in soil samples.

Front Cover: Development and Validation of Reverse‐Phase High‐Performance Liquid Chromatography Method for Estimation of Itraconazole through Hydroxypropyl Methylcellulose Acetate Succinate based Polymeric Films using Quality by Design principles

DOI: 10.1002/sscp.202100037 The cover picture shows a reverse phase HPLC method development for Itraconazole using principles of analytical QbD with surface response curves indicating the results of multivariate analysis in present method conditions, where the retention time (RT) was impacted with mobile phase concentration and flow rate with negligible impact of pH of mobile phase and a non-linear correlation amongst the experimental variables. On the other hand, the response for theoretical plates indicated a linear correlation with the pH of water used in the mobile phase without much impact of other variables. To evaluate the suitability of the test method for rapid screening of formulations, polymeric amorphous dispersion films were prepared for Itraconazole using the solvent evaporation method at different drug concentrations and using two different grades of polymer HPMCAS. It was observed that amongst the drug concentrations and polymer grades studied, the highest clarity of the film was achieved at 10% drug loading with HPMCAS 912G which also resulted in a rapid and complete drug dissolution in less than 30 minutes indicative of supersaturation and prevention of recrystallization after formation of the polymeric amorphous dispersion film.

Measurement of optimal flow rate in gradient elution liquid chromatography

Method development in gradient LC relies upon the selection of a solvent time program and a mobile phase flow rate. The flow rate, optimal for gradient separation cannot be inherently predicted by the isocratic value optimal for a given analyte, and rather should be identified independently to ensure the highest separation performance of gradient analysis. The optimal flow rate (Fopt) is defined herein as the solvent volumetric flow rate (F) maximizing the separation (Δs) of a predetermined peak-pair or the separation capacity (sc) of the entire LC analysis. The theoretical background and the experimental technique of measurement of Fopt in gradient elution analysis were considered and experimentally demonstrated. The technique of measuring Fopt is based on translatable changes of F where the product FtG (tG is the gradient time) was the same for all values of F. The Fopt was found as F corresponding to the maximum in Δs or in sc.

High-speed countercurrent chromatography for isolation and enrichment of betacyanins from fresh and dried leaves of Atriplex hortensis L. var. "Rubra".

Betacyanins and their decarboxylated derivatives from fresh and dried edible leaves of Atriplex hortensis L. var. "Rubra" were fractionated for the first time by high-speed countercurrent chromatography. Pigments present in fresh leaf extract were separated in systems: ethanol - acetonitrile - n-propanol - ammonium sulphate - water (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (tail-to-head mode) and tert-butyl methyl ether - n-butanol - acetonitrile - water with 0.7% heptafluorobutyric acid (2:2:1:5, v/v/v/v) (head-to-tail mode). The mobile phase flow rate was 2mL/min and the retention of the stationary phase was 79.8 and 75.2%, respectively. Pigments from dried leaves were separated in a similar ion-pair system with heptafluorobutyric acid in different volume proportions 1:3:1:5 (head-to-tail mode) and the flow rate of the mobile phase 3mL/min. The stationary phase retention was 64.0%. The application of the countercurrent chromatography for the fractionation of betacyanins from leaves of Atriplex hortensis enabled to isolate and pre-concentrate the pigments for further low- and high-resolution liquid chromatographic-tandem mass spectrometric detection. This study revealed the presence of 10 betacyanins in fresh and 16 in dried leaves of Atriplex hortensis. Two compounds were not previously identified in the whole Amaranthaceae family. Additionally, instead of (iso)amaranthin, celosianin and its epimer were dominant betacyanins in the Atriplex hortensis.