C18 Column Research Articles

Development and Validation of HPLC-UV Method for the Determination of Levothyroxine in Orodispersible Minitablets for Pediatric Application

Background: Thyroid hormones play an important role in cognition and brain development. The determination of the content of levothyroxine, as well as related substances and dissolution test analysis, should be carried out by methods that are selective and highly sensitive due to the low concentration used in low dose orodispersible minitablets Objectives: This study aims to develop and validate an analytical method by HPLC-UV for the quantification of levothyroxine and its related substances in pharmaceutical formulations; looking forward to being this method suitable for a future dissolution test analysis using tandem mass spectrometry detector. Methods: A Hypersil GOLD C18 (100 x 2.1 mm, 3 μm) column was used with 25°C column temperature, 5 μL injection volume, 0.3 mL/min flow rate and detection at 225 nm. The mobile phase consisted of methanol: 0.05% formic acid (55:45). The developed method was validated for specificity, linearity, precision, accuracy and robustness. Results: The method is linear within the range of 2-20 μg mL-1 (R2=0.9982), which makes the method suitable for the evaluation of levothyroxine in pharmaceuticals formulations. LOQ was 0.17 μg/mL (0.85 ng on column) and LOD 0.05 μg/mL (0.25 ng on column) of LT4. Therefore, in terms of efficiency (1671), retention factor, k (6.79), Tailing factor, T (1.09) and resolution, Rs (5.11) the chromatographic method was found to be suitable according to USP 43. Conclusion: The HPLC UV method was found to be linear, specific, precise, accurate and robust, therefore it is suitable for the quality control of levothyroxine in pharmaceutical ODMTs..

Simultaneous determination of 21 perfluorinated and polyfluoroalkyl substances in plant oils by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry

Edible plant oils are a key component of the daily human diet, and the quality and safety of plant oils are related to human health. Perfluorinated and polyfluoroalkyl substances (PFASs) are pollutants that can contaminate plant oil through the processing of raw materials or exposure to materials containing these substances. Thus, establishing a sensitive and accurate analytical method for the determination of PFASs is critical for ensuring the safety of plant oils. In this study, a method based on acetonitrile extraction and solid phase extraction purification combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) was developed for the simultaneous determination of 21 PFASs, including perfluorocarboxylic acids, perfluoroalkyl sulfonic acids, and fluorotelomer sulfonic acids, in edible plant oils. The chromatographic conditions and MS parameters were optimized, and the influences of the extraction solvents and purification method were systematically studied. Plant oil samples were directly extracted with acetonitrile and purified using a weak anion-exchange (WAX) column. The 21 target PFASs were separated on a reversed-phase C18 chromatographic column and detected using a triple quadrupole mass spectrometer with an electrospray ionization source. The mass spectrometer was operated in negative-ion mode. The target compounds were analyzed in multiple reaction monitoring (MRM) mode and quantified using an internal standard method. The results demonstrated that the severe interference observed during the detection of PFASs in the co-extracted substances was completely eliminated after the extraction mixture was purified using a WAX column. The 21 target PFASs showed good linearity in their corresponding ranges, with correlation coefficients greater than 0.995. The limits of detection (LODs) and limits of quantification (LOQs) of the method were in the range of 0.004-0.015 and 0.015-0.050 μg/kg, respectively. The recoveries ranged from 95.6% to 115.8%, with relative standard deviations (RSDs) in the range of 0.3%-10.9% (n=9). The established method is characterized by simple sample pretreatment, good sensitivity, high immunity to interferences, and good stability, rendering it suitable for the rapid analysis and accurate determination of typical PFASs in edible plant oils.

An ultra-fast green ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of zotizalkib in human liver microsomes.

Zotizalkib (ZTK, TPX-0131) is a fourth-generation highly effective inhibitor of wild-type anaplastic lymphoma kinase (ALK) and ALK-resistant mutations that can penetrate the central nervous system. It exhibited greater potency compared to all five officially approved ALK inhibitors. The aim of this study was to develop a rapid, accurate, eco-friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for measuring the concentration of ZTK in human liver microsomes (HLMs). The validation aspects of the current UHPLC-MS/MS methodology in the HLMs were conducted in accordance with the bioanalytical method validation standards specified by the US Food and Drug Administration. ZTK and encorafenib were separated using an Agilent C8 column (Eclipse Plus) and an isocratic mobile phase. The calibration curve for the developed ZTK exhibited a linear relationship within the concentration range of 1-3000ng/mL. The results from the Analytical Green-ness Metric Approach program (0.76) suggested that the created method demonstrated a significant degree of environmental sustainability. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of ZTK were determined to be 15.79min and 51.35mL/min/kg, respectively that suggests the ZTK exhibits characteristics similar to those of a medication with a high extraction ratio. These approaches are crucial for the progress of novel pharmaceutical development, especially in improving metabolic stability.

Detection of trace levels of selected allergic chemicals in commercial hair dyes in local market of Jeddah, Saudi Arabia

Hair dyes are now commonly used in Saudi Arabia, and other nations across the world, to cover up grey hairs and/or to modify the natural color of the hair. The principal ingredients of hair dyes are aromatic amines and phenols and which most likely cause allergic reactions, irritated skin, and even cancer in certain cases. Thus, the current study reports the separation and subsequent identification of four chemical constituents namely p-Phenylenediamine (PPD), 3-aminophenol (3AP), resorcinol (RES), and 2-methylresorcinol (2MRS) in 26 commercial hair dyes that represent ten famous brands available in the local market of Jeddah city, Saudi Arabia. High-performance liquid chromatography (HPLC) technique was successfully employed to identify the four chemical compounds using methanol-aqueous solution (10:90 v/v) containing diethanolamine (0.05 %) and ammonium acetate (0.02 M) at pH 5.2 as a proper mobile phase. The column was 150 × 4.6 mm ID and 5 µm Eclipse Plus C18 column and the retention times were 1.26, 3.16, 4.99 and 6.01 min for PPD, 3AP, RES, and 2MRS, respectively. The four standards PPD, 3AP, RES, and 2MRS at 254 nm displayed excellent linear calibration plots in the concentration range 5–100 µg/mL with acceptable correlation coefficients (R2) of 0.9964, 0.9980, 0.9952 and 0.9961, respectively. The low limits of detection (LOD) and quantification (LOQ) based on the standard deviation of the response and the slopes of the plots were found to be equal 7.17, 5.39, 8.33, and 7.47 µg/mL and 23.91, 17.97, 27.75, 24.89 µg/mL for PPD, 3AP, RES, and 2MRS, respectively.

The development of 177Lu-DOTA-CC-PSMA following a unified “Click Chemistry” protocol of synthesizing metal nuclide-conjugated radiopharmaceuticals

BackgroundCurrently, the synthesis pathway of metal nuclide-labeled radiopharmaceuticals is mainly divided into two steps: first, connecting the chelator with the target molecule, and second, labeling the metal nuclide to the chelator. However, the second step of the reaction to label the metal nuclide requires high temperature (90–100 °C), which tends to denature and inactivate the target molecule, leading to loss of biological activities, especially the targeting ability. A feasible solution may be the click chemistry labeling method, which consists of reacting a metal nuclide with a chelating agent to generate an intermediate and then synthesizing a radiopharmaceutical agent via the click chemistry intermediate and the target molecule-alkyne compound. In this study, through the click chemistry of 177Lu-DOTA-N3 with prostate-specific membrane antigen (PSMA)-alkyne compound, 177Lu-labeled PSMA-targeted molecular probe was synthesized and evaluated for its potential to be cleared from the bloodstream and rapidly distributed to tissues and organs, achieving a high target/non-target ratio. 177Lu-PSMA-617 was utilized as an analogue for comparison in terms of synthesizing efficiency and PSMA-targeting ability.ResultsA novel 177Lu-labeled PSMA radioligand was successfully synthesized through the click chemistry of 177Lu-DOTA-N3 with PSMA-alkyne compound, and abbreviated as 177Lu-DOTA-CC-PSMA, achieving a radiochemical yield of 77.07% ± 0.03% (n = 6) and a radiochemical purity of 97.62% ± 1.49% (n = 6) when purified by SepPak C18 column. Notably, 177Lu-DOTA-CC-PSMA was characterized as a hydrophilic compound that exhibited stability at room temperature and commendable pharmacokinetic properties, such as the superior uptake (19.75 ± 3.02%ID/g at 0.5 h) and retention (9.14 ± 3.16%ID/g at 24 h) within xenografts of 22Rv1 tumor-bearing mice. SPECT/CT imaging indicated that radioactivity in both kidneys and bladder was essentially eliminated after 24 h, while 177Lu-DOTA-CC-PSMA was further enriched and retained in PSMA-expressing tumors, resulting in the high target/non-target ratio.ConclusionThis study demonstrated the potential of click chemistry to unify the synthesis of metal radiopharmaceuticals, and 177Lu-DOTA-CC-PSMA was found for rapid clearance and appropriate chemical stability as a PSMA-targeted radioligand.

Box-Behnken Design employed stability demonstrating RP-HPLC method development of sorafenib tosylate for rapid and sensitive quantification

Sorafenib tosylate (SFN) is a tyrosine kinase inhibitor used clinically to treat liver, kidney, and thyroid carcinoma. This research aims to accurately quantify SFN using a quality-by-design (QbD) approach with RP-HPLC. Chromatographic settings were optimized using a Box-Behnken design, measuring responses such as retention time, tailing factor, theoretical plate, and peak area. At the same time, independent variables were flow rate, mobile phase composition, and wavelength. A C18 column (4.6 × 250 mm, 5 µm) served as the stationary phase, with a mobile phase of methanol and 0.1% o-phosphoric acid in a 41:59 v/v ratio. SFN detection occurred at 267 nm in isocratic mode at a flow rate of 1.1 mL min−1. Method validation followed International Council for Harmonization guidelines, yielding a coefficient of determination (R2) of 0.9998, indicating linearity in the 5-25 µg mL−1 range. Results showed detection (LOD) and quantification (LOQ) limits of 0.04 and 0.12 µg mL−1, respectively. Additionally, the method demonstrated precision, accuracy, and robustness consistent with ICH criteria. Overall, this simple, accurate, rapid, and robust RP-HPLC method is suitable for routine SFN analysis in various formulations.

A new analytical RP-HPLC method development and validation for the estimation of Nilutamide in bulk form and marketed pharmaceutical dosage form

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Nilutamide, in its pure form as well as in tablet dosage form. Chromatography was carried out on a Symmetry C18 (4.6 x 150 mm, 5 µm) column using a mixture of Methanol and water (45: 55 % v/v) as the mobile phase at a flow rate of 0.8 ml/min, the detection was carried out at 260 nm. The retention time of the Nilutamide was 2.379 ± 0.02 min respectively. The method produce linear responses in the concentration range of 24-120 mg/ml of Nilutamide. The method precision for the determination of assay was below 2.0 % RSD. The method is useful in the quality control of bulk and pharmaceutical formulations. The method was validated for accuracy, precision, linearity, robustness, ruggedness and LOD & LOQ of standard solution. The developed RP-HPLC method was found to be accurate, precise, linear, and robust and was successful applied to a pharmaceutical tablet formulation for qualitative estimation of Nilutamide in Bulk form and Marketed Pharmaceutical Dosage forms.

New stability indicating RP-HPLC methods for the determination of related substances and assay of trametinib acetic acid: a mass balance approach.

New stability indicating related substances and assay methods for trametinib acetic acid by RP-HPLC have been developed and validated through forced degradation and mass balance studies for the first time. In related substances (RS) method, trametinib acetic acid was successfully separated from its six related substances namely, cyclopropanamide impurity, desiodo trametinib, desacetyl trametinib, trione acetamide intermediate, trione intermediate, and trione PTSA intermediate using YMC-Triart C18 (150 × 4.6mm, 3µm) column. Orthophosphoric acid (0.15%) in water was used as buffer. Gradient elution was programmed using mobile phase-A (buffer and acetonitrile mixture in 80:20 v/v ratio) and mobile phase-B (buffer and acetonitrile mixture in 20:80 v/v ratio). Acetonitrile and methanol mixture (1:1 v/v) was used as diluent. Flow rate, injection volume, column temperature, and wavelengths were kept as 0.8mL/min, 10µL, 55°C, and 240nm, respectively. Desacetyl trametinib and cyclopropanamide impurity were identified as potential degradation impurities in acid and base degradation conditions, respectively. Assay method specific to above six related substances was also developed. Assay of forced degradation samples was also determined, and the mass balance was established by adding total impurities formed in RS method to assay of trametinib acetic acid.

Evaluation of the Genotoxic Impurities of Selpercatinib Through HPLC and LCMS/MS Identification of Selpercatinib Stress Degradation Products

The current investigation entails the characterization of seven degradation products (DPs) formed in different stress conditions of selpercatinib employing liquid chromatography–tandem mass spectrometry (LCMS/MS). Additionally, high-performance liquid chromatographic (HPLC) method for precise quantifying genotoxic impurities of selpercatinib. To explore the stability profile of selpercatinib, it was subjected to forced degradation experiments including acidic, basic, oxidative, photolytic, and thermal stress were conducted. These experiments revealed the degradation of selpercatinib under basic, acidic and photolytic conditions, resulting in the formation of seven distinct DPs. The chromatographic resolution of selpercatinib and its impurities along with DPs was effectively attained on Zorbax C18 (250 mm × 4.6 mm, 5 μm) column using aqueous ammonium acetate and methanol in 70:30 (v/v) at pH 4.5 with 0.1% formic acid as mobile phase, pumped isocratically at 0.9 mL/min and 226 nm wavelength. The approach generates a precise calibration curve that accurately fits within 15-120 μg/mL range for selpercatinib and LOQ (0.015 μg/mL) to 0.12 μg/mL for impurities with acceptable precision, accuracy and recovery. The efficacy of this method was validated through LCMS/MS, which allowed for the verification of the chemical structures of newly generated degradation products of selpercatinib. Hence this approach can appropriate for resolution and quantification of genotoxic impurities of selpercatinib and can also applicable for evaluation stress degradation products.

Analytical method development and validation for simultaneous estimation of seven markers in polyherbal formulation JKC by using RP-HPLC

BackgroundThe RP-HPLC method has been established to simultaneous estimation of seven markers in polyherbal formulation JKC using the C18 (25 × 0.46 cm, i.d,5 µm) column. The mobile phase consisted of methanol: water (80:20) at a flow rate of 1.0 mL/min and observed retention time at 2 to 11 min with sharp points. The marker compounds viz. Andrographolide (AG), Piperine (PP), Picroside-I (P-I), Picroside-II (P-II), α-Cyprone (AC), 6-Shogaol (6S), and 6-Gingerol (6G) were quantified in JKC formulations by HPLC method. Detection was performed at the wavelength (λ) of 229 nm for AG, 343 nm for PP, 279 nm for P-I, 264 nm for P-II, 254 nm for AC, and 280 nm for both 6S and 6G by HPLC–PDA detector.ResultsThe marker compounds in JKC formulations were observed in different retention times (Rt) i.e. AG at 3.060 ± 0.01 min, PP at 5.460 ± 0.03 min, P-I at 2.789 ± 0.02 min, P-II at 2.553 ± 0.03 min, AC at 10.951 ± 0.02 min, 6S at 6.302 ± 0.03 min, and 6G at 4.111 ± 0.02 min respectively. The proposed method was validated with acceptable linearity (r2 0.9995–0.9999), precision, robustness, ruggedness, and accuracy (RSD < 2%) under optimum conditions. The limit of detection and quantification of bioactive markers were as: AG (1.386; 4.200 ppm), PP (2.033; 6.161 ppm), P-I (2.822; 8.553 ppm), P-II (2.538; 7.691 ppm), AC (0.269; 0.815 ppm), 6G (0.158; 0.480 ppm), 6S (0.188; 0.569 ppm). The amount (mg/g) of bioactive markers detected and estimated in plants and formulation were as: AG (41.282 ± 0.48; 10.06 ± 0.18), PP (53.81 ± 0.25, 13.82 ± 0.37 in PN, PL; 4.27 ± 0.07), P-I (15.97 ± 0.01; 0.48 ± 0.003), P-II (63.24 ± 0.35; 2.31 ± 0.006), AC (0.42 ± 0.01; 0.36 ± 0.006), 6G (0.71 ± 0.03; 0.16 ± 0.001), and 6S (2.64 ± 0.09; 0.12 ± 0.004) respectively. Method was found to be rugged and robust. The results found for all the validation parameters were within the limits according to ICH guidelines.ConclusionThe proposed method is fast, precise, economic, and specific and used for the simultaneously quantifiable analysis of seven major bioactive markers in the ingredients (herbs) and the JKC formulations.

Box-Behnken design in optimization of the green liquid chromatographic method for the quantification of Afatinib in drug product: AQbD approach

The aim of this research work was to quantitatively determine the tyrosine kinase inhibitor, Afatinib, in tablets using an eco-friendly chromatographic method. The development of a green liquid chromatographic method using analytical quality-by-design (AQbD) approach principles to estimate Afatinib via Box-Behnken design. A simple and effective green liquid chromatographic method was developed and validated. The analysis was done on the Zodiac C18 column by isocratic elution mode. Mobile phase composed of pH 3.0 potassium dihydrogen phosphate buffer and ethanol in the ratio of 65:35%V/V. The flow rate was set to 0.6 mL/min. with an injection volume of 10 µL and at detection wavelength of 254 nm. The optimized method was found to be specific, linear, precise, accurate, and robust. Established linearity in the range of 24.6 to 73.9 µg/mL. The developed method exhibited excellent greenness. Based on the greenness assessment tools, the analytical eco-score value of 94 and the pictograms of NEMI, Modified NEMI, GAPI, Complex GAPI, AGREE, and AGREE prep expressed the method’s greenness. Hence, the AQbD approach based green method and the validation data indicate that the method can be employed in routine analysis for the determination of Afatinib in pharmaceutical dosage form in quality control laboratory use.

Simultaneous quantification of baloxavir marboxil and its active metabolite in human plasma using UHPLC-MS/MS: Application to a human pharmacokinetic study with different anticoagulants

Baloxavir marboxil (BXM) is a cap-dependent nucleic acid endonuclease inhibitor, which exerts its antiviral effects after being metabolized to its active form baloxavir acid (BXA). Ethylenediamine tetra-acetic acid (EDTA) and heparin are the two most used anticoagulants in clinical blood sample collection to estimate drug levels in plasma. However, compared to heparin plasma, there is a lack of clinical pharmacokinetic data of BXA using EDTA anticoagulant tubes for blood collection. In the present study, an efficient, rapid, and sensitive ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous quantification of BXM and its active metabolite BXA in human plasma with its isotopic baloxavir-d5 (BXA-d5) as internal standard (IS). Plasma samples (50 μL) were undergone using acetonitrile containing 0.1 % formic acid a precipitant. Chromatographic separation was achieved by a Waters XBridge®C8 (2.1 mm × 50 mm, 2.5 µm) column. The gradient mobile phase was 0.1 % formic acid in water (A, pH 2.8) and 0.1 % formic acid in acetonitrile (B) and delivered at a flow rate of 0.6 mL/min for 4.5 min. BXM and BXA were monitored using a positive electrospray triple quadrupole mass spectrometer (TRIPLE QUAD™ 6500+) via multiple reaction monitoring mode. The mass-to-charge ratios (m/z) were 572.2→247.0, 484.2→247.0 and 489.2→252.0 for BXM, BXA, and BXA-d5 (IS). Calibration curves exhibited excellent linearity in the range of 0.1–10 ng/mL for BXM (r2 > 0.996), and 0.3–300 ng/mL for BXA (r2 > 0.998). Within-run and between-run precisions in coefficients of variations were less than 11.62 % for BXM and less than 7.47 % for BXA, and accuracies in relative error were determined to be within −7.78 % to 5.70 % for BXM and −6.67 % to 8.56 % for BXA. Extraction recovery efficiency was 92.76 % for BXM, 95.32 % for BXA, and 99.26 % for BXA-d5, respectively. The matrix effect of BXM and BXA was in line with the requirements, where the relative deviation of the accuracy was less than 6.67 % and the precision was less than 6.69 %. The validated efficient and simple UHPLC-MS/MS method was successfully used in the pharmacokinetic study of BXM and BXA in healthy human volunteers with K2EDTA and heparin tubes for blood collection. EDTA might compete with BXA for chelating metal ions and thereby decrease the plasma ratio in whole blood, leading to approximately 50 % lower measurement of pharmacokinetic parameters as compared with those obtained from heparin plasma anticoagulant tubes.

Development and validation of an LC-MSMS method for the quantitation of pacritinib; application of kinetics in rabbits

BackgroundAccurate and selective LC/ESI-MSMS method development and validation for the quantitation of pacritinib is the primary goal of this study to perform kinetic studies in the healthy rabbit. MethodsChromatographic resolution was accomplished with a hypersil/ODS (50 mm × 4.6 mm, 3 μ) analytical C18 column and a mobile phase composition of 0.1% formic acid and ACN in the proportion of 25:75 with a 0.6 ml/min flow of the mobile phasic system from the analytical column. The method was employed by monitoring the established ionic transitions of m/z-473.25/98.09 for Pacritinib and 506.18/57.12 for the internal standard (Amprenavir) in multiple reaction monitoring. ResultsThe calibration plot regression line was y = 0.0002× + 0.007, with a correction coefficient (r2) of 0.9989. The CV outcomes for the matrix effect at low-QC and high-QC levels were 4.79% and 4.91%, respectively. The percentage average recoveries for Pacritinib in High-QC (12.70 μg/ml), MQC (8.50 μg/ml), and Low-QC (1.19 μg/ml) were 95.87%, 103.64%, and 94.32%, respectively. The obtained values were found between 2.98 and 5.07% for the QC (1.19, 8.50, and 12.70 μg/ml) samples. The established procedure was subjected to kinetics study of Pacritinib after oral administration in rabbits. Cmax, Tmax, and T1/2, of the Pacritinib tablets were 247.25 ± 3.32 ng/ml, 6.0 ± 0.03 h, and 12.24 ± 0.53 h, respectively. AUC0-∞ infinity for Pacritinib tablets was 1691.74 ± 3.67 ng h/ml. ConclusionAfter oral administration of Pacritinib to healthy rabbits, pharmacokinetic characteristics were presented, and the established technique was effectively verified.

Estimating Octanol-Water Partition Coefficients of Novel Brominated Flame Retardants by Reversed-Phase High-Performance Liquid Chromatography and Computational Models.

Legacy brominated flame retardants, including polybrominated diphenyl ethers (PBDEs), have been classified as persistent organic pollutants and replaced with novel brominated flame retardants (NBFRs). The octanol-water partition coefficients (log KOW) of NBFRs have been computationally estimated, but the log KOW values provided by these methods can differ by 1 to 3 orders of magnitude. Given the importance of this parameter in fate and toxicity models, we indirectly measured the log KOW values of eight NBFRs by their capacity factor (k') on a reversed-phase high-performance liquid chromatography (HPLC) C18 column by isocratic elution and compared these measured values with those estimated by nine computational models. Log KOW values were obtained for the NBFRs 1,2-bis(2,4,6-tribromophenoxy) ethane, pentabromobenzene, pentabromoethylbenzene, pentabromotoluene, 2-ethylhexyl 2,3,4,5-tetrabromobenzoate, allyl 2,4,6-tribromophenylether, 2,3-dibromopropyl-2,4,6-tribromophenyl ether, and bis(2-ethylhexyl) tetrabromophthalate. A training set of phthalates, polychlorinated biphenyls, PBDEs, and halogenated benzenes were chosen to obtain the log k'-log KOW calibration for the NBFRs. The computational models KowWIN, XLogP3, EAS-E Suite, COSMOtherm, DirectML, and Abraham polyparameter linear free energy relationships all predicted the log KOW values of the calibration compounds to within 1 order of magnitude without significant bias. The median of these models predicted log KOW values for the calibration compounds that were close to those known in the literature with root mean square error (RMSE) = 0.224 and for the NBFRs that were close to those measured by HPLC (RMSE = 0.334). Environ Toxicol Chem 2024;43:2105-2114. © 2024 SETAC.

A simple and sensitive high-performance liquid chromatographic method combined with fluorescence detection for bioanalysis of scopoletin in rat plasma: Application to a pharmacokinetic study.

Scopoletin, a coumarin class natural phytoalexin, is present in medicinal plants such as noni (Morinda citrifolia). It exhibits diverse pharmacological properties, including antioxidant, anti-hyperuricemic, and anti-inflammatory effects. The objective of this study was to develop a novel HPLC-fluorescence (HPLC-FL) method for the quantitative analysis of scopoletin in the plasma and to investigate its pharmacokinetics in rats. Sample preparation involved a methanol-based protein precipitation method, and chromatographic separation was conducted using a C18 column with an isocratic mobile phase composed of water and acetonitrile containing 0.1% trifluoroacetic acid. The eluent was detected using an FL detector set to optimized excitation/emission wavelengths of 337/453 nm. Method validation encompassed assessments of selectivity, linearity (1-500 ng/mL), precision, accuracy, recovery, matrix effect, and stability in accordance with the prevailing Food and Drug Administration (FDA) guidelines. The developed method was successfully applied for pharmacokineticstudy in rats. To the best of our knowledge, this study is the first application of a simple and sensitive HPLC-FL method for the quantification of scopoletin in a pharmacokinetic study. This method offers a promising alternative for preclinical pharmacokinetic investigations with appropriate modifications and validations and holds potential for clinical applications.

Separation of weak acids, neutral compounds, and permanent anions using sequential elution liquid chromatography with tandem columns

This paper discusses the development of an analytical method by an alternative separation approach, sequential elution liquid chromatography (SE-LC), to separate permanently charged ions (anions), weak acids, and neutral compounds using anion exchange and reversed-phase columns in tandem. SE-LC separates classes of compounds by group by employing two or more elution modes. Advantages to using SE-LC over conventional HPLC are a greater peak capacity and a reduced separation disorder. Importantly, the same HPLC as used for a conventional HPLC separation may be used to afford a successful SE-LC separation. Mobile phase selection and gradient optimization are integral for a successful SE-LC class separation of permanent anions, weak acids, and neutral compounds and will be discussed in detail in this paper. The most successful (best resolution and repeatability) SE-LC separation was achieved by applying isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange (SAX) column. Repeatability (RSD) in the retention times and peak areas of the analytes was less than 0.25 % and 1.5 %, respectively.

An HPLC-UV Method to Assess Human Plasma 25(OH)D3.

The aim of this study was to validate an HPLC-UV method to assess vitamin D status by determining the linearity and precision of the 25-hydroxyvitamin D3 (25(OH)D3) calibration curve, the limits of detection, quantitation and robustness of the method, and its accuracy. A second stock solution of 25(OH)D3 was prepared (500 ng/mL), and working dilutions (5, 10, 20, 30, 40, and 50 ng/mL) were prepared for a calibration curve. The HPLC equipment had a UV-Vis diode-array detector and utilized an AcclaimTM 120 C18 column (5 µm, 4.6 × 250 mm) with a flow rate of 1.2 mL/min, a column temperature of 30 °C, and the standards and samples were maintained at 4 °C, with an injection volume of 100 µL. Detection of 25(OH)D3 was determined at 265 nm, with a retention time of 4.0 min. The validation was conducted according to the FDA Validation of Analytical Procedures: Guidance for Industry. Vitamin D was extracted from plasma samples using acetonitrile (ACN)-0.1% formic acid (2:1 v/v), and the percentage of recovery was calculated. The proposed method conditions gave excellent linearity (R2 = 0.9989) and the linearity coefficient was R2 > 0.99 for 25(OH)D3. The detection and quantification limits were 1.1703 ng/mL and 3.5462 ng/mL, respectively. Decreasing or increasing the reading temperature by 1 °C decreased the response units (AU) of vitamin D, 25(OH)D3. When the current flow rate decreased by 0.2 mL/min (1.0 mL/min), the retention time increased to 4.913 min, whereas an increase of 0.2 mL/min of the proposed flow rate (1.4 mL/min) decreased the retention time to 3.500 min. The percentage of recovery varied from 92.2% to 97.1%. The proposed method to quantify a vitamin D metabolite (25(OH)D3) in human plasma samples was reliable and validated.

Penalization and Color Code Technical Approaches for Method Greenness and Whiteness Appraisal in Veterinary Medication: Assay of Toltrazuril Suspension.

Intestinal coccidiosis is a debilitating disease in poultry and livestock, leading to economic impact worldwide. Coccidiosis is prevented and treated in broilers by the inclusion of anticoccidials in feed. Toltrazuril is administered in potable water to treat coccidiosis. Three robust analytical methods for the quantitation of toltrazuril in pure and pharmaceutical formulations are developed. Furthermore, ecological metrics, either penalization- or color-code-based techniques, are applied for the appraisal of assays. First, second-derivative (Δλ; 5 nm) spectrophotometric method is used. Toltrazuril is measured from peak to peak at 244-260 nm within a linearity range of 5-25 μg/mL. The second method is an HPTLC analysis performed on an aluminum sheet of silica gel using ethyl acetate-methanol-ammonium chloride buffer-water (8:1:0.5:0.5, by volume respectively) as the elution phase. Toltrazuril, at a retardation factor of 0.66 ± 0.01, is linearly determined in the range of 1-9 μg/spot at 243 nm. The third method is reversed-phase HPLC with diode array detection, using an Agilent C18 column (5 μm, 4.6 × 150 mm) in isocratic elution mode at 1 mL/min flow rate with a mobile phase of acetonitrile and water in a ratio of 80:20 (v/v). Toltrazuril elutes at a retention time of 2.58 ± 0.1 min and is linearly determined at 243 nm in the range of 0.25-25 μg/mL. Calculated 2D-values and peak areas are highly correlated to their corresponding drug concentrations at coefficients: r > 0.999. All methods were International Council of Harmonization (ICH) validated and applied to the dosage form with satisfactory % recoveries (97-103%). Statistical comparisons versus reported one using t-test and F-test disclose insignificant variation. In examining greenness and whiteness norms, the proposed methods were evaluated and ranked alongside four different reported methods. The proposed methods are green, accurate, and can be applied in routine QC for the determination of toltrazuril in pharmaceutical formulations. Intestinal coccidiosis substantially affects the chicken intestinal tract leading to reduced growth. Toltrazuril is used for the treatment and prevention of intestinal coccidiosis. Three robust, accurate, and precise analytical methods are developed for toltrazuril determination in pure and pharmaceutical formulations. All proposed methods were ecologically assessed and compared with published ones.

Analytical Method Development and Validation of Faricimab Injection by RP-HPLC

A robust and reliable Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative analysis of Faricimab in Vabysmo injections. Faricimab, an IgG1-based bispecific antibody targeting VEGF-A and Ang-2, is used for treating retinal vascular disorders like age-related macular degeneration and diabetic macular edema. The method development involved the use of an Inertsil C18 column (250 x 4.6 mm, 5 µm) with a mobile phase composed of KH2PO4 (pH 4.6) and methanol in a 1:1 ratio, sonicated for 20 minutes. The developed method was evaluated for various validation parameters as per ICH guidelines, including linearity, precision, accuracy, and sensitivity. Linearity was demonstrated within the concentration range of 50-150 µg/ml with a correlation coefficient (R²) of 0.999. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.050 µg/ml and 0.166 µg/ml, respectively, indicating high sensitivity. Precision, expressed as relative standard deviation (RSD), was below 0.1%, confirming the method's reliability. Accuracy was assessed, with mean assay values ranging between 97-101%, and RSD below 0.2%, indicating the method's precision. Robustness tests confirmed the method's resilience against slight variations in analytical conditions such as acetonitrile ratio, flow rate, detection wavelength, and pH. Stability studies showed that Faricimab remains stable under various stress conditions, including exposure to 0.1N HCl, 0.1N NaOH, 30% peroxide, heat, and sunlight, with assay values ranging from 88.93% to 94.07%. The optimized RP-HPLC method ensures good peak shape and resolution, making it suitable for routine quality control of Faricimab in pharmaceutical formulations. The validated method adheres to ICH guidelines, ensuring its reliability for consistent and accurate quantification of Faricimab

Simultaneous estimation of rutin and donepezil through RP-HPLC: implication in pharmaceutical and biological samples.

Aim: AHPLC method was developed and validated for the novel combination of rutin (RN) and donepezil (DNP). Materials & methods: RN and DNP were simultaneously eluted through a C18 column (Ø 150×4.6mm) with a 60:40 v/v ratio of 0.1% formic acid aqueous solution to methanol at 0.5ml/min. Results: The purposed method was found linear, selective, reproducible, accurate and precise with percent RSD less than 2. The limit of quantification for RN and DNP was found 3.66 and 3.25μg/ml, respectively. Conclusion: Validated as per the ICH guidelines, the developed method efficiently quantified RN and DNP co-loaded in DQAsomes (121nm) estimating matrix effect, release profile, entrapment efficiency, loading efficiency and in vivo plasma kinetics.