Elution Research Articles

Improvement and Application HPLC-UV Method for detecting of N-drugs in Pharmaceutical Formulations

The ultraviolet-visible high-performance liquid chromatography (HPLC–UV) coupled method was developed to detect five pharmaceutical compounds: indapamide (INDP), clomipramine (CMI), promethazine HCl (PMH), lisinopril (LSP) and trifluoperazine HCl (TFPH). The mobile phase consisted of 70% acetonitrile and 30% water, ensuring specific retention times (tR) for each compound. Analytical validation parameters were calculated from a calibration curve, including sensitivity, limit of detection (LOD) and linearity of the method. The LOD values of N-drugs were calculated as 6.24 μM for INDP, 2.19 for CMI, 10.57 for PMH, 6.68 for LSP and 1.25 for TFPH. Peak deconvolution or spectral deconvolution of standard solutions of drug mixtures was used to separate overlapping peaks in a dataset and extract information about individuals. Chromatographic separation of the standard mixture of pure compounds achieved a good resolution (R_s), ensuring the effective separation of the mixture components. However, other mixtures did not achieve satisfactory resolution due to retention time interference. Overall, the HPLC–UV method demonstrated good sensitivity and selectivity for the detection of the chosen pharmaceutical compounds. KEYWORDS Chromatogram, deconvolution, limit of detection, pharmaceutical analysis, retention time, sensitivity

Synthesis, impurity profiling, simultaneous NP-HPTLC method development, molecular modelling study and EGFR tyrosine kinase inhibitory profile: An integrated approach to characterize desethynyl erlotinib process impurity

The study presents an integrated approach to characterizing Desethynyl Erlotinib, a process impurity in the synthesis of Erlotinib, a potent EGFR tyrosine kinase (EGFR TK) inhibitor used in lung cancer treatment. A normal phase high-performance thin-layer chromatography (NP-HPTLC) method was developed and validated for the simultaneous profiling of Erlotinib and its Desethynyl Erlotinib impurity. The optimized method utilized ethyl acetate, methanol, and glacial acetic acid (9: 0.5: 0.5 v/v/v) as the mobile phase for effective separation and quantification. The method demonstrated excellent linearity for Erlotinib and its impurity over a concentration range of 200-1200 ng/spot, with R2 values of 0.9979 and 0.9998, respectively. Validation confirmed precision with intra-day and inter-day % RSD values of less than 2% and robustness. The “limits of detection (LOD) and quantification (LOQ)” were 5.18 ng/spot and 15.70 ng/spot for Erlotinib and 7.07 ng/spot and 21.43 ng/spot for the impurity. In-vitro assays against the A549 lung cancer cell line expressing wild-type EGFR tyrosine kinase (WT EGFR TK) showed that the Desethynyl Erlotinib impurity exhibits significant inhibition compared to Erlotinib, suggesting the potential toxicity of the Desethynyl Erlotinib impurity and causing side effects such as diarrhea, skin rashes and interstitial lung disease due to WT EGFR tyrosine kinase (WT EGFR TK) inhibition. Molecular docking and molecular dynamics simulations further corroborated greater stability in the Desethynyl Erlotinib impurity with WT EGFR tyrosine kinase (WT EGFR TK). Clinically, these findings highlight the importance of monitoring and minimizing impurities like Desethynyl Erlotinib to prevent adverse effects and maintain the therapeutic safety of Erlotinib in lung cancer treatment. This research underscores the necessity for rigorous quality control in Erlotinib production to ensure purity and therapeutic effectiveness.

Sustainability assessment of the micellar optimized HPLC method for concomitant quantification of a common triple regimen for cardiovascular disorders: application to marketed formulations and in vitro dissolution testing.

Hypertension and dyslipidemia are two of the most frequently co-occurring cardiovascular risk factors. The combined regimen of hydrochlorothiazide (HCTZ), rosuvastatin (ROS), and losartan (LOS) helped in the successful management of both conditions. This work's objective is to develop an eco-friendly, sensitive, simple, and reliable chromatographic method for the simultaneous estimation of HCTZ, ROS, and the LOS ternary mixture in their pure form, and pharmaceutical formulations. Utilizing sodium dodecyl sulphate (SDS) in combination with polyoxyethylene-23-lauryl ether (Brij-35) could be a greener option with minimal environmental impact in liquid chromatographic separation techniques. Therefore, a micellar HPLC protocol was optimized and validated employing a mobile phase comprising a mixture of micelles of SDS (0.15 mol L-1) and Brij-35 (0.03 mol L-1) with 0.01 M monobasic potassium phosphate buffer, pH 3.5. The cited drugs were detected at 230 nm within five minutes, using a symmetry RP-C18 column, an isocratic elution mode, and a flow speed of 1 mL min-1. Method validation was successfully performed as stated by ICH. The suggested HPLC approach was effectively applied to determine the mentioned drugs in three pharmaceutical preparations. It was also employed for monitoring their in vitro release pattern using two innovator tablet formulations, applying the USFDA guidelines. The applied procedure was evaluated and compared with previously published HPLC approaches using various metrics, including the Eco-Scale Scoring method (AES), the Green Analytical Procedure Index (GAPI), and the Analytical Greenness Calculator (AGREE) for greenness, the most current Blue Applicability Grade Index (BAGI) for blueness, and the more inclusive RGB 12 algorism for whiteness assessment.

Pharmacognostic analysis and antimalarial evaluation of quercetin in Ilex umbellulata bark using HPTLC, in vitro screening, molecular docking, and network pharmacology

BackgroundThe bark of Ilex umbellulata is traditionally used for the treatment of many diseases such as malaria. Despite its traditional relevance, the pharmacognostic parameters and pharmacological properties remained unexplored. In this study, we aim to develop the missing pharmacognostic parameters with modern analytical techniques and carry out multi-step computational studies to study the antimalarial potential of I. umbellulata.ResultsThe bark was 2–6 mm thick, composed of different colored layers, and was bitter-sweet in taste. Powdered microscopy revealed the presence of starch granules, calcium oxalate crystals, cork cells, trichomes, and fibers. Physicochemical properties such as ash values (total, acid-insoluble, and water-soluble), extractive values (petroleum ether, chloroform, ethyl acetate, methanol, aqueous, 80% MeOH), moisture content, swelling index, fluorescence, and pH of the bark were determined. FT-IR fingerprint profiling of petroleum ether, chloroform, ethyl acetate, methanol, aqueous, and 80% MeOH extracts revealed characteristic bands at different wavelengths that are indicative of the presence of certain functional groups. HPTLC fingerprint profiling with a mobile phase of hexane: ethyl acetate: formic acid (4.5:5.5:0.5 v/v) revealed 9 characteristic peaks. With a mobile phase of toluene: ethyl acetate: formic acid (5:4:0.2 v/v), the validated TLC densitometric studies revealed the presence of 2.07 mg of quercetin (Rf = 0.477 ± 0.005) in 100 mg of 80% MeOH bark extract of I. umbellulata. JazQSAR web tool previously developed by us predicts the IC50 of quercetin against Plasmodium falciparum as 3.88 ± 0.35 µM, which was not far from the practically observed value for quercetin. Multi-target molecular docking with a validated docking protocol revealed that quercetin could potentially interact with 20 proteins of P. falciparum that are highly expressed during the schizont and trophozoite stages. Network pharmacology studies revealed that quercetin could potentially alleviate malaria mainly by inhibiting pro-inflammatory response through the action of IL-4, IL-10, and IL-13 and by triggering the immune system.ConclusionsThe pharmacognostic parameters of I. umbellulata bark may be used as quality control parameters to aid in identification and authentication and to prevent adulteration. The results obtained from the multi-target molecular docking and network pharmacology studies support the use of I. umbellulata as a traditional herbal remedy against malaria.

Method development for simultaneous separation and identification of different therapeutic drugs by humic acid-based high performance liquid chromatographic column

Simultaneous quantitative and qualitative identification of drugs is an increasing demand by pharmaceutical industries. Present study deals with development of efficient, reliable and simultaneous separation of five drugs belonging to different therapeutic groups (i.e., Ornidazole (anti-bacterial/anti-fungal), Oxaprozin (anti-inflammatory), Fusidic acid (antibiotic), Pantoprazole and Omeprazole (acid reducers)) as first application by a newly developed humic acid appended aminopropyl silica filled chromatographic column. The extracted soil humic acid was immobilized on aminopropyl silica by amide bond formation. The material was characterized by fourier-transform infrared and scanning electron microscopic techniques. The separation method was developed by varying mobile phase composition, pH and flow rate, resulting optimum conditions were found to be 30% acetonitrile, 5.5 pH at 1 mL/min flow rate. Method was validated according to ICH guidelines. The results suggested higher accuracy, reproducibility, and linearity of column. Moreover, satisfactory values for limit of detection and limit of quantification justified the sensitivity of developed column. The column was successfully applied for determination of each drug in their commercial formulations. Finally, the efficiency of developed column was also compared with commercially available column. The results revealed that developed column is suitable for quality control and routine analysis of the cited drugs in their pharmaceutical dosage form.

Development of a green and rapid ethanol-based HPLC assay for aspirin tablets and feasibility evaluation of domestically produced bioethanol in Thailand as a sustainable mobile phase

Abstract Despite the growing trend of using ethanol as a greener alternative to hazardous solvents like methanol and acetonitrile in high-performance liquid chromatography (HPLC) analysis, no ethanol-based assay has been developed for aspirin tablets, nor has the potential of domestically produced bioethanol as a mobile phase been explored – until now. This study introduces a novel ethanol-based HPLC assay for aspirin and assesses the feasibility of using locally produced bioethanol. The optimized method, featuring a 40% (v/v) ethanol–water mobile phase adjusted to pH 3.6 with glacial acetic acid, with a 15-cm C18 column at 40°C and UV detection at 237 nm, successfully separated aspirin and its degradation product, salicylic acid, in 5 min. It demonstrated excellent linearity (0.1–0.6 mg·mL−1, r² = 0.9997), sensitivity (LOD = 0.01 mg·mL−1, LOQ = 0.03 mg·mL−1), accuracy, and precision, with no interference from excipients. The method’s performance on commercial aspirin tablets aligned with official pharmacopoeial methods. In a case study using fuel-grade bioethanol (>99.5% purity) derived from sugarcane molasses or cassava from local manufacturers in Thailand, chromatographic and analytical results matched those obtained with imported ethanol, validating its effectiveness. This innovative approach not only reduces costs and dependence on imported solvents but also supports local bio-circular-green economies and promotes greener, more sustainable analytical practices.

The Prevalence of Xylazine in Patient Urine Samples That Were Positive for Fentanyl in Western New York.

Xylazine, a veterinary sedative, is increasingly being discovered in the illegal drug supply across the United States and is associated with overdose fatalities. Not approved for human use, xylazine poses life-threatening risks, particularly when used in conjunction with opioids such as fentanyl. This study evaluates the prevalence of xylazine in urine samples that screened positive for fentanyl. The evaluation involved measuring the parent drug xylazine and its metabolites, xylazine glucuronide and hydroxy-xylazine, as well as finding their correlation with fentanyl and norfentanyl concentrations in patient samples. Samples were collected over a one-month period. Urine samples were analyzed for fentanyl, norfentanyl, and 3 xylazine-specific analytes (i.e., xylazine, xylazine glucuronide, and hydroxy-xylazine). Briefly, reverse-phase chromatographic separation was performed on a Bonshell Phenyl-Hexyl column utilizing a binary mobile phase gradient. The eluents were detected through positive electrospray ionization and multiple reaction monitoring analysis on a triple quadrupole mass spectrometer. Out of a total of 230 urine samples, 184 were confirmed positive for fentanyl. Xylazine was detected in 56 out of the 184 fentanyl-positive samples, accounting for 30% of the fentanyl-positive confirmatory test. Xylazine (or its metabolites) was not observed in fentanyl-negative samples. Furthermore, the parent xylazine drug was detected in all 56 samples, whereas the metabolite glucuronide and hydroxy forms were only detected in 28 and 6 samples, respectively. This study estimated a xylazine prevalence of 30% in fentanyl-positive urine samples within our local Western New York population. This study represents the first report within our clinical population.

Gel-Based Electrolytes for Organic Electrochemical Transistors: Mechanisms, Applications, and Perspectives.

Organic electrochemical transistors (OECTs) have emerged as the core component of specialized bioelectronic technologies due to their high signal amplification capability, low operating voltage (<1 V), and biocompatibility. Under a gate bias, OECTs modulate device operation via ionic drift between the electrolyte and the channel. Compared to common electrolytes with a fluid nature (including salt aqueous solutions and ion liquids), gel electrolytes, with an intriguing structure consisting of a physically and/or chemically crosslinked polymer network where the interstitial spaces between polymers are filled with liquid electrolytes or mobile ion species, are promising candidates for quasi-solid electrolytes. Due to relatively high ionic conductivity, the potential for large-scale integration, and the capability to suppress channel swelling, gel electrolytes have been a research highlight in OECTs in recent years. This review summarizes recent progress on OECTs with gel electrolytes that demonstrate good mechanical as well as physical and chemical stabilities. Moreover, various components in forming gel electrolytes, including different mobile liquid phases and polymer components, are introduced. Furthermore, applications of these OECTs in the areas of sensors, neuromorphics, and organic circuits, are discussed. Last, future perspectives of OECTs based on gel electrolytes are discussed along with possible solutions for existing challenges.

High throughput quantification of oil degradation using high-performance thin layer chromatography

India, the largest consumer of vegetable oil, generates massive amounts of wastewater during its production, adversely affecting the aquatic ecosystem. Various methods are used to degrade vegetable oils. Conventionally, oil degradation was quantified by measuring a sample’s end products, such as free fatty acid (FFA) or glycerol. None of these techniques measures intermediate compounds (monoglycerides or diglycerides), which leads to erroneous oil degradation calculations. We propose a high-performance thin-layer chromatography (HPTLC) based method to quantify the percentage degradation of soybean oil. The mobile phase consists of n-hexane, ethyl acetate, acetic acid (9:1:0.1, v/v/v) and spots scanned at 196 nm. Soybean oil in water (1%, v/v) degraded using immobilized lipase at 37 °C temperature, pH 7, and 180 rpm for 6 h. The change of bonds in FTIR qualitatively confirms the degradation of oil. The proposed method was validated by comparing the quantity of FFA obtained from the HPTLC chromatogram (22.62 ± 0.07%, w/w) with the data calculated from acid value (21.98 ± 0.56%, w/w). The percentage degradation was calculated as 45.67 ± 0.67 (w/w). The present study shows that the developed method is a consistent, precise, reproducible, accurate, and rapid analytical technique for quantifying degraded oil in water.

Protocol for the Determination of Total Iodine in Iodized Table Salts Using Ultra-High-Performance Liquid Chromatography

Potassium iodate and potassium iodide are commonly fortified in iodized table salt, which must be continuously monitored to maintain quality. Our study reported an optimized detection method for total iodine in iodized table salt using 0.5 M sodium bisulfite as the reducing agent. The iodized table salt (0.5 g) was dissolved in 0.5 M sodium bisulfite solution prior to injection in ultra-high-performance liquid chromatography (UHPLC) coupled with a diode array detector using a weak anion-exchange column (2.1 mm × 150 mm, 5 μm). Iodide was eluted at 9.92 ± 0.06 min (λ = 223 nm) when an isocratic mobile phase of 1:1 (v/v) methanol/120 mM phosphate buffer mixed with tetrasodium pyrophosphate (pH 3.0) was running at 0.20 mL/min (15 min). Iodide was detected as total iodine from 10.0 to 50.0 mg/kg with a limit of detection (LOD) of 1.2 mg/kg and a limit of quantification (LOQ) of 3.7 mg/kg. The method was validated with relative standard deviations (RSDs) of 4.2%, 0.4%, 1.6%, and 0.8% for accuracy, repeatability, intermediate precision, and robustness, respectively. The determination of total iodine was successful on six (6) samples (n = 3), which recovered 87.2–106.9% of iodate and iodide spike. Thus, this study provides a validated protocol for the determination of total iodine in iodized table salt using 0.5 M sodium bisulfite.

Development and Validation of an Analytical Procedure for the Determination of Caffeic Acid in Nonea rossica Herb by HPLC

INTRODUCTION. According to the literature, alcoholic extracts of Nonea rossica Steven have anticoagulant, antimicrobial, and antifungal properties, which may be due to the presence of caffeic acid and its derivatives in the plant. The standardisation of this herbal drug requires the development of an analytical procedure for the quantitative determination of caffeic acid, and the most promising method for this is high-performance liquid chromatography (HPLC).AIM. This study aimed to develop and validate an analytical procedure for the quantitative determination of caffeic acid in Nonea rossica herb by HPLC.MATERIALS AND METHODS. The study focused on the aerial parts of Nonea rossica plants collected in a steppificated meadow in the Novosibirsk Region during the flowering stage. The study used an Agilent 1100 Series HPLC system equipped with a Zorbax SB-C18 column (150 × 2.1 mm, 3.5 μm) and a detector operating at a wavelength of 330 nm.RESULTS. The authors selected chromatographic conditions to obtain one peak per one molecular form of caffeic acid, acceptable resolution of the extract components, and the maximum intensity of the analyte peak. The gradient elution conditions developed in this study were as follows: a two-component mobile phase, including a 0.01 M aqueous solution of KH2PO4 with a pH of 2.70 (Solvent A) and methanol (Solvent B), and a flow rate of 0.25 mL/min. Under these conditions, caffeic acid eluted at 13.5 minutes, and the total analysis time was 60 minutes. The analytical procedure was validated for its specificity, analytical range, detection limit, quantitation limit, accuracy, repeatability, and intralaboratory precision.CONCLUSIONS. The developed and validated analytical procedure for the quantitative determination of caffeic acid in Nonea rossica herb by HPLC provides results with a relative standard deviation of ≤5.0%. The analytical procedure can be used to standardise the herbal drug Noneae rossicae herba for further use in medicinal practice.

Two chromatographic methods for analyzing paracetamol in spiked human plasma with its toxic metabolite, N-acetyl parabenzoquinone imine and its antidote, N-acetyl-l-cysteine

Acetaminophen, also known as paracetamol (APAP), is a highly utilized pharmaceutical agent on a global scale, particularly in the field of pediatrics. Regrettably, an overdose of APAP, resulting from the predominant oxidation, has the potential to trigger acute liver injury. The study’s goal was to find an easy, accurate, and selective way to measure APAP, N-acetyl para benzoquinone imine (NAPQI) (an APAP metabolite that is harmful), and N-acetyl-l-cysteine (NAC) (an antidote). Two different chromatographic methods were used. The HPTLC method, which used silica gel 60 F254 as a stationary phase and a developing liquid made up of methanol, ethyl acetate, and glacial acetic acid (8:2:0.2, v/v/v) and a UV detection at 254 nm. The HPLC method was developed using a mobile phase consisting of water, methanol, and formic acid in a proportion of (70:30:0.15, v/v/v). The stationary phase used in the approach was a C18 column. Analytes quantification was established utilizing a UV detector operating at a wavelength of 254 nm. The present methods make it possible to measure the amount of APAP in plasma samples. When it comes to pharmacokinetics or medication levels in children’s plasma, for example, this may be also very helpful. The current methods can quantify NAPQI, which is helpful in figuring out drug concentrations in individuals with APAP intoxication diagnoses. Additionally, the current approaches can estimate NAC as an antidote; as a result, this study is a complete study because it can analyse drug, toxic metabolite, and antidote in one analytical run. Using the innovative blue applicability grade index software, which measures the practicality of procedures, both methodologies were compared with a reported methods. Additionally, the achievement of the eco-friendliness profile of the designed procedures was assessed. Both techniques passed the ICH validation tests.

New Standardized Procedure to Extract Glyphosate and Aminomethylphosphonic Acid from Different Matrices: A Kit for HPLC-UV Detection

Background: Glyphosate has been extensively used as herbicide since the early 1970s. The daily exposure limit is set at 0.3 mg/kg bw/d in Europe and 1.75 mg/kg bw/d in the USA. Among its derivatives, aminomethylphosphonic acid is the most stable and abundant. Understanding their biological effects then requires reliable methods for quantification in biological samples. Methods: We developed and validated a fast, low-cost, and reliable chromatographic method for determining glyphosate and aminomethylphosphonic acid concentrations. The validation included following parameters: specificity, selectivity, matrix effect, accuracy, precision, calibration performance, limit of quantification, recovery, and stability. Sample extraction employed an anion exchange resin with elution using hydrochloric acid 50.0 mmol/L. For HPLC analysis, analytes were derivatized, separated on a C18 column with a mobile phase of phosphate buffer (0.20 mol/L, pH 3.0) and acetonitrile (85:15), and detected at 240 nm. Results: The method demonstrated high reliability and reproducibility across various matrices. Its performance met all validation criteria, confirming its suitability for quantifying glyphosate and aminomethylphosphonic acid in different biological and experimental setups. Conclusions: This method can offer a practical resource for applications in experimental research, medical diagnostics, quality control, and food safety.

A generalizable methodology for predicting retention time of small molecule pharmaceutical compounds across reversed-phase HPLC columns.

Quantitative structure retention relation (QSRR) is an active field of research, primarily focused on predicting chromatography retention time (Rt) based on molecular structures of an input analyte on a single or limited number of reversed-phase HPLC (RP-HPLC) columns. However, in the pharmaceutical chemistry manufacturing and controls (CMC) settings, single-column QSRR models are often insufficient. It is important to translate retention time across different HPLC methods, specifically different stationary phases (SP) and mobile phases (MP), to guide the HPLC method development, and to bridge organic impurity profiles across different development phases and laboratories. In response to this need, we present a novel approach for retention time transfer across SPs and MPs, without requiring pre-existing Rt data on the target column. To achieve this, we developed an RP-HPLC based Genentech Multi-column Retention Time (GMCRT) database containing 51 small molecule pharmaceutical compounds analyzed on twenty SPs and multiple pH levels. The database incorporated the SP selectivity parameters from Hydrophobic Subtraction Model (HSM) - hydrophobicity (H), steric hindrance (S), hydrogen-bond acidity (A), hydrogen-bond basicity (B), ionic interaction (C) under two different pHs (2.8 and 7) and ethylbenzene (EB) retention factor. Two machine learning approaches, partial least squares (PLS) and artificial neural networks (ANN) were found to improve accuracy of Rt prediction on new SPs compared to the direct mapping approach that have been previously published, especially when the RP-HPLC columns have significant selectivity difference. As a comparison, our approach does not require pre-existing retention data on the target SPs and it is generalizable to any RP-HPLC columns with a set of known column selectivity parameters (https://www.hplccolumns.org/). The generalizability is achievable not only via the available retention data correlation among the twenty commonly-used RP-HPLC columns in GMCRT, but also via the retrainable mechanism of our ML models by adding Rt of the compounds of interest on the source columns into GMCRT, followed by predicting Rt on the target column. Thus, we propose a new QSRR framework that incorporates the physiochemical properties of SPs and MPs and makes the retention time prediction transferable across SPs and MPs. Such a framework is expected to open up possibilities for developing more comprehensive and generalizable models, and streamline RP-HPLC method development and lifecycle management across various pharmaceutical CMC development phases.

Retention mechanism on phosphodiester stationary phases in HILIC and purely aqueous mobile phase, Part I: The problem of hold-up volume determination.

Non-destructive chromatographic methods were used to determine the hold-up volumes of four self-packed columns containing embedded phosphate groups. The stationary phases are named Diol-P-C10, Diol-P-C18, Diol-P-Benzyl and Diol-P-Chol. The hydrophobicity of organic ligands bound to the phosphate group increases in the benzyl< decyl < octadecyl <cholesterol direction. Using a stochastic model of inverse size exclusion chromatography with a wide pore size distribution, the hold-up volume, interstitial volume and pore volume of the columns were determined. Using the method of minor disturbances, the thermodynamic hold-up volume of the columns was calculated and the excess isotherm was determined for acetonitrile-water mobile phases. The results show that these stationary phases can be used effectively in RPLC and HILIC. When using hold-up volume markers, it was found that the composition of the mobile phase has to be taken into account. In the case of protic solvents, inorganic salts are excluded from the pores and their elution volume is not the total hold-up volume. Rather, in the case of aprotic mobile phase, retention occurs due to the adsorbed water layer.

Development and validation of a UHPLC-MS/MS method for the quantitative analysis of trans-ISRIB in human plasma.

The integrated stress response (ISR) is a cellular defense mechanism activated under stress conditions. When the ISR is activated, it slows the production of proteins, the building blocks that cells need to function. Trans-integrated stress response inhibitor (trans-ISRIB) is a compound that can reverse the effects of ISR activation, showing promise for treating neurodegenerative diseases. The preclinical and clinical evaluation of trans-ISRIB necessitates a reliable analytical method. This study presents the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative analysis of trans-ISRIB in human plasma, conforming to the U.S. FDA's guidelines for bioanalytical method validation. The method developed utilizes a liquid-liquid extraction procedure to prepare plasma samples with a spiked internal standard (IS). The extracts containing trans-ISRIB and the IS were dried under nitrogen, reconstituted in the mobile phase, and separated on a Waters XSelect HSS T3 column under isocratic conditions with a mobile phase containing 0.1% acetic acid in 70% methanol aqueous solution at a flow rate of 0.500mL/min. Detection and quantification were accomplished using a positive electrospray ionization tandem mass spectrometer (ESI+-MS/MS) operated in multiple-reaction-monitoring (MRM) mode. The method demonstrated a linear calibration range for trans-ISRIB concentrations from 0.500 to 1.00x103 nM, with high specificity, precision, accuracy, and recovery. This method addresses a significant analytical gap, offering a robust tool for quantifying trans-ISRIB in human plasma. Chemical compounds studied in this article: 2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide (trans-ISRIB) (CAS # 1597403-47-8); 2-(4-chlorophenoxy)-N-(2-{[(4-chlorophenoxy)acetyl]amino}ethyl)acetamide (CAS # 327071-30-7).

A simple HPLC-UV method for monitoring therapeutic adherence in pulmonary arterial hypertension.

A considerable percentage of ineffective treatment in pulmonary arterial hypertension (PAH) may be related to subtherapeutic dosage or non-adherence. The aim of the study was to develop a simple analytical method suitable for plasma determination of selected drugs: riociguat (RIO), bosentan (BOS) and macitentan (MAC) administered to PAH patients. An isocratic HPLC-UV system (Spectra Physics - Shimadzu) with a manual injector (50μL loop) was applied. Chromatographic analysis was performed using a Suplecosil LC-CN column (150×4.6mm, 5μm) protected with a Supelguard precolumn at room temperature. The separation was carried out using the mobile phase: CH3CN:H2O:0.5M KH2PO4:85% H3PO4 (172:324.2:3.7:0.1, v/v) at a flow rate of 1.8mL/min. Ethyl acetate (4mL) was used for 10-min liquid-liquid extraction from 0.4mL alkalized plasma sample. Detection was performed at λ=245nm chosen as a compromise between signal intensity and matrix interference. The analytes were eluted at retention times of 4.4min (RIO), 5.4min (BOS), 8.9min (MAC) and 7.8min for gallopamil (internal standard, GAL). The method was found linear and calibrated in the ranges: 5-1000ng/mL for RIO, 10-2000ng/mL for BOS and 20-2000ng/mL for MAC, with r2 of 0.9991 for RIO, 0.9983 for BOS, and 0.9949 for MAC, respectively. Within the given ranges, the method ensured reliable results with the required precision and accuracy: ≤15% (≤20% for LLOQ). There was no significant carryover effect. The method has been successfully used in pilot study on adherence in patients treated for PAH, enabling monitoring of RIO, BOS and MAC. Drug concentrations were assessed in samples taken before (C0) and 3h after drug administration (C3). For RIO, BOS and MAC, the developed method was suitable for both C0 and C3 samples, allowing steady-state drug determination if used. The presented method can be recommended to laboratories equipped with basic HPLC apparatus as an attractive analytical tool for both TDM and adherence studies.

Development and validation of a RP-HPLC method for simultaneous determination of cimetidine, metoprolol tartrate and phenol red for intestinal perfusion studies.

A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5mM)-acetonitrile mixture as mobile phase and C18 column (Inertsil ODS-3; 5µm, 4.6×250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10min. Total run time for analysis was 10min and the injection volume was 20µL. Detection of compounds was performed at 207nm. Under optimum HPLC conditions, retention times were 4.03min for CIM, 6.99min for MT and 8.49min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary in-situ intestinal perfusion studies. In conclusion, samples obtained from in-situ intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.

Insights into the separation, enantioseparation and recognition mechanisms of methamphetamine isotopologues on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography.

Isotopologues resulting from the labelling of molecules with deuterium have attracted interest due to the isotope effect observed in chemistry and biosciences. Isotope effect may also play out in noncovalent interactions and mechanisms leading to intermolecular recognition. In chromatography, differences in retention time between isotopologues, as well as between isotopomers have been observed resulting in two different elution sequences (isotope effects): the normal isotope effect when heavier isotopologues retain longer than lighter analogues, and the inverse isotope effect featuring the opposite elution order. Although several cases of deuterium isotope effects have been reported so far, the molecular bases of these phenomena remain unclear. We report the separation of isotopologues of methamphetamine (N-methyl-1-phenylpropan-2-amine) (MET), featuring different number and location of deuterium substituents, on an achiral column by high-performance liquid chromatography (HPLC), as well as the simultaneous separation of isotopologues and their enantiomers on some polysaccharide-based chiral columns. The effects of the number and location of deuterium substituents introduced in MET's structure, of the surface chemistry of the adsorbent, and the mobile phase composition and pH on the retention and separation of isotopologues and their enantiomers were examined. In several cases, the effect of temperature was also evaluated, and the thermodynamic quantities associated with isotopologue adsorption, separation, and enantioseparation were also calculated. To elucidate the molecular bases of the experimental observations, quantum mechanics calculations were performed focusing on the vibrational degree of freedom calculated for models of isotopologue-selector complexes. On this basis, zero-point vibrational energies were computed as useful descriptors to differentiate computationally between deuterated isotopologues. Using HPLC as experimental technique with the following dual function: 1) separation of MET isotopologues and their enantiomers; 2) exploring noncovalent interactions underlying their separation. The degree of deuteration, location of deuterium substituents, and mobile phase pH play key roles in isotopologue separations. By integrating experimental and computational analyses, noncovalent interactions underlying normal and inverse isotope effects were deconvoluted, highlighting the contribution of hydrogen bond, hydrophobic and dispersive forces in isotopologue and enantiomer separation.

Ion exchange chromatography of biotherapeutics: Fundamental principles and advanced approaches.

Ion exchange chromatography (IEX) is an important analytical technique for the characterization of biotechnology-derived products, such as monoclonal antibodies (mAbs) and more recently, cell and gene therapy products such as messenger ribonucleic acid (mRNA) and adeno-associated viruses (AAVs). This review paper first outlines the basic principles and separation mechanisms of IEX for charge variant separation of biotherapeutics, and examines the different elution modes based on salt or pH gradients. It then highlights several recent trends when applying IEX for the characterization of biotechnology-derived products, including: i) the effective use of pH gradients, ii) the improvement of selectivity by using organic solvents in the mobile phase, multi-step gradients, or by combining ion pairing and ion exchange, and iii) the increase in analytical throughput using ultra-short columns or automated screening of conditions. The review also discusses the incorporation of IEX into multidimensional liquid chromatography setups, integrating it with other chromatographic dimensions for the analysis of complex biotherapeutic products. It also covers the coupling of IEX with mass spectrometry (MS), ion mobility spectrometry (IMS), and multi-angle light scattering (MALS) to identify the various species contained in complex biotherapeutic samples. In conclusion, IEX is considered today as an essential technique in the analytical toolbox for the characterization and quality control of biotechnology-derived products. It offers a unique separation mechanism and can be coupled with highly informative detectors, such as MS and MALS.