Mobile Phase Flow Rate Research Articles

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

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

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Sensitive, precise fingerprint profiling for monosaccharide analysis of Bacillus Calmette-Guérin polysaccharide and nucleic acid isolates

A sensitive and precise HPLC-DAD method with pre-column PMP derivatization was established and validated, for analyzing the polysaccharides in Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN) isolates, after acid hydrolysis. And the HPLC fingerprint profiling was used to analyze its monosaccharide composition. The monosaccharide concentration-peak area calibration curve was of good linearity (R2 > 0.99), over the range of 0.016–0.08 mg/mL for mannose or 0.24–1.20 mg/mL for glucose, with high recovery of 93–105 % for quality control samples. The intra-day RSD values of mannose and glucose concentration were less than 2.5 % and 2.1 %, respectively, and their inter-day RSD values were less than 4.3 % and 2.2 %, respectively, and remained stable for up to 14 days. This method also remained durable against changes in chromatographic parameters, but it's susceptible to the flow rate of mobile phase. Additionally, the method was applied to analyze the content of mannose and glucose in 22 batches BCG-PSN powder and 17 batches BCG-PSN injection. The results showed that the HPLC-DAD fingerprint spectra of all the BCG-PSN powder and BCG-PSN injection samples had a high degree of similarity, with the similar indexes up to 0.999 and 0.998, respectively. The HPLC-DAD method with pre-column PMP derivatization is highly rapid, effective, visual, and accurate for determination of monosaccharide contents. The validated method was successfully applied to the analysis of polysaccharide in both BCG-PSN powder and injection.

Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA

Modified messenger RNA (mRNA) represents a rapidly emerging class of therapeutic drug product. Development of robust stability indicating methods for control of product quality are therefore critical to support successful pharmaceutical development. This paper presents an ion-pair reversed-phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimised method could be used for separation and analysis of large RNA, sized up to 1000 nucleotides. Column temperature, mobile phase flow rate and ion-pair selection were each studied and optimised. Baseline separations of the model RNA ladder sample were achieved using all examined ion-pairing agents. We established that the optimised method, using 100 mM Triethylamine, enabled the highest resolution separation for the largest fragments in the RNA ladder (750/1000 nucleotides), in addition to the highest overall resolution for the selected modified mRNA compound (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analysing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with ribonucleases. Our results showed that the formed degradation products, which appeared as shorter RNA fragments in front of the main peak, could be well monitored, using the optimised method, and the relative stability of the mRNA under the various stressed conditions could be assessed.

Exploring Negative Chemical Ionization of Per- and Polyfluoroalkyl Substances via a Liquid Electron Ionization LC-MS Interface.

Per- and polyfluoroalkyl substances (PFASs) are a class of aliphatic manufactured compounds comprising fluoro-chemicals with varied functional groups and stable carbon-fluorine bonds. They are defined as "forever chemicals" due to their persistent and bioaccumulative character. These substances have been detected in various environmental samples, including water, air, soil, and human blood, posing significant health hazards. High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) is typically employed for the analysis of PFASs. Negative chemical ionization (NCI) is generally coupled to gas chromatography (GC) and offers high selectivity and sensitivity for compounds containing electronegative atoms, such as PFASs. The liquid electron ionization (LEI) interface is an efficient mechanism developed to robustly couple a liquid flow rate from an LC system to an EI or a CI source. This interface has been successfully utilized for pesticide determination in UHPLC-LEI-CI in negative ion mode (NCI). This work aims to evaluate different parameters involved in the ionization of PFASs analyzed in LC-LEI-NCI and subsequently develop a method for their detection in real samples. The parameters considered for this study include (i) a comparison of different CI reagent gases (methane, isobutane, and argon); (ii) the use of acetonitrile as both the chromatographic solvent and CI reagent gas; (iii) the presence of water and formic acid as chromatographic mobile phase components; and (iv) the mobile phase flow rate. The optimal combination of these parameters led to promising results. Tentative fragmentation pathways of PFASs in NCI mode are proposed based on the dissociative electron capture mechanism.

Simultaneous determination of four non paraben preservatives (benzyl alcohol, phenoxyethanol, sodium benzoate, and salicylic acid) in shampoos by an isocratic HPLC method

Salicylic acid (SA), sodium benzoate (SB), phenoxyethanol (PO), and benzyl alcohol (BA) are “non paraben” preservatives commonly used in cosmetic products like shampoos, and often in combination of at least two compounds to assure their antimicrobial effectiveness at low concentrations. In this study, an HPLC-DAD method was developed and validated for simultaneous determination of SA, SB, PO, and BA in commercial shampoos. The method was carried out using an YMC Triart C18 (250 × 4.6 mm, 5 µm) column with a mixture of methanol and 0.02 M solution of phosphate buffer pH 2.7 (35 : 65, v/v) as mobile phase. The flow rate of mobile phase was maintained at 1.0 mL per minute, the injection volume was 20 µL and the UV detection was done at 208 nm for BA and at 230 nm for SA, SB and PO. The method was fully validated according to requirements on analytical method performance of AOAC International and validation results confirmed its reliability and suitability for intended application. The method was employed to determine the four “non paraben” preservatives in 18 commercial shampoos, finding the actual content of these substances in all samples conformed to their regulatory limits.

Preparation and evaluation of two chiral stationary phases based on imidazolyl-functionalized bromoethoxy pillar[5]arene-bonded silica

Chiral pillar[5]arene-based mesoporous silica, an emerging class of chiral structure, possesses excellent characteristics such as abundant chiral active sites, encapsulated cavity and excellent chiral modification, which make them a promising candidate as new chiral stationary phases (CSPs) in enantioseparation. In this study, two imidazole-containing (S)-1-(4-phenyl-1H-imidazol-2-yl)ethanamine and (S)-Histidinol were respectively modified to bromoethoxy pillar[5]arene-bonded silica to construct new chiral stationary phases (sPIE-BP5-Sil and sHol-BP5-Sil) for the separation and analysis of enantiomers. The separation conditions such as mobile phase composition, flow rate and temperature were optimized. Under optimal conditions, both sPIE-BP5-Sil and sHol-BP5-Sil showed good separation performance for different types of enantiomers. Interestingly, sPIE-BP5-Sil and sHol-BP5-Sil showed better enantioselectivity for chiral aromatic compounds and chiral aliphatic compounds, respectively. This enantioseparation result was closely related to the presence of additional aromatic rings and abundant hydroxyl groups in the side chains of the two chiral groups. In addition, the enantioseparation process was further studied by molecular docking simulation. Therefore, this work provided a new strategy for the preparation and application of imidazolyl-derived pillar[5]arene-based chiral stationary phases, which can be efficiently used for screening and separating enantiomers.

Determination of chromatographic conditions for quantitative assessment of active components in complex nasal spray after manufacturing and expiry date

The aim of the work is the development of chromatographic conditions, the study of the validation characteristics of the method of quantitative determination of phenylephrine hydrochloride, nitrofural, lidocaine hydrochloride and diphenhydramine hydrochloride, panthenol, povidone in the joint presence in the nasal spray by a complex method of liquid chromatography with UV detection. Evaluation of the quantitative content of active components after manufacturing and during the shelf life.
 Materials and methods. Agilent 1260 liquid chromatographs, equipped with a diode-matrix detector from the company "Agilent technologies", USA. Chromatographic columns 250×4.6 mm in size, filled with octadecylsilyl silica gel for chromatography (Zorbax StableBond SB-Aq, Agilent company), mobile phase A - phosphate buffer solution pH 7.0 - acetonitrile P (1650:350), mobile phase B – acetonitrile P; elution mode – gradient; mobile phase flow rate – 1.0 ml/min; detection wavelengths – 220 nm (for panthenol, phenylephrine, povidone, diphenhydramine) and 235 nm (for nitrofural and lidocaine).
 Results. Chromatographic separation conditions were developed for the co-presence determination of six target substances: panthenol, phenylephrine hydrochloride, nitrofural, povidone, lidocaine hydrochloride and diphenhydramine hydrochloride. The suitability of the technique for this task was confirmed by determining the validation characteristics. The methodology at the appropriate level is characterized by specificity, linearity, correctness and convergence in the range of application for panthenol (range 20.33-38.26 mg/ml, ΔZ=0.93 ≤ max ΔZ=3.20, a=0.63 max a=5.12, r = 0.9978 min r= 0.9924), phenylephrine hydrochloride (range 1,70-3,21 mg/ml, ΔZ=0.51 ≤ max ΔZ=3.20, a=0.15 max a=5.12, r = 0.9984 min r= 0.9924), nitrofural (range 0.137-0.257 mg/ml, ΔZ=0.91 ≤ max ΔZ=3.20, a=0.032 max a=5.12, r = 0.9987 min r= 0.9924) povidone (range 20,44-38,50 mg/ml, ΔZ=0.23 ≤ max ΔZ=3.20, a=2,33 max a=5.12, r = 0.9942 min r= 0.9924), lidocaine hydrochloride (range 6,80-12,81 mg/ml, ΔZ=0.34 ≤ max ΔZ=3.20, a=0.66 max a=5.12, r = 0.9988 min r= 0.9924), diphenhydramine hydrochloride (range 1,36-2,56 mg/ml, ΔZ=0.20 ≤ max ΔZ=3.20, a=0.15 max a=5.12, r = 0.9980 min r= 0.9924). There are no significant changes when stored at 25 °C for 6 months.
 Conclusions. An analytical method of quantitative determination of the component composition in an extemporaneous nasal spray by a complex method of high-performance liquid chromatography has been developed. The determined validation parameters confirm the correctness of the methodology. The chemical stability of the dosage form is observed for 6 months

Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers.The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 μm, 160 Å) using isocratic elution and MS-compatible mobile phase.The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Application of a multivariate optimization strategy to validate an RP-HPLC analytical method for determining hydroxychloroquine sulphate in pharmaceutical products and in forced degradation studies

AbstractIn this study, a multivariate optimization strategy was used to develop and validate a simple, rapid, accurate, cost-effective, and stability-indicative RP-HPLC analytical method for quantifying hydroxychloroquine sulphate (HCQ) in coated tablets. The validation conditions involved isocratic elution mode, using a mixture of buffer solution at pH 2.2 and methanol (74:26, v/v) as the mobile phase, an Agilent® reverse phase column, model Zorbax Eclipse Plus C18 (250 cm × 4.6 mm × 5 μm), a flow rate of 1.3 mL min−1, column temperature 40 °C and detection at 343 nm. The method showed linearity in the range of 4–44 μg mL−1, with a correlation coefficient (R) of 0.9998. Recovery obtained average values of between 99.71 and 100.84% and precision with average RSD values of <2%. The robustness demonstrated by assessing the effect of seven variables (pH of the mobile phase buffer; percentage of methanol; filter brand; mobile phase flow rate; wavelength; column temperature and sample agitation time), with effect values for each variable lower than the calculated value of s√2 (1.43), showed that none of these factors had a significant influence on the analytical response. The method was applied to samples of the reference medicine Plaquinol® 400 mg and similar Reuquinol 400 mg, nomenclature established by the National Health Surveillance Agency (Anvisa), law no. 978 of 10 February 1999, purchased from local pharmacies. Results showed advantages and benefits in relation to the official method and those reported in the literature. The application of the multivariate strategy, the choice of methanol, in a lower proportion in the organic phase, due to its low toxicity, economy and easier availability compared to acetonitrile, and the other organic solvents used was a promising and important alternative for the analytical method. Furthermore, the use of reversed stationary phase, common in quality control laboratories, provided an analyte retention time of 4.595 min, demonstrating good performance and speed in routine analyses.

Alidation of an analytical method by HPLC for the quantification of yangambin from the Ocotea duckei species

Ocotea duckei, known as Louro-de-cheiro, belongs to the Lauraceae family and presents lignoid yangambine (YAN) as the main plant marker. This work aimed to develop and validate an analytical method by high performance liquid chromatography for the quantification of YAN. The sample used was the crude ethanolic extract (CEE) obtained from aerial parts. In the developed method, a C18 column was used. The mobile phase was composed of acetonitrile and water (45:55), whereas the method parameters included mobile phase flow rate at 0.8 mL/min, oven temperature at 40°C, and monitoring at 205 nm. In the validation, the parameters of selectivity, linearity, precision, accuracy, robustness, limits of detection and quantification were evaluated. As a result, the developed method is in accordance with the guidelines for validation of analytical methods and presented satisfactory chromatographic parameters for YAN determination. Thus, the present analytical methodology can be applied in the quality control of O. duckei raw materials.

Stereoisomeric separation of the chiral herbicide profoxydim and residue method development in rice by QuEChERS and LC–MS/MS

A method for the simultaneous determination of the four stereoisomers of the chiral herbicide profoxydim in rice and husk was developed using the QuEChERS method and LC-tandem mass spectrometry. Four polysaccharide-based chiral stationary phase columns were evaluated. All four stereoisomers were successfully separated on a Chiracel OJ-3R column. The effects of mobile phase, modifiers, mobile phase flow rate and temperature on the separation were also investigated. Different QuEChERS methods were compared for the development of an optimized sample preparation procedure. The method, following SANTE guidelines, showed excellent linearity (R2 ≥ 0.99), the LODs were below 4.0 µg kg−1, and the LOQs did not exceed 12.5 µg kg−1. The overall average recoveries at three levels (12.5, 25.0 and 250 µg kg−1) ranged from 76.77 % to 106.53 %, with RSD values less than 7 %. The method is demonstrated to be convenient and reliable for the routine monitoring of profoxydim stereoisomers in rice and husk.

UPLC METHOD DETERMINATION AND QUANTIFICATION OF BELUMOSUDIL AND ITS IMPURITIES IN THE HUMAN PLASMA SAMPLES

This current analysis demonstrates the development of the related substances (RS) method for separating and quantifying BSL and its related substances from plasma using the UPLC technique. The solid phase extraction (SPE) method was employed to extract the belumosudil (BSL) and related impurities from plasma. This SPE method has been shown to have high recovery values for BSL and its related impurities. The method employed a column from Waters, specifically the X-Bridge BEH C18 150x4.6mm, 3.5µm, with a mobile phase flow rate (0.8 mL/min) and a UV-wavelength (250 nm). In this method, BSL and its impurities show good peak shapes and high resolutions. This developed method shows high sensitivity, accuracy, and precision values. While performing the stability studies, the drug was stable and it showed less than 4.53% degradation. Six different concentration solutions (BSL (25-250µg/mL) and impurities (3-18µg/mL)) were prepared for constructing linearity plots. The obtained linearity plot shows the accepted correlation coefficient (R2) value (≥0.999). The Limit of detection (LOD) and Limit of quantification (LOQ) were determined using the signal-to-noise (S/N) ratio method. This UPLC method was validated according to ICH guidelines.

Optimizing Nicotine Extraction and Analysis Method from Tobacco Agrowaste Extract

Tobacco cultivation is prevalent in Indonesia and contributes significantly to the economy. However, it has negative impacts on social, health, and environmental conditions. The tobacco waste generated is classified as pre-harvest and post-harvest waste, which can be utilized to extract nicotine and recycle essential nutrients. Nicotine has various biological activities and potential health benefits. The extraction of nicotine from tobacco waste is a pressing issue to provide a valuable resource for various industries and reduce the environmental harm caused by burning tobacco waste. The study optimized the HPLC conditions for the detection of nicotine, including the mobile phase composition and flow rate, using a UV detector and a C18 column. The optimal eluent composition was Acetate Buffer: Methanol: Acetonitrile with a ratio of 30:50:20, and the optimal flow rate was 0.2 mL min-1. Additionally, it was found that the nicotine content of tobacco stem samples was higher than that of tobacco dust samples. Overall, this study provides valuable information on the extraction and analysis of nicotine in tobacco samples using HPLC, which can have important implications on developing sustainable tobacco production practices to minimize the negative impacts of tobacco cultivation on social, health, and environmental conditions.

Evaluation of phenolic and anthraquinone profiles of Polygonacaeae family from Turkey

The purpose of this research is to compare the content of phenolic compounds (catechin, esculetin, polydatin, rutin, resveratrol, quercetin) and anthraquinones (aloe-emodin, emodin, chrysophanol, physcion) in Polygonum cognatum Meissn. and Rumex crispus L. plant taxa from the flora of Turkey. Extraction parameters such as solvent, solvent ratio, and time for soxhlet method were optimized, and the most effective extraction was carried out for eight hours with methanol-water (3:1 v/v). The development and validation of an HPLC-DAD method allowed for the simultaneous quantification of phenolic compounds and anthraquinones in the plant extracts. The analytical method conditions were defined through an investigation of various parameters, including mobile phase composition, flow rate, and column temperature. The method was validated with good linearity, precision, sensitivity, and recovery. The LOD and LOQ values of the compounds were in the range of 0.032–0.457 µg/mL and 0.106–1.523 µg/mL, respectively. The findings of the study showed that this analytical method was successfully applied to identify and measure the phenolic and anthraquinone compounds in Polygonum cognatum Meissn. and Rumex crispus L. plants.

Reverse Phase Liquid Chromatography for Cetrimide Determination in Pure and Pharmaceutical Preparations

An effective, duplicable, and quick method to determine the purity and formulating form of Cetrimide (CE) have studied and modified utilizing the Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The method was based on the separation of the CE using a reverse phase HPLC Shimadzu LC–20 A, Japan, and Phenomenex C18 column (15 × 4.6mm, 5.0μm). The process was carried out by using the mobile phase ammonium acetate solution 0.1M (AA), methanol (MeOH), and the methyl cyanide in the ratio of (15:60:25 V/V/V), a flow rate of 1.0mL/min, at pH = 3, with 10μL sample loop and the UV - spectrophotometry detector was set at 280nm. However, for the optimum conditions the mobile phase composition, wavelength, pH, and flow rate were studied. The calibration carve has obtained in the range of 10-1000mg/L and the correlation coefficient is 0.9985. The average recovery of the standard drug was 97.56%. The LOD was 5mg/L and LOQ is 16.5mg/L for CE. The process was used successfully to determine the CE in the pharmaceutical forms with the average recovery of 95.61 and 96.02%.

A QbD Assisted Bioanalytical Method Development and Validation for Simultaneous Estimation of Montelukast and Bilastine by LC-MS Technique

This study describes how to use internal standards (IS) under the Analytical Quality by Design (AQbD) methodology to measure montelukast and bilastine in artificial rabbit plasma. The protein precipitation method was used for sample preparation and extraction. The chromatographic analysis was performed on the processed materials. The measurement range of the calibration was between 2 to 40 ng/mL for Bilastine and 1 to 20 ng/mL for montelukast. Optimizing mobile phase composition, flow rate, and pH were used to determine peak area and retention time. A significant reduction in method variability was observed as a result of the method of experimental design during optimization, and this enhanced the stability of the technique. A validation study demonstrated linearity, accuracy, precision, selectivity, sensitivity, and stability for estimating bilastine and montelukast in rabbit plasma using the developed method. The chemical composition of the drug was not altered during tests on its stability in human plasma. Overall, the investigations showed that the established approach is straightforward, reliable, and affordable for use in regular pharmacokinetic and bioequivalence research.

Modification of gradient HPLC method for determination of small molecules' affinity to human serum albumin under column safety conditions: Robustness and chemometrics study

In the early stages of drug discovery, beyond the biological activity screening, determining the physicochemical properties that affect the distribution of molecules in the human body is an essential step. Plasma protein binding (PPB) is one of the most important investigated endpoints. Nevertheless, the methodology for measuring %PPB is significantly less popular and standardized than other physicochemical properties, like lipophilicity. Here, we proposed how to modify protocols presented by Valko into column safety conditions and evaluated their robustness using fractional factorial design. For robustness testing, four factors were selected: column temperature, mobile phase flow rate, maximum isopropanol concentration in the mobile phase, and buffer pH. Elaborate methods have been applied for the analysis of HSA affinity for three groups of antibiotic-oriented substances that vary in chemical structure: fluoroquinolones, sulfonamides, and tetrazole derivatives. Furthermore, based on the reversed-phase chromatography the workflow of pilot studies was proposed to select molecules that have high affinity to HSA and can not be eluted from the HSA column using the concentration of organic modifier recommended by the column manufacturer.

Development and validation of a stability-indicating ultra-high-performance liquid chromatography method for the estimation of ibrutinib and trace-level quantification of related substances using quality-by-design approach.

A new ultra-high-performance liquid chromatography method was developed using quality-by-design principles for quantifying trace-level impurities of ibrutinib. The method utilized an ACQUITY UPLC BEH C18 column with a mobile phase consisting of equal parts of 0.02 M formic acid in water and 0.02 M formic acid in acetonitrile. The critical method parameters, including mobile phase pH, column temperature, and flow rate, were optimized using the design of experiments. Statistical analysis revealed the impact of these parameters on critical quality attributes. Perturbation and response surface plots illustrated the individual and interactive effects of the parameters. The optimal parameter levels were determined to be pH,2.5; column temperature, 28°C; and flow rate, 0.55 mL/min. Confirmation experiments demonstrated the method's robustness, with the separation of impurities and unknown degradation products within a 5-min runtime. The optimized ultra-performance liquid chromatography method was validated according to ICH guidelines. The method exhibited linear response within the range of 0.025-100 μg/mL for ibrutinib and 0.0187-0.225 μg/mL for impurities (r2 > 0.9995), with limits of detection/limits of quantification of 0.01/0.025 and 0.015/0.0187 for ibrutinib and four impurities, respectively. Recoveries for the drug and impurities ranged from 92.69 to 102.7%, and precision was below 2% and 8% relative standard deviation for ibrutinib and impurities, respectively.

Combined continuous nanoparticle synthesis with chromatographic size classification

In this paper, we report a combination of the continuous flow synthesis of gold nanoparticles (AuNPs) with subsequent purification and narrowing of the particle size distribution (PSD) by size-exclusion chromatography (SEC) by adapting the flow rates of synthesis and classification. First, we show scalability of chromatographic classification with respect to column dimension and the absence of irreversible nanoparticle adhesion on the column material. Two different syntheses lead to a large and widely distributed and a small and narrowly distributed AuNP dispersion, which are classified by a semipreparative column. The PSDs of individual fractions are characterized by analytical SEC. The broadly distributed AuNP dispersion was classified into three fractions with distinct PSDs. For the narrowly distributed AuNPs, the separation is almost independent of the mobile phase flow rate: coarse and fine fractions with almost identical PSDs and separation efficiency curves are observed irrespective of the flow rate. Even NP samples with narrow PSDs can be classified into multiple fractions with tailored PSDs while simultaneously removing dissolved impurities from the dispersion. With our study, we demonstrate the potential of a direct combination of continuous NP synthesis with chromatographic classification for the optimization of final PSDs and the simultaneous purification of nanoparticulate dispersions.