Mobile Phase Solvent Research Articles

Application of quality by design in the development of HPTLC Method for the estimation of ceritinib in bulk and synthetic mixture

The aim of the present work is to develop and validate the High-Performance Thin Layar Chromatography method for the estimation of Ceritinib in bulk and synthetic mixture using the Analytical Quality by Design (AQbD) approach as per ICH Q8 (R2). The AQbD is used to develop a precise HPTLC method. The AQbD parameter elements like QTMP (Quality Target Method Profile- Ceritinib, method type, instrument requirement, sample characteristics, synthetic mixture preparation) CMA—Critical method attributes (chloroform ± 1 ml, methanol ± 1 ml, triethylamine ± 0.05), and Risk assessment (the Ishikawa (fishbone) diagram) were carried out for the development of the HPTLC method for Cerinitib. The Box-Behnken design was utilized for the optimization of chromatographic conditions in HPTLC. The method utilized an aluminum HPTLC plate pre-coated with silica gel G60 F254 as a stationary phase along with chloroform: methanol: triethylamine (8.9: 1.6: 0.07 v/v/v) as a mobile phase. ANOVA results and perturbation plot indicates that methanol and triethylamine had more impact than chloroform on the retardation factor. The retardation factor was found to be 0.37. The absorbance mode at 277 nm was used for the densitometric study. It was found that the linearity ranged from 20 to 100 ng/band, with a correlation coefficient of 0.998. No interference was observed from excipients and mobile phase solvents. The results indicate that the AQbD approach is effectively applied to optimize the retardation factor and the method is appropriate for routine laboratory analysis.

Method Development and Validation of HPTLC Method for Simultaneous Estimation of Piperine, Bergapten, Plumbagin and Lupeol in <i>Chitrakadi Vati</i>

Background: Chitrakadi Vati is an Ayurvedic formulation containing several phytoconstituents. A reliable method for simultaneous quantification of its key components is essential for quality control and standardisation. Aim: To develop and validate a High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous determination of four key phytoconstituents in Chitrakadi Vati: Piperine, Bergapten, Plumbagin and Lupeol. Methods: An HPTLC method was developed using silica gel 60F254 plates with a mobile phase of Petroleum ether: Ethyl acetate: Toluene: Formic acid (7:2:1:0.1 v/v/v/v). The method was validated for linearity, accuracy, precision, specificity, robustness, Limit of Detection (LOD) and Limit of Quantitation (LOQ). Robustness was further evaluated using a 2³ factorial design, examining the effects of mobile phase volume, saturation time, and solvent front on retention factors. Results: The method demonstrated good linearity (R² = 0.9972-0.9989), precision (%RSD <2%), accuracy (97.68-102.80 % recovery) and specificity. LOD and LOQ ranged from 100.8-325 ng/band and 305.5-985 ng/band, respectively. The factorial design revealed that mobile phase volume and solvent front significantly impacted retention factors, while saturation time had minimal effect. Conclusion: The developed and validated HPTLC method proved effective for the simultaneous estimation of Piperine, Bergapten, Plumbagin and Lupeol in Chitrakadi Vati. The comprehensive validation, including the Design of Experiments for robustness evaluation, ensures the method’s reliability for quality control applications.

Formation of Micelles by Nonionic Detergent Molecules Leads to the Breakthrough Peak in Reversed-Phase Ultraperformance Liquid Chromatography (UPLC).

A peculiar phenomenon known as "breakthrough" occurs under reversed-phase ultraperformance liquid chromatography (UPLC) conditions and has been under scrutiny for decades. This effect takes place when a large volume of analyte solution, prepared in a solvent with an eluotropic strength significantly higher than that of the initial mobile phase solvent, is injected. According to the literature, under specific experimental conditions, a substantial portion of solutes is carried by the mobile phase and detected near the dead time of the chromatographic system. This phenomenon is typically observed when the injected volume of a particular analyte is sufficiently large. However, the underlying physicochemical principles governing this phenomenon have remained elusive. We present evidence demonstrating that breakthroughs can occur even when injecting a sample of a neat solvent devoid of any solute. By mass spectrometric analysis, we identified the breakthrough peak to represent the nonionic detergent Triton. When columns are equilibrated with water, Triton molecules, present as impurities in filtered water, accumulate on the nonpolar stationary phase. Upon the introduction of a solvent with a stronger elution strength, Triton molecules retained on the stationary phase are removed. As detergents, these Triton molecules aggregate into micelles featuring a hydrophobic inner core and a hydrophilic outer shell. These hydrophilic micelles are carried by the polar mobile phase and detected as the breakthrough peak at the dead time of the chromatographic system. When analytes are present, a portion of the injected solutes is captured by the micelles and transported with the breakthrough plug. This assertion was verified and confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis of a methanolic solution of perfluorooctanoic acid (PFOA). The mass spectra corresponding to the breakthrough plug featured a peak for the PFOA anion (m/z 413) in addition to those for Triton.

Moment analysis method for determination of rate constants of solute permeation across interface of spherical molecular aggregates by means of high-performance liquid chromatography

A moment analysis method was developed for the study of solute permeation at the interface of spherical molecular aggregates. At first, new moment equations were developed for determining the partition equilibrium constant (Kp) and permeation rate constants (kin and kout) of solutes from the first absolute (μ1A) and second central (μ2C) moments of elution peaks measured by using high-performance liquid chromatography (HPLC). Then, the method was applied to the analysis of mass transfer phenomena of three solutes, i.e., hydroquinone, resorcinol, and catechol, at the interface of sodium dodecylsulfate (SDS) micelles. HPLC data were measured by using an ODS column and an aqueous phosphate buffer solution (pH = 7.0) as the mobile phase solvent. Pulse response experiments were conducted while changing SDS concentration (5 - 20 mmol dm−3) in the mobile phase under the conditions that the surface of ODS stationary phase was dynamically coated by SDS monomers. In order to demonstrate the effectiveness of the moment analysis method using HPLC, the values of Kp, kin, and kout were determined for the three solutes as 35 - 69, 2.4 × 10−8 - 1.4 × 10−6 m s−1, and 7.0 × 10−10 - 2.1 × 10−8 m s−1, respectively. Their values increase with an increase in the hydrophobicity of the solutes. The method has some advantages for the study of interfacial solute permeation of molecular aggregates. For example, neither immobilization nor chemical modification of both solute molecules and molecular aggregates is required when elution peaks are measured by using HPLC. Interfacial solute permeation takes place in the mobile phase without any chemical reaction or physical action on molecular aggregates. The values of Kp, kin, and kout were analytically determined from those of μ1A and μ2C by using the moment equations. The results of this study must contribute to the dissemination of an opportunity for studying the interfacial solute permeation of molecular aggregates to many researchers because of extremely high versatility of HPLC.

A robust ultra performance liquid chromatography method for dacarbazine quantification in nanosponge formulation: A quality by design approach

A new, user-friendly Ultra Performance Liquid Chromatography (UPLC) method was developed using Analytical Quality by Design (QbD) for precise analysis of Dacarbazine in both bulk powder and Dacarbazine-loaded Nanosponges. This method follows ICH guidelines and uses a statistically designed approach to optimize the separation conditions for Dacarbazine. A central composite design was implemented to identify the ideal combination of organic solvent (mobile phase composition) and flow rate. The UPLC method was successfully optimized for rapid separation (1.3 minutes) for Dacarbazine using a mobile phase consisting of 70% Methanol: 30% Orthophosphoric acid (0.1%) at a flow rate of 1.0 mL/min, using a Hypersil C18 column. The method exhibited excellent linearity with a correlation coefficient value of 0.997 across a concentration range of 4-20 μg/mL, accuracy (drug recovery of 96-100%) and sensitivity (LOD 1.15 μg/mL, LOQ 3.50 μg/mL) and it successfully quantified the drug in the formulated Dacarbazine-loaded Nanosponges. This QbD-driven UPLC method offers a valuable tool for reliable Dacarbazine quality control in pharmaceuticals providing rapid analysis, high accuracy, and low detection limits.

A New HPLC-Fluorescence Method for the Simultaneous Determination of Fluticasone Propionate and Salmeterol Xinafoate in Their Solutions Involving Schiff-Base Derivatization with Hydrazine

A partially validated high-performance liquid chromatographic method with UV and fluorescence detection was developed to determine fluticasone propionate (FP) and salmeterol xinafoate (SX) simultaneously in their solutions. RP-HPLC (Hypersil C18 column, 250 mm × 4.6 mm, 5 µm) was used for chromatographic separation employing an isocratic mobile phase consisting of methanol, acetonitrile and water (50/20/30, v/v/v) at a flow rate of 0.8 mL.min–1. With the aid of the Schiff-base reaction, the hydrazine derivative of FP was prepared to enhance the fluorescence signal of FP. The method was employed at an excitation maximum of λ = 246, 262 and 298 nm for FP, SX and FP derivatives, respectively. The method is accurate, precise and linear (R2 ≥ 0.999) in the range of 0.246–86.957 µg×mL–1 for FP in its derivatives mixture and 0.156–50.00 µg×mL–1 for SX. The calculated LOD values are 0.081 and 0.052 µg×mL–1, respectively. It was noted that the fluorescence detection sensitivity of FP and SX is better than that of UV (LOD = 0.396 and 0.254 µg×mL–1 for FP and SX, respectively). The advantage of the validated HPLC-Fluorescence method compared with expensive and sophisticated LC-MS methods is its simplicity. When compared with other HPLC-UV/Fluorescence methods, the proposed method includes simple chromatographic conditions (no column oven nor high flow rate) and uses non-buffered mobile phase solvents; it is the first HPLC-Fluorescence method for the simultaneous determination of SX and FP. Moreover, the Fluorescence signal of FP was enhanced by derivatization with hydrazine. This was confirmed by computational and Mass spectrometric analysis.

LC and LC-MS/MS Studies for Identification and Characterisation of Related Substances and Degradation Products of Abrocitinib

In the pharmaceutical industry, Related Substances (RCs), impurities or Degradation Products (DPs) are associated with the Active Pharmaceutical Ingredient (API) in the final drug product. These compounds must be within permissible limits for safe therapeutic use for consumers. Hence there is a need to quantify these compounds using an appropriate analytical method. No method is reported in the literature for quantification of these compounds in abrocitinib which is a medical drug prescribed to treat severe atopic dermatitis in adults. This study includes the optimisation of stability indicating the HPLC method for resolution and subsequent quantification of abrocitinib RCs and structural characterisation of stressinduced DPs of abrocitinib. The method was optimised by varying mobile phase solvents, pH, flow rate and wavelength of the detector. The finalised conditions were validated and applied for the resolution and evaluation of stress-induced DPs. The stress was induced in abrocitinib pure drug HCl (0.1M), NaOH (0.1M), hydrogen peroxide (3%), 80°C in an air oven and 254nm in a UV chamber. The generated DPs were structurally characterised with the LCMS experiment. Abrocitinib and DPs along with known RCs were resolved on ACE Ultra Core Super C18 250mm column using 0.9mL/min gradient flow of methanol (Solvent A), acetonitrile and buffer (Solvent B). The resolved compounds were detected through a UV detector at 295nm and a mass detector at NMR positive mode. The method identifies 5.85min, 3.13min, 6.60min and 4.38min respectively for abrocitinib, related compounds 1 to 3 respectively with acceptable system suitability. A very high correlate (< 0.999) linear graph was achieved within 5 to 30 μg/mL concentration level for abrocitinib and 0.05 to 0.3μg/ mL for related compounds. Three DPs with molecular mass and formula of C10H13N5 (203.2 g/mol), C11H14N4 (202.2 g/mol) and C13H19N5O2S (309.3 g/mol) in acid-induced stress study and two DPs of C12H21N5O2S (299.3 g/mol) and C11H19N5O2S (285.3 g/mol) in peroxide-induced stress study and one DP of C10H14N4 (190.2 g/mol) in base induced stress study were successfully characterised. The method proposed in this study can effectively resolve unknown degradation products, and known related compounds along with abrocitinib and is hence applicable for quality control analysis of abrocitinib.

Antioxidant Activity and Isolation of Bioactive Compounds from Seaweed of Silpau (Dyctyosphaeria sp)

Seaweed is the key to developing habitat in deep sea, coastal, and estuarine environments. Seaweed, humans, and animals have been interrelated since the beginning, especially as a food source of fiber, protein, vitamins, antioxidants, and bioactive compounds. Natural antioxidants are seen as a superior and safe method of fighting free radicals. Sources of antioxidants usually come from land plants and rarely from seaweed. Silpau is a seaweed and a local food ingredient in Maluku Province, Indonesia, and has the potential as an antioxidant. Antioxidant and bioactive compounds are needed by humans and animals as antibodies to fight various negative influences from the environment. This research aimed to isolate and characterize active antioxidant compounds and the toxicity of silpau extracts. The extraction of the active compound begins with a single maceration method for 3 x 24 hours with methanol solvent, processing with the partition extraction method for subfraction separation using column chromatography with the silica gel stationary phase and the mobile phase solvent n-hexane: ethyl acetate with a ratio (50: 1 ~ 1: 1) gradient. The sample toxicity was tested using the Brine Shrimp Lethality Test method. The trial activity used the Free Radical Reduction method with 2,2-diphenyl-1-pikrilhidrazil as a reagent. This research succeeded in isolating one bioactive compound from the glare green macroalgae. Based on the phytochemical analysis and FT-IR spectrophotometer, sub-fraction IV was identified as a triterpenoid class of compounds that has an IC50 value of 126,582 μg / ml and is considered an antioxidant.

The Solvation Shell of Small Solutes in Aqueous-Organic Solvent Mixtures and Its Implications for Reversed-Phase Liquid Chromatography.

Reversed-phase liquid chromatography (RPLC) operates with water-organic solvent (W-OS) mobile phases where preferential solvation (PS) of solutes is likely. To investigate the relevance of the solute solvation shell in the mobile phase for RPLC retention, we combine data from molecular dynamics simulations of small, neutral solutes (six analytes and two dead time markers) in W-methanol (MeOH) and W-acetonitrile (ACN) mixtures with corresponding retention data obtained on an RPLC column over a wide range of W/OS ratios. Data derived from Kirkwood-Buff integrals show PS by the OS for analytes vs low or negative PS for dead time markers. W-ACN mixtures generate a higher amount of PS than W-MeOH mixtures, which contributes to the higher eluent strength of ACN in RPLC. Difference spatial distribution functions reveal anisotropic solvation shells with OS excess at hydrocarbon elements and W excess at functional groups, predicting that retention by the hydrophobic stationary phase is favored by hydrocarbon elements and limited by functional groups. Analysis of solute-solvent hydrogen bonds pinpoints the hydrogen-bond requirements toward W as the retention-limiting factor. The relation between the solute solvation shell and retention confirms the importance of W-OS and solute-W hydrogen bonding for RPLC retention.

Greener and Whiter Analytical Chemistry Using Cyrene as a More Sustainable and Eco-Friendlier Mobile Phase Constituent in Chromatography.

Cyrene (dihydrolevoglucosenone) was evaluated for the first time as a potential sustainable mobile phase solvent in reversed-phase chromatography. As a benign biodegradable solvent, Cyrene is an attractive replacement to classical non-green organic chromatographic solvents such as acetonitrile and a modifier, co-eluent to known green solvents such as ethanol. Compared to ethanol, Cyrene is less toxic, non-flammable, biobased, biodegradable, and a cheaper solvent. A fire safety spider chart was generated to compare the properties of Cyrene to ethanol and show its superiority as a greener solvent. Cyrene's behavior, advantages, and drawbacks in reversed-phase chromatography, including the cut-off value of 350 nm, elution power, selectivity, and effect on the column, were investigated using a model drug mixture of moxifloxacin and metronidazole. A monolithic C18 (100 × 4.6 mm) column was used as a stationary phase. Different ratios of Cyrene: ethanol with an aqueous portion of sodium acetate buffer mobile phases were tested. A mobile phase consisting of Cyrene: ethanol: 0.1 M sodium acetate buffer pH 4.25 (8:13:79, v/v/v) was selected as the most suitable mobile phase system for separating and simultaneously determining metronidazole and moxifloxacin. The greenness and whiteness of the method were evaluated using the qualitative green assessment tool AGREE and the white analytical chemistry assessment tool RGB12. Further potentials of Cyrene as a solvent or modifier in normal phase chromatography, liquid chromatography-mass spectrometry, and supercritical fluid chromatography are discussed.

B-310 High Through-put Online Solid Phase Extraction of Immunosuppressive Drugs From Whole Blood on Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) in a Tertiary Hospital Laboratory

Abstract Background Cyclosporine A and Tacrolimus are immunosuppressive drugs that are commonly used in solid organ transplant and are characterized by a relatively narrow therapeutic index, potential drug interactions and significant intra- and inter-individual variability. Therapeutic drug monitoring is therefore beneficial for optimizing dosage regimens, preventing rejection of the graft and reducing toxicities in transplant recipients receiving immunosuppressive drug therapy. A commercial assay (RECIPE ClinMass LC-MS/MS Advanced kit) for simultaneous measurement of immunosuppressants in whole blood using LC-MS/MS methodology has been adapted for assay performance evaluation in our laboratory on the Agilent 1290 Infinity II liquid chromatograph coupled to an Agilent 6470 triple quadrupole mass spectrometer. Our study evaluated the analytical performance of Cyclosporine A and Tacrolimus on the RECIPE assay and compared it against our existing in-house LC-MS/MS assay. Methods The RECIPE ClinMass® LC-MS/MS Advanced Immunosuppressants assay kit consist of mobile phase solvents, precipitation reagents, stable isotopic labeled internal standards, solid phase extraction and analytical columns, lyohilized whole blood calibrators and quality controls. Drug analytes were extracted from whole blood using single-step protein precipitation followed by on-line solid phase extraction on the LC, eluted on isocratic chromatography and detected on positive ESI MS ionisation mode. The RECIPE assay run time is under 3 min. Performance validation parameters included within/total assay imprecision, linearity, recovery, limits of detection (LOD)/quantification (LOQ) and carry-over. Method correlation studies using fresh patient whole blood specimens (n = 212 Cyclosporine A; n = 120 Tacrolimus) were performed on existing in-house assay on Agilent 6460 LC-MS/MS and the RECIPE assay on Agilent 6470 LC-MS/MS systems. Results Within run and total imprecision for Cyclosporine A and Tacrolimus on the RECIPE assay were determined to be ≤4.4% and ≤7.5% on the Agilent 6470 LC-MS/MS. The assay demonstrated linearity across the analytical measurement range up to 1500 ug/L for Cyclosporine A and up to 55 ug/L for Tacrolimus. Analytical recoveries obtained for both drugs ranged between 93%–100%. The LODs were assessed to be 13.0 ug/L and 0.70 ug/L for Cyclosporine A and Tacrolimus respectively. LOQs were assessed to be 22.7 ug/L and 1.30 ug/L for Cyclosporine A and Tacrolimus respectively. Results of carry-over studies were insignificant. Participation in the CAP Immunosuppressive Drugs External Quality Assessment Survey showed good agreement with LC-MS/MS peers. Method correlation with in-house assay demonstrated good agreement: (1) Cyclosporine A: Passing Bablok regression RECIPE = 0.94 (In-house) + 17.2, Bland Altman mean bias of +2.0%, Spearman correlation coefficient of 0.985; (2) Tacrolimus: Passing Bablok regression of RECIPE = 1.03 (In-house) - 0.2, Bland Altman mean bias of -0.2%, Spearman correlation coefficient of 0.988 Conclusion The RECIPE ClinMass® LC-MS/MS Immunosuppressants assay performed within manufacturer’s specifications except for LOD/LOQs which were determined at higher concentrations. The assay is adaptable to contemporary LC-MS/MS systems and confer benefits from rapid turnaround time (<3 min), simplified specimen preparation procedures and ready prepared reagents. The advantages allow enhancements in workflow efficiency and ease of staff training in our laboratory and the assay is suitable for routine clinical use in high workload clinical laboratories.

Validated ultraviolet high-performance liquid chromatography method for post-mortem 5-hydroxy-indoleacetic acid measurement in human cerebrospinal fluid

Objectives: The objective of this study was to develop an ultraviolet high-performance liquid chromatography (UV-HPLC) method for the measurement of 5-hydroxyindoleacetic acid (5-HIAA) in human cerebrospinal fluid (CSF) as a potential biomarker for neurological and psychiatric illnesses, including depressive disorders with suicidal behavior. Materials and Methods: The study utilized CSF samples from individuals brought for medicolegal autopsy. The 5-HIAA concentration was measured using a UV-HPLC method with three mobile-phase solvents. The most effective mobile phase solvent was then used to measure 5-HIAA in the CSF samples. The materials used in the study included CSF samples obtained from individuals brought for medico-legal autopsy, UV-HPLC equipment, and mobile phase solvents, including 5-hydroxyindole-3-acetic acid (Merck/sigma), acetonitrile, concentrated formic acid, concentrated acetic acid, methanol, and phosphoric acid. Statistical Analysis: This was done using R Studio (version 4.2.0). Results: The study found that the UV-HPLC method utilizing formic acid (0.05–0.1%): acetonitrile in a 1:9 as mobile phase was the most effective for measuring 5-HIAA in human CSF. The method exhibited excellent linearity, accuracy, and precision. Conclusion: The study concludes that the developed UV-HPLC method is reliable and effective for measuring 5-HIAA in human CSF. Measuring 5-HIAA levels in CSF can serve as a potential biomarker for neurological and psychiatric illnesses, including depressive disorders with suicidal behavior. This method is promising for clinical and forensic practice to diagnose suicidal cases. Further research is needed to determine the clinical significance of these findings and the potential for broader application in psychiatry. This article helps to give a practical, cost-effective methodology to detect CSF 5-HIAA.

Exploring the Impact of Organic Solvent Quality and Unusual Adduct Formation during LC-MS-Based Lipidomic Profiling.

Liquid chromatography-mass spectrometry (LC-MS) is the key technique for analyzing complex lipids in biological samples. Various LC-MS modes are used for lipid separation, including different stationary phases, mobile-phase solvents, and modifiers. Quality control in lipidomics analysis is crucial to ensuring the generated data's reliability, reproducibility, and accuracy. While several quality control measures are commonly discussed, the impact of organic solvent quality during LC-MS analysis is often overlooked. Additionally, the annotation of complex lipids remains prone to biases, leading to potential misidentifications and incomplete characterization of lipid species. In this study, we investigate how LC-MS-grade isopropanol from different vendors may influence the quality of the mobile phase used in LC-MS-based untargeted lipidomic profiling of biological samples. Furthermore, we report the occurrence of an unusual, yet highly abundant, ethylamine adduct [M+46.0651]+ that may form for specific lipid subclasses during LC-MS analysis in positive electrospray ionization mode when acetonitrile is part of the mobile phase, potentially leading to lipid misidentification. These findings emphasize the importance of considering solvent quality in LC-MS analysis and highlight challenges in lipid annotation.

Extraction and quantification of melatonin in cornelian cherry (Cornus mas L.) by ultra-fast liquid chromatography coupled to fluorescence detector (UFLC-FD)

AbstractWild edible plants (WEPs) can be widely found in the world and defined as native species that grow naturally in their natural habitat. They have become part of the traditional food as human diet and used in folk medicine to treat diseases. They are very rich in terms of nutraceuticals. Melatonin is a natural hormone providing several benefits for human health. It has functions such as regulating growth and development and increasing tolerance to environmental stress factors in plants. It is stated that the serum melatonin level in humans increases after intake of foods containing melatonin. This study examined the presence of melatonin in wild grown cornelian cherry fruits by UFLC-FD and determined suitable extraction and chromatographic conditions. The optimum mobile phase, excitation/emission wavelength, and extraction solvent were determined as methanol: water: acetic acid, 275/345 nm, and methanol: water: HCl, respectively. Melatonin content in fruits ranged from 130.82 to 201.84 ng g−1 in fresh fruit.

Analytical Purity Determinations of Universal Food-Spice Curcuma longa through a QbD Validated HPLC Approach with Critical Parametric Predictors and Operable-Design’s Monte Carlo Simulations: Analysis of Extracts, Forced-Degradants, and Capsules and Tablets-Based Pharmaceutical Dosage Forms

Applications of analytical quality by design (QbD) approach for developing HPLC (High Performance Liquid Chromatography) methods for food components assays, and separations of complex natural product mixtures, are still limited. The current study developed and validated, for the first time, a stability-indicating HPLC method for simultaneous determinations of curcuminoids in Curcuma longa extracts, tablets, capsules, and curcuminoids' forced degradants under different experimental conditions. Towards separation strategy, critical method parameters (CMPs) were defined as the mobile phase solvents' percent-ratio, the pH of the mobile phase, and the stationary-phase column temperature, while the peaks resolution, retention time, and the number of theoretical plates were recognized as the critical method attributes (CMAs). Factorial experimental designs were used for method development, validation, and robustness evaluation of the procedure. The Monte Carlo simulation evaluated the developing method's operability, and that ensured the concurrent detections of curcuminoids in natural extracts, commercial-grade pharmaceutical dosage-forms, and the forced degradants of the curcuminoids in a single mixture. The optimum separations were accomplished using the mobile phase, consisting of an acetonitrile-phosphate buffer (54:46 v/v, 0.1 mM) with 1.0 mL/min flow rate, 33 °C column temperature, and 385 nm wavelength for UV (Ultra Violet) spectral detections. The method is specific, linear (R2 ≥ 0.999), precise (% RSD < 1.67%), and accurate (% recovery 98.76-99.89%), with LOD (Limit of Detection) and LOQ (Limit of Quantitation) at 0.024 and 0.075 µg/mL for the curcumin, 0.0105 µg/mL and 0.319 µg/mL for demethoxycurcumin, and 0.335 µg/mL and 1.015 µg/mL for the bisdemethoxycurcumin, respectively. The method is compatible, robust, precise, reproducible, and accurately quantifies the composition of the analyte mixture. It exemplifies the use of the QbD approach in acquiring design details for developing an improved analytical detection and quantification method.

Montmorillonite-based polymethacrylate composite monoliths as stationary phase materials for food and pharmaceutical analysis in capillary liquid and gas chromatography

This work relates to the preparation of novel and promising stationary phases containing inorganic-organic composites for capillary liquid and gas chromatography. A naturally occurring montmorillonite was introduced to polymethacrylate monoliths, then used under different conditions of GC and HPLC at the same time. The performance of the columns was evaluated for the separation of alkane and alkylbenzene series in GC and capillary HPLC, respectively. While the bare monoliths failed to separate the model analytes, montmorillonite-based polymethacrylate allowed a full separation of the mixtures with Rs≥1.42. The columns were applied for the determination of myrcene and limonene isomers in the peel extracts of some fruits using GC, and for the analysis of active ingredients including aspirin, vitamin-C, caffeine, and ibuprofen extracted from common drugs using capillary HPLC. In GC, fast separation was achieved in 1.0 min with Rs of 6.53. The columns exhibited the best efficiency for myrcene with 20,900 plates/m. Using the capillary HPLC columns, the active ingredients were resolved in 10 min with Rs≥5.72. The efficiency values located between 12,800–21,700 plates/m in all cases. The developed methods were found to be linear in the range of 0.10–10.0 and 0.20–180 μg/mL for GC and HPLC, respectively. In comparison with commercial columns, the results in GC methods reveal that, despite their much shorter length, the prepared columns proved a faster separation with higher efficiency and comparable detection limits and chromatographic resolution. The prepared HPLC capillaries exposed lower run times and detection limits with comparable efficiency and resolution, and consume fewer samples and mobile phase solvents. The results demonstrate that the montmorillonite-based polymethacrylate composites are applicable as stationary phases for routine analysis and quality control of important fields such as food and pharmaceutical samples.

Naturally Occurring Montmorillonite-Based Polymer Monolith Composites as Stationary Phases for Capillary Liquid and Gas Chromatography

This work is associated with the preparation of capillary chromatographic columns containing inorganic-organic composites comprised of naturally occurring montmorillonite (MMT) clay mineral and polymethacrylate monolithic material. The prepared composites combine the best qualities of both constituents, offering desirable properties for use under the disparate conditions of both GC and HPLC at the same time. The stationary phases were investigated by scanning electron microscopy (SEM), the specific surface area, and thermogravimetric analysis (TGA) and examined in terms of various conditions utilized for GC and HPLC methods. The prepared columns demonstrated an excellent permeability and stability against common chromatographic conditions, such as the eluent type, flow rate, pressure, and temperature. The results confirmed that the addition of small amounts of MMT into the monolith induced significant improvement in the specific surface area, which contributed to the formation of more active sites and enhanced the retention of analytes. The registered column backpressures did not exceed 980 kPa and 16,500 kPa for the prepared GC and HPLC columns, respectively. The prepared columns were subjected to the separation of various interesting compounds possessing different chemistries and polarities, including alkanes, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), alcohols, ketones, phenols, some common organic solvents, and isomeric mixtures. Under the optimal conditions, the efficiency of the columns fell between 4900–38,500 plates m−1 for GC and 3400–58,800 plates m−1 for capillary HPLC applications. In all cases, the measured chromatographic resolution was more than 1.38, with excellent an peak symmetry and low tailing factors. In comparison with the most commonly used commercial columns, the polysiloxane open tubular column for GC and silica-based C18 packed column for HPLC, the prepared GC columns demonstrated a faster separation with a higher efficiency, comparable resolution and tailing factors, and lower consumption of carrier gas. Regarding the capillary columns prepared for HPLC, the chromatographic experiments exposed a much lower run time with a comparable efficiency and resolution and drastically lower consumption of mobile phase solvents and samples. The results demonstrate that the MMT-based polymethacrylate monolith composites are applicable as novel and promising separation media for analyzing various mixtures of interest in different fields, such as petrochemical and environmental samples.

Identification and trend analysis of organic cationic contaminants via non-target screening in suspended particulate matter of the German rivers Rhine and Saar

Non-target screening of suspended particulate matter (SPM), collected from the German rivers Rhine and Saar, was conducted with the goal of identifying organic, permanent cationic contaminants and of estimating their temporal trends over an extended period. Therefore, annual composite samples of SPM, provided by the German Environmental Specimen Bank, were extracted and analyzed with high resolution LC-QToF-MS/MS. To facilitate the identification of substances belonging to the class “permanent cations”, prioritization methods were applied utilizing the physicochemical properties of these compounds. These methods include both interactions of the analyte molecules with cation exchange resins and analyzing mass deviations when changing from non-deuterated to deuterated mobile phase solvents during LC-MS analysis. By applying both methods in a combined approach, 123 of the initially detected 2695 features were prioritized, corresponding to a 95% data reduction. This led to the identification of 22 permanent cationic species. The organic dyes Basic Yellow 28 and Fluorescent Brightener 363 as well as two quaternary ammonium compounds (QACs) were detected in environmental samples for the first time to best of or knowledge. The other compounds include additional QACs, as well as quaternary tri-phenylphosphonium compounds (QPC/TPP). In addition to identification, we determined temporal trends of all compounds over a period of 13 years and assessed their ecotoxicological relevance based on estimated concentrations. The two QACs oleyltrimethylammonium and eicosyltrimethylammonium show significant increasing trends in the Rhine SPM and maximum concentrations in the Saar SPM of about 900 and 1400 µg/kg, respectively. In the case of the dyes, constant trends have been observed at the end of the studied period, but also maximum concentrations of 400 µg/kg for Basic Yellow 28 in 2006 and 1000 µg/kg for Fluorescent Brightener 363 in 2015, potentially indicating a strong ecotoxicological risk.

Phytochemical evaluation of the marketed drakshasava formulation by spectroscopic &amp; chromatographic methods

Background: Drakshasava is a general health tonic, carminative and blood purifier. It is used in the treatment of anemia, digestive &amp; cardiac disorders. A review of the literature suggested that modern analytical methods are not reported for the phytochemical evaluation of drakshasava. Objective: To report phytochemical evaluation of Drakshasava Formulation by Spectroscopic &amp; Chromatographic Methods. Materials and methods: Total phenolic, flavonoid, sugar and reducing sugar content in marketed drakshasava formulations were estimated by a UV-Vis spectrophotometer. The markers and extract of drakshasava formulations I &amp; II were scanned by FTIR spectrophotometer individually to find the common bands of the vibrational spectra of markers and formulations. The markers and extract of drakshasava formulations I &amp; II were subjected to HPLC analysis. Results: UV-Vis spectrophotometer analysis revealed that total phenolic, flavonoid, total sugar, &amp; total reducing sugar of marketed drakshasava formulations were within the limit of ayurvedic pharmacopeia. The gradient mobile phase solvent A (HPLC grade water containing 0.85% w/v ortho phosphoric acid) and solvent B (acetonitrile) were optimized by considering the resolution, peak shape, and symmetry. Conclusion: The evaluation parameters reported in this article could be used as a standard reference for quality management of marketed drakshasava formulations.

Selection of Stationary Phase and Mobile Phase in High Performance Liquid Chromatography

Selection of ideal mobile phase and stationary phase is very important to get accurate separation of mixtures or impurities as a whole. There indeed are parameters which are explained in this article, important to be kept in mind while a method development of HPLC method. But as such there is no list of such parameters and their accurate limits can be applied while method development. In this article, there have been mention of certain parameters which are generally looked for, and not exact, but optimal application of such has been discussed. When we talk about stationary phase, parameters which we tend to optimize very often include mostly commonly the pH of the analyte as well the mobile phase used and stability of the column packing material over a range of temperature. Choosing of stationary phase greatly depends on the nature of analyte. Stationary phase will depend and vary simultaneously if analyte is lipophilic or an ionic compound. In order to increase separation and increase mass transfer there have been new addition to column packing material apart from the widely used silica. New advances result into greater column loading, better flow, reduced plate height and reduced back pressure. Leaving alone stationary phase, mobile phase selection is also a task of its own. Very commonly used mobile phase solvents include acetonitrile, methanol, THF, water and buffer of salts like acetate, phosphate, etc. Apart from these traditional choices, there have been use of pressurized hot water and ionic liquids as mobile phases which are termed as “Environmental-friendly” because these solvents are devoid of any organic counterpart or organic modifier in them, which produces harmful fumes on getting heated. Apart from the environmentally friendly mobile phase options, we have some specific chemicals to be used when chemical property of the analyte is specific.