Chromatography Methodology Research Articles

Guilty by dissociation: Part A: Development of a rapid Ultra-High Performance Liquid Chromatography (UHPLC)-MS/MS methodology for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS)

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (tG = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Generic reversed‐phase ultra‐high‐pressure liquid chromatography methodology developed by using computer‐assisted modeling for streamlined performance evaluation of a wide range of stationary phase columns

AbstractSelection of column and mobile phase eluent/buffer is a crucial step in chromatographic method development. The introduction of new column technologies requires a continuous performance evaluation enabling the deployment of improved chromatographic screening workflows. Consequently, the development of a universal method that could be used across different column chemistries and mobile phase pH ranges would be highlighted as advantageous. Herein, we introduce a single generic probe developed via software‐based modeling that delivers excellent results for the separation of a standard multicomponent mixture across a wide range of stationary phases under different pH and temperature conditions. This approach has been applied to interrogate the performance and stability of multiple columns (C18, Cyano, Phenyl Hexyl, PFP, Polar‐embeded, and other stationary phases) using an automated ultra‐high‐pressure liquid chromatography screening system illustrating its practicality and effectiveness.

A Greener Stability-Indicating High-Performance Thin-Layer Chromatography Approach for the Estimation of Topiramate.

Despite various reported analytical methods for topiramate (TPM) analysis, greener analytical approaches are scarce in literature. As a consequence, the objective of the current research is to design a normal-phase stability-indicating high-performance thin-layer chromatography (SI-HPTLC) methodology for TPM analysis in marketed tablet dosage forms that is rapid, sensitive, and greener. TPM was derivatized densitometrically and analyzed at 423 nm in visible mode with anisaldehyde-sulfuric acid as the derivatizing agent. The greener SI-HPTLC technique was linear in the 30–1200 ng band−1 range. In addition, the suggested SI-HPTLC methodology for TPM analysis was simple, rapid, cheaper, precise, robust, sensitive, and environmentally friendly. The greener SI-HPTLC method was able to detect TPM along with its degradation products under acid, base, and oxidative degradation conditions. However, no TPM degradation was recorded under thermal and photolytic stress conditions. TPM contents in commercial tablet dosage forms were recorded as 99.14%. Using 12 different principles of green analytical chemistry, the overall analytical GREEnness (AGREE) score for the greener SI-HPTLC method was calculated to be 0.76, confirming the proposed normal-phase SI-HPTLC method’s good greener nature. Overall, these results demonstrated that the suggested SI-HPTLC technique for TPM measurement in pharmaceutical products was reliable and selective.

Comprehensive Analysis of Plant Lipids Using Sub-2-μm Particle CO2-Based Chromatography Coupled to Mass Spectrometry.

Lipids play an essential role in plants, and historically manipulating their levels and composition has been an important target for metabolic engineering. A variety of analytical techniques, many based on mass spectrometry, have been utilized for lipid profiling, but the analysis of complex lipid mixtures still poses significant analytical challenges. Recent advances in technology have revived the supercritical fluid chromatography (SFC) as a promising separation technique for lipid analysis. Utilization of sub-2-μm particle columns improves the separation efficiency and robustness of the SFC systems. The combination of SFC with sub-2-μm particle separation, commonly referred as ultra-performance convergence chromatography, has been successfully used for separation of both polar and neutral lipids. In this chapter, we present a simple method for lipid class separation using Sub-2-μm particle CO2-based chromatography coupled to mass spectrometry. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation of lipid classes or individual lipids within class.

Efficient Synthesis of Potential Impurities in Levonadifloxacin (WCK 771).

Levonadifloxacin (WCK 771) is a novel broad-spectrum anti-methicillin-resistant Staphylococcus aureus (MRSA) agent recently launched in India. Five process impurities and one degradation impurity were synthesized as reference standards for their quantification by high-performance liquid chromatography (HPLC) methodology in drug substance and drug product. These compounds are not easily commercially available. The synthesis and characterization of these impurities are discussed in detail.

A Rapid HPLC-UV Protocol Coupled to Chemometric Analysis for the Determination of the Major Phenolic Constituents and Tocopherol Content in Almonds and the Discrimination of the Geographical Origin.

Reversed phase-high-pressure liquid chromatographic methodologies equipped with UV detector (RP-HPLC-UV) were developed for the determination of phenolic compounds and tocopherols in almonds. Nineteen samples of Texas almonds originating from USA and Greece were analyzed and 7 phenolic acids, 7 flavonoids, and tocopherols (−α, −β + γ) were determined. The analytical methodologies were validated and presented excellent linearity (r2 > 0.99), high recoveries over the range between 83.1 (syringic acid) to 95.5% (ferulic acid) for within-day assay (n = 6), and between 90.2 (diosmin) to 103.4% (rosmarinic acid) for between-day assay (n = 3 × 3), for phenolic compounds, and between 95.1 and 100.4% for within-day assay (n = 6), and between 93.2–96.2% for between-day assay (n = 3 × 3) for tocopherols. The analytes were further quantified, and the results were analyzed by principal component analysis (PCA), and agglomerative hierarchical clustering (AHC) to investigate potential differences between the bioactive content of almonds and the geographical origin. A decision tree (DT) was developed for the prediction of the geographical origin of almonds proposing a characteristic marker with a concentration threshold, proving to be a promising and reliable tool for the guarantee of the authenticity of the almonds.

Simultaneous Enantiomeric Separation of Carfentrazone-Ethyl Herbicide and Its Hydrolysis Metabolite Carfentrazone by Cyclodextrin Electrokinetic Chromatography. Analysis of Agrochemical Products and a Degradation Study.

The different activity and toxicity that the enantiomers of agrochemicals may have requires the development of stereoselective analytical methodologies enabling the individual determination of each enantiomer. The aim of this work was to develop the first Electrokinetic Chromatography methodology enabling the simultaneous enantiomeric separation of carfentrazone-ethyl herbicide and its hydrolysis metabolite carfentrazone. The use of an anionic cyclodextrin as chiral selector (captisol at 2.5% (w/v)) in a 25 mM acetate buffer, at a temperature of 30 °C, and an applied voltage (reverse polarity) of −30 kV, allowed the simultaneous separation of the four enantiomers of the two compounds studied in 6.8 min with enantiomeric resolutions of 5.0 for carfentrazone-ethyl and 5.1 for carfentrazone. Analytical characteristics of the developed method were evaluated and found adequate to achieve the quantitation of carfentrazone-ethyl and carfentrazone. Analysis of a commercial herbicide formulation showed the potential of the method for the quality control of these agrochemical products. Degradation studies for carfentrazone-ethyl revealed that no significant degradation took place in cleaned sand samples while a significant but not stereoselective degradation took place in soils for the whole period of time considered (seven days).

Size exclusion chromatography of aromatic compounds in high-boiling petroleum samples

Knowledge of aromatic compounds in petroleum samples is of paramount interest from processing and environmental viewpoints. Considering the complexity of such samples, a selective analytical strategy viz. aromatic specific size exclusion chromatographic methodology using amino-bonded silica phase, was adopted to obtain the average molecular weights of distilled cuts, residue and the corresponding crude oil. The method was found to be suitable from middle distillates up to the residue. It was found that the increase in boiling temperature of distilled cuts was reflected in an increase in high molecular weight aromatic species. The increase in the molecular weight of aromatic species is linked to the increase in alkylation as verified by studying the elution pattern of alkylated aromatic compounds. The accuracy of the molecular weights obtained from the size exclusion chromatographic method depends on the elution profile of aromatic species in petroleum samples. It was found that the size and shape of the parent aromatic compounds negatively influence the size exclusion elution profile. However, such influence can be assumed to be negligible as all the aromatic species of each distilled cut, residue, and crude oil eluted in the permeation zone of the chromatogram. This method has the potential to be used in the second dimension of a 2D-LC method where the aromatic compounds are separated based on the size (or alkylation). The first dimension will separate compounds based on the number of aromatic rings, thereby eliminating the interference of parent aromatic compounds of different sizes in the total elution profile.

Ecotoxicity evaluation of tetramethrin and analysis in agrochemical formulations using chiral electrokinetic chromatography

The separation of the four isomers of tetramethrin was performed for the first time using a cyclodextrin-micellar electrokinetic chromatography methodology. Using sodium deoxycholate and 2-hydroxypropyl-β-CD as chiral selectors, tetramethrin isomers were separated with resolution values of 1.7 and 1.1 for trans- and cis-isomers, respectively, in analysis times lower than 12.5 min. Once developed and optimized, the analytical method was applied to the analysis of an antiparasitic commercial formulation and to the evaluation of the stability and ecotoxicity of tetramethrin. Using measured concentrations, the stability was assessed at enantiomeric level and the ecotoxicological parameters on Daphnia magna were determined. Tetramethrin presents toxicity on aquatic microinvertebrates, with EC50 (t = 72 h) of 1.8 mg/L. The acute toxicity of tetrametrin was attributed to the trans-1 enantiomer. The first evidence of oxidative stress-mediated mode of action for tetramethrin on Daphnia magna is reported in the present work.

Futuristic review on progress in force degradation studies and stability indicating assay method for some antiviral drugs

Force degradation studies of drug substance give perceptive knowledge about the intrinsic stability of the molecule as well as possible degradants which formed during the shelf life of drug and thus, aid within the successive development of its stable formulation. A number of analytical methods with hyphenated techniques are required for the identification, determination and characterization of degraded product and impurities produce during different conditions of stress studies; Chromatographic methodology play a vital role in the field of impurity and degradation profiling .This review summarizes the current regulatory requirements guidelines for the laboratory performance of forced degradation and its application for the development of stability indicating method. There are number of strategies have been implemented for the quantitative assessment of antiviral drugs. This study will provide detailed literature on stability- indicating HPLC/ RP-HPLC approaches for the development and validation of various antiviral drugs.

Non-mercaptalbumin is significantly associated with the coronary plaque burden and the severity of coronary artery disease

Human non-mercaptalbumin (HNA), oxidized form of serum albumin, has been reported as a useful marker in oxidative stress-related diseases; however, few reports have examined the association between HNA and the severity of coronary artery disease (CAD). The present study evaluated whether the HNA fraction is correlated with coronary artery stenosis in 140 patients considered to have a high risk of CAD or who were suspected of having acute coronary syndrome. The severity of CAD was defined by the number of stenotic coronary vessels and a severity score system (the Gensini score). HNA measurements were performed using our newly established high-performance liquid chromatography methodology. The results had shown that HNA was significantly increased in patients with three-vessel disease, compared with those without CAD or with single-vessel disease (p = 0.025), and was positively correlated with the Gensini score (ρ = 0.421, p < 0.001). A multivariate analysis showed that the number of stenotic vessels was an independent and significant factor associated with HNA (ρ = 1.246, p = 0.012). A logistic regression analysis showed that HNA was a strong predictor of multivessel CAD (odds ratio, 1.12; 95% confidence interval, 1.020–1.229; p = 0.017). These findings indicate that the measurement of HNA could be clinically practical for predicting the severity of coronary artery stenosis.

Overview of ion chromatographic applications for the analysis of nuclear materials: Case studies

AbstractAccurate, precise, and rapid analytical monitoring of various nuclear materials is essential for the smooth functioning of nuclear reactors. Ion chromatography (IC) has emerged as an effective analytical tool for simultaneous detection of different ions in a wide range of materials used in the nuclear industry. The major advantages over other techniques include superior selectivity and sensitivity for detection of anions and cations, wide dynamic range, and speciation studies of ions. This article provides an overview of different ion chromatographic methodologies developed for the analyses of various nuclear materials such as fuel, control rods, moderator, coolant, and process streams. Comparison of various analytical aspects of IC over the other routine techniques reveals the ease and multidimensional capability of the technique. An insight is given to the modern variations in the field such as coupling of IC with other techniques for the characterization of nuclear matrices, implementation of capillary IC in terms of miniaturization, and so on. The information presented herein will serve as a very useful resource for investigators in the field of characterization of nuclear materials.

Simple TLC-spectrodensitometric method for studying lipophilicity and quantitative analysis of hypoglycemic drugs in their binary mixture.

A selective and simple salting-out-assisted thin-layer chromatographic methodology was developed for the simultaneous determination of two oral hypoglycemic drugs, dapagliflozin (DAPA) and metformin (MET) in their pure forms, tablets and spiked human plasma samples. Silica gel 60 F254 plates were used in the separation of the two drugs using a mobile phase consisting of 0.5m (NH4 )2 SO4 and methanol (3:7, v/v). The plates were scanned in the reflectance mode at λmax = 237 nm. The obtained retardation factor (Rf ) values for DAPA and MET were 0.77 ± 0.02 and 0.25 ± 0.02, respectively. The thin-layer chromatography method was validated according to International Conference on Harmonization guidelines. The peak areas were linearly increased with the increases in concentrations of 45-1,000 and 50-1,500 ng/band for DAPA and MET, respectively. Moreover, the method was applied to estimate the molecular lipophilicity parameters of DAPA and MET via retention data. The suggested method was efficiently utilized for the analysis of DAPA and MET in pharmaceutical tablets and plasma samples with recoveries 98.4-100.4 and RSDs in the ranges of 1.4-2.6 and 2.2-3.0% for DAPA and MET, respectively.

HPLC Method Validated for Quantification of Fluconazole Co-Encapsulated with Propolis Within Chitosan Nanoparticles.

The online version contains supplementary material available at 10.1007/s12088-021-00954-2.

A sensitive and practical ELISA for analyzing naringenin in pummelo and herb samples

Naringenin, a flavonoid compound found in pummelo, is a key biological active compound in some traditional Chinese medicines, including Citri reticulatae pericarpium, Citri reticulatae pericarpium viride, Aurantii fructus immaturus, and Aurantii fructus. These Chinese medicinal preparations are the peels or immature fruits of certain citrus species. Aiming at detecting naringenin in complex matrices such as pummelo and traditional Chinese medicines, we put forward a sensitive and practical indirect competitive enzyme-linked immunosorbent assay (icELISA) based on anti-naringenin monoclonal antibodies (anti-Nar-mAbs). The median inhibitory concentration (IC50) was 4.43 ng/mL, and the working range was 1.15–15.81 ng/mL. The findings of the icELISA for the analysis of naringenin in pummelo and herb samples had a good correlation with the ultra performance liquid chromatography (UPLC) methodology and showed good accuracy and reproducibility. These data demonstrated that the developed icELISA is reliable, accurate, and suitable for detecting naringenin in pummelo and traditional Chinese medicines.

SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata.

Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur-rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII-HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII-HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6mg/mL to 20.4mg/mL was studied using small angle X-ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII-HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII-HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII-HbIII) complex is not present.

Implementation of an analytical method for the simultaneous determination of greenhouse gases in a reservoir using FID/µECD gas chromatography

ABSTRACT The greenhouse gas emissions of CH4, N2O and CO2 from a tropical reservoir and a tributary river that supplies it were measured from 2015 to 2019 using a GC-FID/µECD method. The chromatographic technique was used with a configuration of a methanizer and two specific columns, by direct injection of the gaseous samples using the floating static chamber as a field sampling method. The parameters analysed for the chromatographic methodology validation were selectivity, linearity, detection limit and quantification, bias (as a practical assessment of trueness), precision and uncertainty. These parameters were found to be satisfactory. The results showed a low deviation of the analysed data throughout the period evaluated.

Using continuous chromatography methodology to achieve high-productivity and high-purity enrichment of charge variants for analytical characterization

Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants.

Implementation of Analytical Quality-by-Design for Developing a Robust HPLC Method for Quantitative Estimation of Voriconazole: Application in Drug Formulations

The current studies endeavour to develop and validate a simple, sensitive and robust reversed- phase (RP) high-performance liquid chromatography (HPLC) methodology for quantitative estimation of voriconazole (VRC), following systematic principles of Analytical Quality-by-Design (AQbD). A comprehensive risk assessment plan, followed by factor screening studies employing Taguchi design, was executed to select “vital few” critical method parameters, influencing critical analytical attributes. Subsequently, response surface optimisation studies were conducted on the identified critical method parameters, viz., mobile phase ratio and flow rate, with chosen critical analytical attributes, viz., peak area, theoretical plate count, retention time and peak tailing, employing a Central Composite Design. The design space, demarcating the optimal method operating conditions, was identified using graphical optimisation and subsequently validated employing Monte Carlo simulations. The optimal solution consisted of acetonitrile and acetic acid solution (0.05 %, pH 4) as mobile phase (50:50 v/v), flow rate of 1 mL/ min with a detection wavelength of 256 nm, exhibiting linearity between 0.1 and 50 μg/mL in methanol and Hanks balanced salt solution. Validation studies confirmed precision (97.78-103.42 %), accuracy (98.99-102.34 %), and suitability of the developed analytical method for effective quantification of VRC with high sensitivity (LOD: 0.031 μg/mL; LOQ: 0.093 μg/mL). The validated analytical technique was successfully ratified for quantification of VRC in drug nanoformulations too. In a nutshell, the application of AQbD and Monte Carlo simulations aided in developing a robust analytical method for the estimation of VRC per se as well as in drug nanoformulations.

Screening the elite chemotypes of Gloriosa superba L. in India for the production of anticancer colchicine: simultaneous microwave-assisted extraction and HPTLC studies

BackgroundGloriosa superba L. (Colchicaceae) is a high-value medicinal plant indigenous to Africa and Southeast Asia. Its therapeutic benefits are well-established in traditional medicines including Ayurveda. It is well known for its natural bioactive compound colchicine which exhibits a wide range of pharmacological activities i.e. rheumatism, gout and was also introduced into clinical practices. The increasing demand as well as its illegal harvesting has brought this valuable plant under threatened category.MethodsThe present investigation describes a microwave assisted extraction (MAE) strategy coupled with a densitometric-high performance thin layer chromatographic (HPTLC) methodology for the analysis of colchicine from 32 different populations of G. superba. A Box-Behnken statistical design (3 level factor) has been employed to optimize MAE, in which power of microwave, time of irradiation, aqueous ethanol and pH were used as independent variables whereas colchicine was used as the dependent variables. Chromatography was carried out on Silica gel 60 F254 TLC plates with toluene: methanol, 85:15 (v/v) being used as solvent system. Densitometric measurement was performed at λ=254 nm following post-derivatization (10% methanolic sulphuric acid).ResultsOptimal conditions for extraction to obtain the maximum colchicine yield was found to be 7.51 mg g− 1 which was very close to be predicted response 7.48 mg g− 1 by maintaining microwave power (460 W), irradiation time (6.4 min), aqueous ethanol-30, pH -3. Colchicine content ranged between 2.12–7.58 mg g− 1 among 32 G. superba populations in which only three chemotypes viz. GS- 1, GS- 3, and GS- 2 collected from West Bengal and Sikkim, respectively exhibited maximum yield of colchicine.ConclusionTherefore, this newly developed optimized MAE coupled with HPTLC densitometry methodology not only quantifies colchicine in order to identify elite chemotypes of G. superba, but it also encourages in selecting high yielding populations of the plants for industrial use and economic boost for the farmers. This validated, simple and reproducible HPTLC protocol is being used for the first time to estimate colchicine from natural populations of G. superba obtained from 32 different geographical regions of India.Graphical abstract