Mobile Phase Of Acetonitrile Research Articles

On the fractionation of lignin oligomers by stepwise gradient reversed-phase liquid chromatography

With the increased interest in lignin valorization, the analytical challenge to separate a complex mixture of a vast number of phenolics has made chromatography an indispensable step in lignin analysis. High-resolution separations, such as gas chromatography, reversed-phase liquid chromatography and supercritical fluid chromatography have typically been targeting low-molecular-weight compounds, while larger lignin oligomers have received less attention. These compounds have proven to be difficult to separate due to the inherent complexity of the high-molecular-weight fraction of lignins, in fact, even high-resolving linear reversed-phase gradients elute them as one wide zone. To tackle this, in this study we show that a crude fractionation of lignin oligomers can be achieved by applying stepwise reversed-phase gradients. A commonly employed reversed-phase system with water:acetonitrile mobile phase is evaluated for this task. Special attention was devoted to uncovering the molecular level explanation of the retention phenomenon. Our results indicate that separation is mainly governed by reversed-phase retention phenomena without any major exclusion or viscosity-related effects, shown by great fits to linear retention models (R2avg=0.9599 for five different oligomers) and apparent differences in retentivity between different stationary phases. The influence of the gradient shape was demonstrated by the comparison of stepwise gradients with different number and frequency of steps, leading to the conclusion that gradients with a low number of steps yield fewer, but better resolved fractions, while finer multi-step gradients can be used to distinguish more fractions.

RGB Trichromatic Whiteness Assessment of Bio Analytical Chromatographic Tool Using Fluorescence for Quantitation of Semaglutide: Application to Pharmaceutical Preparations and Spiked Plasma.

Semaglutide (SEMG) is one of the most widely used and trending medications to treat type II diabetes and obesity.This work aimed to develop a liquid chromatography with spectroflourimetric detection (HPLC-flourimetry) analysis of SEMG in both its tablet dosage form and plasma. The power of fluorescence detection coupled with HPLC proved its capability as a bioanalytical tool to assay SEMG in plasma samples owing to its simplicity and sensitivity which reached below the Cmax of SEMG. Separation was done using a C18 column with mobile phase of acetonitrile and water acidified with orthophosphoric acid (pH 3.5) (1.41 × 10-5M) in isocratic mode in ratio 57:43 and 1mL/min flow rate after extraction using protein precipitation. Detection was carried out at λ excitation of 238nm and λ emission of 416 and 307nm for SEMG and the internal standard, respectively. Evaluation of greenness of the proposed method was done using AGREE (Analytical GREEnness Metric Approach), ComplexGAPI (Complementary Green Analytical Procedure Index) & the new algorithm RGB 12 model (Red-Green-Blue). They showed that these methods can be a greener alternative with acceptable sensitivity for analysis of SEMG. The developed seven min-assay was validated per ICH as well as FDA bio analytical methods' guidelines to prove its applicability for routine sample analysis and future pharmacokinetic studies.

A validated high-performance liquid chromatography-ultraviolet method for the determination of valproic acid derivatives in pharmaceutical formulations with a microbiological suitability evaluation

Valproic acid and its derivatives are common drugs used in the treatment of epilepsy, a result of CNS disorders. Because the molecular structure of valproic acid, as a fatty acid, does not contain any chromophore groups, its peak response demanded special conditions to be detected in HPLC, ensuring the best precision results. Still, the current approach illustrates simple, validated procedures for conduction. The conducted chromatographic system consists of the BDS Hypersil C8, 150 × 4.6 mm, 5 µm column using a mobile phase of acetonitrile: phosphate buffer (4:6) at a detection wavelength of 215 nm at room temperature. A full method validation study was conducted and approved to ensure precise, repeatable, and accurate results by implementing system suitability parameters. In microbiological terms, a suitability test is used to validate microbiological testing methods, ensuring their efficacy despite potential interference from antimicrobial properties in the tested materials. The HPLC-UV developed and validated analytical method was evaluated, and it was found to be sensitive for use in the detection of low concentrations of methylparaben, propylparaben, and valproate with an optimum run time of six minutes. limit of detection (LODs) were statistically estimated and found to be 2.27 µg/mL, 39.77 ng/mL, and 1.84 µg/mL for methylparaben, propylparaben, and valproate, respectively. Additionally, the method demonstrated a high recovery of methylparaben, propylparaben, and valproate with an excellent closed-accuracy range (98.84% - 101.34%). An excellent regression coefficient (r) of 0.99924, 0.99998, and 0.99997 for methylparaben, valproate, and propylparaben, respectively, was achieved. Assay determination of various pharmaceutical dosage forms in the local market was implemented, yielding admirable results.

Stability-Indicating HPLC Method Development and Validation for the Simultaneous Determination of Vildagliptin and Dapagliflozin in Pharmaceutical Dosage Form.

Vildagliptin (VD) and Dapagliflozin (DP) were determined simultaneously by RP-HPLC using an ultraviolet (UV) detector, a Hypersil Gold C18 (250 × 4.6 mm) column, 5 µm and a mobile phase of acetonitrile: water (adjusted with O-Phosphoric acid to pH 3) in ratio 40: 60% v/v. The estimation wavelength was chosen to be 213 nm. VD and DP were shown to have retention times of 2.9 and 8.3 minutes, respectively. Q2 (R1) ICH guidelines were followed during the validation process. VD and DP correlation coefficient R2 values are 0.9993 and 0.999, respectively. While DP’s linearity range is 1 to 6 µg/mL, VD’s is 10 to 60 µg/mL. The precision and accuracy studies show %relative standard deviation (%RSD) below 2%. Recovery% was assessed to meet the ICH Q2 (R1) guidelines criteria. The presented research on forced degradation showed stability indicating studies. The findings also demonstrated that the suggested method is suitable for determining VD and DP precisely and accurately.

Development and Validation of an LC-MS/MS Assay for the Quantitation of MO-OH-Nap Tropolone in Mouse Plasma: Application to In Vitro and In Vivo Pharmacokinetic Studies.

A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of MO-OH-Nap tropolone (MO-OH-Nap) in mouse plasma. MO-OH-Nap is an α-substituted tropolone with anti-proliferative properties in various cancer cell lines. Detection and separation of analytes was achieved on an ACE Excel C18 (1.7 µm, 100 × 2.1 mm, MAC-MOD Analytical, Chadds Ford, PA, USA) column with mobile phase consisting of 0.05% trifluoroacetic acid in water (mobile phase A) and 0.05% trifluoroacetic acid in acetonitrile (mobile phase B), with an isocratic elution of 15:85% (A:B) at a total flow rate of 0.25 mL/min. The LC-MS/MS system was operated at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combination of 249.10 > 202.15 m/z for MO-OH-Nap and 305.10 > 215.05 m/z for the internal standard (IS), BA-SM-OM. The MS/MS response was linear over a concentration range of 1 to 500 ng/mL with a correlation coefficient (r2) of ≥0.987. The within- and between-batch precision (%RSD) and accuracy (%Bias) were within acceptable limits. The validated method was successfully applied to determine MO-OH-Nap metabolic stability, plasma protein binding, and bio-distribution studies of MO-OH-Nap in CD-1 mice.

Development of Quality Standard and a Comprehensive Quality Control Study of Bharngyadi Kvatha Churna: An Ayurvedic Formulation

Background: Bharngyadi Kvatha Churna (BKC) is an important polyherbal formulation mentioned in the Ayurvedic Formulary of India (AFI) for the treatment of intermittent fever and chronic fever but lacks the presence of standardised quality control parameters. Aims: The aim of the present study was to develop a quality standard of BKC and to carry out a comprehensive quality control study, including the evaluation of physicochemical parameters, safety parameters, elemental content, secondary metabolites, and selected phytochemical analysis. Methods: In HPTLC analysis, piperlongumine was quantified in BKC using a silica gel-coated aluminium plate and developing solvent of toluene: acetone (6:4 v/v). In HPLC analysis, piperine was quantified using acetonitrile: water (80: 20 v/v) mobile phase, and ellagic acid was quantified using water (0.05% KH2PO4): acetonitrile (70: 30 v/v) mobile phase. ICP-OES was used to determine elemental content. The estimation of secondary metabolites like total sugars, tannins, flavonoids, alkaloids, and saponins was also carried out using a UV-Vis spectrophotometer. The volatiles present in the volatile oil of BKC were analysed using the gas chromatography-mass spectrometry technique. Results: The physicochemical parameters namely, loss on drying, total ash, acid-insoluble ash, alcohol- soluble extractive, water-soluble extractive, pH (10% aq. susp.), and volatile oil ranged from 10.56% to 11.06%, 4.67% to 5.76%, 0.73% to 1.78%, 7.56% to 8.2%, 12.04% to 13.06%, 5.87% to 5.96%, and 0.091% to 0.097%, respectively. The amount of piperlongumine in BKC ranged from 0.7688 mg/g to 0.9902 mg/g. The amount of piperine and ellagic acid ranged from 0.1180% to 0.1362% and 0.0016% to 0.0034%, respectively. Oleyl alcohol is the major volatile phytochemical present in BKC. Conclusions: The developed standardised quality control parameters of BKC would aid the herbal industry in developing BKC with requisite quality.

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 – 78.2 °C for 10–100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1–3 °C, and denaturation power increases in the order MeOH<EtOH<MeCN. Next, we investigated the RNA duplex melting using selected chromatographic techniques and within the 10–90 °C column temperature range. Melting temperature obtained with size exclusion chromatography in 25 mM sodium phosphate buffer was ∼ 70 °C, about 5 °C lower compared to DSC values. The apparent melting temperature for a reversed-phase chromatography experiment was < 10 °C for 10 mM ammonium acetate mobile phase with acetonitrile eluent. Ion-pair reversed-phase with 10 mM triethylamine, 100 mM hexafluoroisopropanol and MeCN as eluent yielded Tm 49.3–54.9 °C. Hydrophilic interaction chromatography experiments with 10 mM ammonium acetate and ∼ 50 % acetonitrile mobile phase showed high duplex stability (Tm ∼ 80 °C). The observed chromatographic Tm values suggest that the formation of an organic/aqueous solvent layer on the chromatographic sorbent surface affects the duplex stability more significantly than the composition of the mobile phase alone.

Unified and Versatile Multiplex Platform for Expedited Method Development and Comprehensive Characterization of Therapeutic Peptides.

Agile analytical approaches are needed for fast and comprehensive characterization of peptide drug candidates. In this study, a unified and versatile multiplex platform was developed to expedite method development and enable the routine determination of multiple quality attributes simultaneously. The platform integrates the automation of size exclusion chromatography (SEC), reversed phase liquid chromatography coupled to reversed phase liquid chromatography (RPLC-RPLC), and hydrophilic interaction liquid chromatography hyphenated to charged aerosol detection (HILIC-CAD). Various therapeutic peptide constructs, including macrocyclic peptides and disulfide constrained peptides, across different lots were studied. The effect of the mobile phase acetonitrile content on the impurity profiles was systematically studied using two SEC columns. A prototype MaxPeak Premier SEC 125 Å column packed with BEH PEO particles achieved the separation of impurities (>2.0% area), whereas no impurities could be observed with an ACQUITY UPLC Protein BEH SEC 125 Å column packed with BEH diol particles. Comprehensive impurity profiling and expedited method development was performed utilizing RPLC-RPLC. Each peptide was analyzed by a combination of 12 conditions in the second dimension, using four columns with octadecyl, phenyl-hexyl, and cyano bonded phases, and three mobile phases with various solvents, modifiers, and pH compositions. Additionally, a HILIC-CAD method was developed for the quantification of TFA, commonly present in peptide products.

The effects of herbicide application on the properties of agricultural soil in Algeria

Phytosanitary products, particularly herbicides, are essential for weed control in agriculture. This study examined the concentration and persistence of Pinoxaden residues in different soil textures. Seventy-two soil samples from two farms in Tiaret, Algeria, were analyzed using liquid chromatography (HPLC) to measure herbicide residues. The analysis used a mobile phase of acetonitrile and methanol (50/50, V/V) with a retention time of 2 minutes. Results indicated significant differences in residue persistence based on soil texture. Clay-loam soils showed higher residue persistence, averaging 6.01 × 10^-4 ± 8.7 × 10^-5 μg/g, while sandy soils had lower persistence, averaging 6.4 × 10^-5 ± 5.9 × 10^-6 μg/g. This underscores the impact of soil characteristics on herbicide behavior and the need for tailored management strategies. Despite herbicides' benefits, their environmental impacts are concerning. Persistent residues can contaminate soil and harm ecosystems. Raising awareness among stakeholders about these risks is essential. The study recommends sustainable alternatives, such as biological control methods, to reduce dependency on chemical herbicides. Biological controls offer eco-friendly solutions, promoting ecosystem balance and reducing agriculture's ecological footprint. Implementing crop rotation and other integrated pest management (IPM) strategies can enhance weed control effectiveness while decreasing reliance on chemical herbicides. These practices mitigate environmental risks and improve soil health and agricultural productivity. In conclusion, while herbicides are indispensable in modern agriculture, responsible use and management are crucial for environmental sustainability and the long-term viability of agricultural systems. By adopting sustainable practices and innovative technologies, it is possible to balance agricultural productivity with environmental protection.

Determination of ivermectin in plasma and whole blood using LC-MS/MS.

Ivermectin is a widely used drug for the treatment of helminthiasis and filariasis worldwide, and it has also shown promise for malaria elimination through its potent mosquito-lethal activity. The objective of this study was to develop and validate a high-throughput and sensitive method to quantify ivermectin in plasma and whole blood samples, using automated sample extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Phospholipids were removed in patient whole blood (100 µl) and plasma (100 µl) samples using a 96-well plate Hybrid-solid phase extraction technique. Ivermectin and its isotope-labelled internal standard (ivermectin-D2) were separated on an Agilent Poroshell 120 EC-C18 50mm × 3.0mm I.D. 2.7µm, using a mobile phase of acetonitrile: ammonium formate 2 mM containing 0.5% formic acid (90: 10, v/v). Detection was performed using a triple quadrupole mass spectrometer in the positive ionization mode. The method was validated in the concentration range 0.970 - 384 ng/ml in both plasma and whole blood matrices. Intra- and inter-batch precisions during the validation were below 15%. There was no carryover or matrix effects detected. Ivermectin is a stable compound and results showed no degradation in the different stability tests. The validated method proved to have high sensitivity and precision, good selectivity and to be suitable for clinical application or laboratory quantification of ivermectin in plasma or whole blood samples.

A dual methodology employing ion-pair chromatography and built-in UV spectrophotometry for quantifying recently approved combination of mometasone and indacaterol in a novel combined metered dose inhaler: assessing the greenness, carbon footprint, blueness, and whiteness

Developing analytical techniques that align with green and sustainable chemistry principles is crucial in today's scientific landscape. This work introduces two innovative approaches for the simultaneous quantification of indacaterol (IND) and mometasone (MOM), a recently approved combination therapy for chronic obstructive pulmonary disease. These methods—rapid isocratic ion pair chromatography (IPC) and UV–visible spectrophotometry—demonstrate improved environmental sustainability, cost-effectiveness, and versatility compared to existing techniques. The optimized 4-min IPC method achieved excellent resolution (retention times 2.18 ± 0.1 min for IND and 3.95 ± 0.1 min for MOM), peak symmetry, and sensitivity. It utilizes a low-cost ion pair mobile phase of acetonitrile and acidified water containing 0.025% sodium dodecyl sulfate (50:50% v/v), making it suitable for laboratories with standard chromatographic instruments. The spectrophotometric approach offers two procedures: first derivative and ratio derivative methods. These serve as simplified, low-cost alternatives for resource-limited laboratories without access to advanced instruments. Both techniques feature simplified protocols that minimize extraction and fractionation steps. Comprehensive validation confirmed outstanding accuracy (98–102%) and precision (%2 <). Sustainability assessments using ComplexGAPI, AGREE, carbon footprint, BAGI, and RGB12 tools demonstrated enhanced environmental performance compared to existing methods. The IPC and spectrophotometry methods achieved greenness scores of 0.81 and 0.85, respectively, surpassing the 0.63–0.67 range of reported techniques. Additionally, they showed lower carbon footprints of 0.035 and 0.022 kg CO2 equivalent emissions per sample, compared to 0.079–0.092 kg for conventional procedures. The application of novel "blueness" and "whiteness" concepts using BAGI and RGB12 algorithms further confirmed superior sustainability, with scores of 87.5 & 90 for blueness and 88.1 & 89.8 for whiteness. Successfully applied to quantify IND and MOM in combined capsules, this work provides a model for eco-friendly pharmaceutical analysis that maintains high analytical reliability while improving sustainability metrics.

The retention features of nitrogen-containing heterocyclic compounds in reversed-phase and hydrophilic HPLC-MS modes

Aromatic five- and six-membered nitrogen-containing heterocyclic compounds are biologically active substances and are widely used in biochemistry and medicine. In addition, such compounds are known as ecotoxicants formed during oxidation processes in industrial wastewater. High-performance liquid chromatography-mass spectrometry is widely used to determine nitrogen-containing heterocycles in various complex mixtures. Octadecyl silica gel is the main sorbent for chromatographic separation. However, octadecyl silica gel does not always enable satisfactory separation in the presence of highly polar isomeric molecules, and more selective chromatographic approaches are required. One of these is hydrophilic chromatography, which facilitates the separation of polar compounds. The aim of the study was to comparatively characterize the retention of a number of five- and six-membered nitrogen-containing heterocyclic compounds during reversed-phase and hydrophilic liquid chromatography with mass spectrometric detection. In addition, spectrophotometric detection at a wavelength of 210 nm was carried out. In the study, we used sorbents based on octadecyl silica gel and unmodified silica gel. High-resolution mass spectrometry was used in the electrospray ionization mode. We studied 19 nitrogen-containing heterocyclic compounds. We used aqueous acetonitrile mobile phases with added aqueous solutions of formic acid and diethylamine as acid-base modifiers. An ammonium formate solution was used as a modifier during hydrophilic chromatography. Reversed-phase chromatography showed that the introduction of 0.1% aqueous formic acid solution increased the ionization and the mass spectrometer signal intensity, but did not always result in a satisfactory separation of isomers upon elution from the octadecyl silica gel surface. The introduction of 0.01% diethylamine solution provided no significant improvement in the separation compared to the mobile phase without modifiers. The use of unmodified silica gel in hydrophilic chromatography mode allowed us to separate isomeric five-membered heterocycles, which was demonstrated by narrow symmetric peaks, but did not result in selective separation of isomeric diazines. Thus, we studied the retention of 19 nitrogen-containing heterocyclic compounds using reversed-phase and hydrophilic liquid chromatography with electrospray ionisation. It was determined that hydrophilic chromatography on unmodified silica gel could be used for satisfactory separation of isomeric five-membered heterocyclic compounds of different classes. For six-membered heterocycles, satisfactory separation was achieved by elution from the surface of octadecyl silica gel using aqueous acetonitrile mobile phase without modifiers.

Penalization and Color Code-Technical Approaches for Methods' 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 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. Firstly, Second-Derivative (Δλ; 5 nm) spectrophotometric method; Toltrazuril is measured from peak to peak at 244-260 nm within a linearity range of 5-25 μg/mL. The second one is a high-performance thin-layer chromatography (HPTLC) analysis performed on an aluminum sheet of silica gel using ethyl acetate, methanol, ammonium chloride buffer, and water (8:1:0.5:0.5) (%V/V) 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 one is Reversed Phase-HPLC-diode array detection, using Agilent column C18 (5 μm, 4.6 x 150 mm) in isocratic elution mode with a mobile phase of acetonitrile and water in a ratio of 80:20 (v/v), respectively, at 1 mL/min flow rate. 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 ICH validated and applied to dosage form with satisfactory % recoveries (97-103%). Statistical comparisons reported one using t-test and F-test disclose insignificant variation. Examining greenness and whiteness norms, proposed methods were evaluated and ranked alongside four different reported methods. The proposed methods are green, accurate, and can be applied in routine quality control for the determination of toltrazuril in pharmaceutical formulations.

Central Composite Design Expert-Supported RP-HPLC Optimization and Quantitative Evaluation of Efonidipine Hydrochloride Ethanolate & Chlorthalidone in Tablet.

Central composite design based RP-HPLC method optimization for the synchronized analysis of Efonidipine Hydrochloride Ethanolate (EFE) and Chlorthalidone (CHL) in tablet. The effective separation was performed using Inertsil ODS C18 column (250 × 4.6mm, 5μm), PDA detector with 0.05M KH2PO4 Buffer (pH4.5): Acetonitrile (40:60%v/v) mobile phase. Independent variables were investigated include the concentration of KH2PO4 (X1) and flow rate of mobile phase (X2). Based on responses obtained (retention time, resolution and tailing factor), the optimum condition selected was X1 = 40% and X2 = 1ml/min. Optimized HPLC condition was validated by assessing validation parameters and it meets the acceptance criteria set by ICH. The linear calibration curve was found to be in the quantity range 6.25-18.75 and 20-60μg/ml Assay of drugs was 100.94 and 100.06% for CHL and EFE. The validated RP-HPLC-PDA method can be used for routine analysis of EFE and CHL in tablet.

Pharmacokinetics of Anti-rheumatic Drugs Methotrexate and Tofacitinib with its Metabolite M9 in Rats by UPLC-MS/MS.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

Development and Validation of New LC-MS/MS Bioanalytical Method of Tremelimumab in Rat Plasma by Using Nivolumab as Internal Standard and Its Application with Pharmacokinetic Studies

Nivolumab was decided to be used as the IS. To separate the drugs, an isocratic mobile phase of acetonitrile (ACN): Ammonium formate containing formic acid (70:30) was adopted and delivered at 1-mL/min, along with a 150 x 4.6 mm x 3.5 μm Alliance, e2695 Luna Phenyl Hexyl column. Drug and IS, both of which display proton adducts approximately m/z 146.3685 to 120.0638 and m/z 143.7695 to 76.7964, might be detected simultaneously using MRM-positive modalities. Over a linearity level of 2.00 to 40.00 ng/mL, the method had a correlation value (r2) of 0.99977. The accuracy and precision of this method during the day ranged from 89.85 to 102.89% and 0.19 to 2.81%, respectively. Tremelimumab was reported to remain stable during three freeze-thaw cycles, benchtop tests, and postoperative stability studies. Cmax and Tmax values were acquired directly from experimental data. Cmax and Tmax averaged 18.024 ng/mL and 6 hours, respectively. At t1/2 of 18 hours, plasma levels began to fall. The obtained AUC024 and AUC0 values were 257 and 257 ng h/mL, respectively.

Green HPLC Enantioseparation of Chemopreventive Chiral Isothiocyanates Homologs on an Immobilized Chiral Stationary Phase Based on Amylose tris-[(S)-α-Methylbenzylcarbamate

Sulforaphane is a chiral phytochemical with chemopreventive properties. The presence of a stereogenic sulfur atom is responsible for the chirality of the natural isothiocyanate. The key role of sulfur chirality in biological activity is underscored by studies of the efficacy of individual enantiomers as chemoprotective agents. The predominant native (R) enantiomer is active, whereas the (S) antipode is inactive or has little or no biological activity. Here we provide an enantioselective high-performance liquid chromatography (HPLC) protocol for the direct and complete resolution of sulforaphane and its chiral natural homologs with different aliphatic chain lengths between the sulfinyl sulfur and isothiocyanate group, namely iberin, alyssin, and hesperin. The chromatographic separations were carried out on the immobilized-type CHIRALPAK IH-3 chiral stationary phase with amylose tris-[(S)-methylbenzylcarbamate] as a chiral selector. The effects of different mobile phases consisting of pure alcoholic solvents and hydroalcoholic mixtures on enantiomer retention and enantioselectivity were carefully investigated. Simple and environmentally friendly enantioselective conditions for the resolution of all chiral ITCs were found. In particular, pure ethanol and highly aqueous mobile phases gave excellent enantioseparations. The retention factors of the enantiomers were recorded as the water content in the aqueous-organic modifier (methanol, ethanol, or acetonitrile) mobile phases progressively varied. U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography retention mechanism on the CHIRALPAK IH-3 chiral stationary phase. Finally, experimental chiroptical studies performed in ethanol solution showed that the (R) enantiomers were eluted before the (S) counterpart under all eluent conditions investigated.

Bioanalytical HPLC method with fluorescence detector for determination of Entresto™ when co-administered with ibuprofen and fexofenadine: a pharmacokinetic study.

Entresto™ (LCZ696) has been approved globally for heart failure management. However, its lifelong use alongside over-the-counter (OTC) drugs like ibuprofen (IBU) and fexofenadine (FEX) necessitates an in-depth investigation of potential pharmacokinetic interactions, as they share the same metabolic and elimination pathways. This study aimed to develop a bioanalytical HPLC method with a fluorescence detector (FLD) to quantify LCZ696 analytes (valsartan, VAL; sacubitril, SAC; and sacubitril active metabolite, LBQ657) in rat plasma. Additionally, an in vivo study was performed to investigate the pharmacokinetic interactions of LCZ696 with IBU and FEX. Utilizing HPLC with a gradient-mode mobile phase of acetonitrile and 0.025 M phosphate buffer (pH 3), the study demonstrated a significant increase in the bioavailability of LCZ696 analytes (VAL and LBQ657) when co-administered with IBU (C max 0.23 ± 0.07 and 0.53 ± 0.21 μg mL-1, respectively) compared to the control (0.17 ± 0.03 and 0.33 ± 0.14 μg mL-1). A more significant increase in C max was noticed with FEX (0.38 ± 0.01 and 0.77 ± 0.18 μg mL-1, respectively). Moreover, a decrease in the clearance (Cl/F) of VAL and LBQ657 was observed (18.05 ± 1.94 and 12.42 ± 2.97 L h-1 kg, respectively) with a more pronounced effect in the case of FEX (30.87 ± 4.29 and 33.14 ± 9.57 L h-1 kg, respectively) compared to the control (49.99 ± 7.31 and 51.19 ± 9.12 L h-1 kg, respectively). In conclusion, our study underscores the importance of cautious administration and appropriate dose spacing of IBU and FEX in patients treated with LCZ696 to prevent elevated serum concentrations and potential toxicity. The novelty of this work lies in its dual contribution: developing a highly sensitive HPLC-FLD method and comprehensively elucidating significant pharmacokinetic interactions between LCZ696 and common OTC drugs.

Simultaneous determination of maltol and seventeen saponins in red and black ginseng by UPLC-PDA wavelength switching method

This study developed a UPLC-PDA wavelength switching method to simultaneously determine the content of maltol and seventeen saponins in red and black ginseng and compared the quality differences of two different processed products of red and black ginseng. A Waters HSS T3 column(2. 1 mm×100 mm, 1. 8 μm) at 30 ℃ was adopted, with the mobile phase of acetonitrile(A) and water containing 0. 1% phosphoric acid(B) under gradient elution, the flow rate of 0. 3 m L·min~(-1), and the injection volume of 2 μL.The wavelength switching was set at 273 nm within 0-11 min and 203 nm within 11-60 min. The content results of multiple batches of red and black ginseng samples were analyzed by the hierarchical cluster analysis(HCA) and principal component analysis(PCA) to evaluate the quality difference. The results showed that the 18 constituents exhibited good linear relationships within certain concentration ranges, with the correlation coefficients(r) greater than 0. 999 1. The relative standard deviations(RSDs) of precision,repeatability, and stability were all less than 5. 0%. The average recoveries ranged from 95. 93% to 104. 2%, with an RSD of 1. 8%-4. 2%. The content determination results showed that the quality of red and black ginseng samples was different, and the two types of processed products were intuitively distinguished by HCA and PCA. The method is accurate, reliable, and reproducible. It can be used to determine the content of maltol and seventeen saponins in red and black ginseng and provide basic information for the quality evaluation and comprehensive utilization of red and black ginseng.

Development of Analytical method by UV and HPLC of Acetazolamide in bulk and tablet dosage form

In present study, on a Jasco V-730 spectrophotometer, the method for estimating acetazolamide was used, with ethanol: distilled water (5:5) serving as the solvent. For acetazolamide, the recovery analysis research indicates recoveries of 102.55%, 102.68%, and 103.35% for various concentrations of the spiked medication. For quantifying acetazolamide in bulk and tablet dosage form, an RP-HPLC method was created and validated with mobile phase acetonitrile: water (80:20). For acetazolamide, the assay (% w/w) was found 98.6 %, 98.5 %, 98.9 %. The flow rate was 1ml/min. The sample volume was 20.00µl. Run time 5 min and retention time was 2.044.