Reversed-phase Ultra-performance Liquid Chromatography Research Articles

190 Metabonomics in Chronic Venous Disease - Local and Systemic Phenotypes

Abstract Aim Chronic venous disease (CVD) describes a spectrum of clinical presentations associated with a poorly functioning venous system, ranging from asymptomatic venous disease to varicose veins, skin changes and venous leg ulceration. These conditions negatively impact on quality of life. There is evidence that ‘local’ blood sampled from lower limb veins contains different biomarkers to systemic blood, particularly with respect to inflammatory and prothrombotic mediators; however, the literature pertaining to metabolic phenotyping platforms is limited. The aim of this study was to assess whether the metabolic signatures in systemic serum differed from the signatures in truncal/varicose vein (local blood) in patients with CVD. Method 64 patients presenting with CVD were recruited to participate in the study following clinical and duplex ultrasound assessment. Blood samples were collected from the antecubital fossa and from the lowermost refluxing vein or varicosity in the leg. Metabolic profiling was performed using Reversed-Phase Ultra Performance Liquid Chromatography (RP-UPLC) lipid profiling analysis. Univariate and multivariate statistical analysis were performed. Results Principal component analysis (PCA) of overall spectral data showed no separation amongst metabolites. Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) revealed preliminary group differences between leg and arm samples, but this was not significant upon CV-ANOVA testing (P = 1) Conclusions The analysis via RP-UPLC did not identify significant differences in the metabolic profile of systemic and local serum samples; however, this may be due to the limited sample size, or the platform employed. Future work will include performing the analysis employing complimentary metabolic phenotyping platforms and a larger sample size.

NEW METHOD FOR THE SIMULTANEOUS ESTIMATION OF SIPONIMOD AND PONESIMOD BY USING ULTRA PERFORMANCE LIQUID CHROMATOGRAPHIC SYSTEM IN ACTIVE PHARMACEUTICAL INGREDIENT FORM

The aim of this study is to create and validate a fast, easy-to-use, affordable, sensitive, and accurate method for measuring Siponimod and Ponesimod in bulk and pharmaceutical products using Reversed-Phase Ultra-Performance Liquid Chromatography (RP-UPLC). A Luna C18 column was used with a running phase composed of 0.1% trifluoroacetic acid: acetonitrile (30:70 v/v) at a flow rate of 1.0 ml/min. UV detection was used at a wavelength of 230 nm. Ponesimod and Siponimod correlation coefficients were found to be 0.999 over a concentration range of 5-75 µg/ml and 0.5-7.5 µg/ml, respectively. Ponesimod and Siponimod have respective retention times of 1.751 and 3.203 minutes. The run time for separating Ponesimod and Siponimod peaks was 5 minutes. The validation results agreed with what's acceptable and had good limits. This method, proposed as a regular analysis and quality control tool for medications that contain these active drugs either individually or in combination, was evident to be a suitable one.

A Chemometric Strategy in the Development of a RP-UPLC Method for the Quantitative Resolution of a Two-Component Syrup Formulation.

A novel chemometric strategy was implemented in the development of a new ultraperformance liquid chromatography method for the quantitative estimation of guaifenesin and pseudoephedrine hydrochloride in a two-component syrup formulation with minimal experimental effort, time and reagent. A full factorial design with three factors was investigated to find optimal working conditions of chromatographic factors (column temperature, flow rate, and 0.1M H3PO4% in mobile phase) that affect the chromatographic separation. Then, optimum experimental conditions providing adequate separation of the analyzed drug substances within the short runtime were determined. Under optimal experimental conditions, the retention times for guaifenesin and pseudoephedrine hydrochloride were obtained as 0.817 and 1.430min, respectively. In the optimized RP-UPLC method, chromatographic response was reported as a linear function of concentration between 5.0 and 80.0μg/mL for guaifenesin and 10.0-90.0μg/mL for pseudoephedrine hydrochloride. The proposed method was carefully validated and successfully applied to quality control and analysis of a cough syrup preparation containing guaifenesin and pseudoephedrine hydrochloride. Consequently, the proposed reversed-phase ultraperformance liquid chromatography method provided an opportunity to quantify relevant drugs with small amount of reagents and short runtime.

Quantification of Lipid and Peptide Content in Antigenic Peptide-loaded Liposome Formulations by Reversed-phase UPLC using UV Absorbance and Evaporative Light Scattering Detection

Antigenic peptide-loaded cationic liposomes have shown promise as cancer vaccines. Quantification of both peptides and lipids is critical for quality control of such vaccines for clinical translation. In this work we describe a reversed phase ultra-performance liquid chromatography (RP-UPLC) method that separates lipids (DOTAP, DOPC and their degradation products) and two physicochemically different peptides within 12 min. Samples were prepared by dilution in a 1:1 (v/v) mixture of methanol and water. Peptide quantification was done via UV detection and lipids were quantified by an evaporative light scattering detector (ELSD), both coupled to the RP-UPLC system, with high precision (RSD < 3.5%). We showed that the presence of lipids and peptides did not mutually influence their quantification. Limit of detection (LOD) and limit of quantification (LOQ), as determined in the ICH guidelines, were 6 and 20 ng for DOTAP, 12 ng and 40 ng for DOPC, 3.0 ng and 8.0 ng for peptide A and 2.4 ng and 7.2 ng for the more hydrophobic peptide B. Finally, lipid degradation of DOTAP and DOPC was monitored in peptide loaded DOTAP:DOPC liposomes upon storage at 4 °C and 40 °C.

Stability-Indicating New RP-UPLC Method for Simultaneous Determination of a Quaternary Mixture of Paracetamol, Pseudoephedrine, Chlorpheniramine, and Sodium Benzoate in (Cold-Flu) Syrup Dosage Form.

A combination of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form is specified for the treatment of common cold and flu symptoms. The functional role of this study is to develop a novel, reliable, and selective stability-indicating reversed-phase ultra-performance liquid chromatography (RP-UPLC) method for simultaneous identification of a quaternary mixture of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form. The specific method is accomplished using an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm), 1.8 µm particle size with pore size 100 Å, utilizing a mixture of purified water-methanol-trifluoroacetic acid (72.5:27.5:1.5, v/v) as the mobile phase at a flow rate of 0.3 mL/min. The column void volume is 1.15 min. UPLC detection is adjusted at 205 nm using a photodiode array detector. Calibration curves are obtained in the linearity ranges: 25-500 µg/mL for paracetamol, 10-50 µg/mL for pseudoephedrine, 0.5-5 µg/mL for chlorpheniramine, and 3-30 µg/mL for sodium benzoate with a correlation coefficient > 0.9992. The mean recovery of the developed method is tested and shows good recovery results between 99-101%; selectivity and forced degradation studies are investigated as per the International Council for Harmonisation Guidelines and no interference is detected due to degradation peaks. The proposed stability-indicating UPLC method for simultaneous determination of the three drugs, paracetamol, pseudoephedrine, and chlorpheniramine, with a preservative sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form is successfully accomplished, developed, and validated, and can be easily used in the analysis of drugs in pure or dosage form. The novelty of the current research work lies in the development of the UPLC method for simultaneous determination of a quaternary mixture of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form.

Detection and Quantification of Two Potential Genotoxic Impurities in Telbivudine Active Pharmaceutical Ingredient by Targeted Ultra Performance LC-MS/MS Analysis

A new selective, rapid and highly sensitive method using reversed-phase ultra-performance liquid chromatography (RP-UPLC) coupled with mass analyzer was developed for the detection and quantification of two genotoxic impurities viz. 4-dimethyl aminopyridine (GTI-1) and 2-bromo-3,5-diacetyl thymidine (GTI-2) in Telbivudine drug substance. The separation of two genotoxic impurities was accomplished using Kromasil C8 column (100 mm × 4.6 mm, 3.5 μm) maintained at 45 ºC using formic acid (0.1%) as buffer solution and gradient grade acetonitrile with 0.5 mL/min flow rate. Both GTIs were quantified using positive electrospray ionization (ESI) mode on MS/MS Analyser. ICH guidelines were followed for validation of genotoxic impurities. % RSD for system precision was found to be 2.71 and 2.94 and the overall %RSD for ruggedness was 3.11 and 2.89, respectively for GTI-1 and GTI-2. Two GTIs and Telbivudine drug were well resolved at retention times: 1.76 min for GTI-1, 3.81 min for Telbivudine and 6.62 min for GTI-2. The linearity relationship holds good in the range of concentrations 0.5 ppm to 7.5 ppm for both the impurities with a correlation coefficient of 0.999. Accuracy ranged from 95.1% to 105.1%. The GTI-1 and GTI-2 were shown, respectively detection limits: 0.07 ppm and 0.09 ppm and quantification limits: 0.22 ppm and 0.27 ppm and the solutions were stable upto 24 h at room temperature. The proposed and validated method is sensitive, reproducible, specific and linear and can be successfully applied for the quantification of both genotoxic impurities in Telbivudine active pharmaceutical ingredient and formulation.

Development and Validation of Novel Rapid Stability Indicating Mass Compatible UPLC Method for the Simultaneous Estimation of Assay and Related Substances of Remdesivir in Bulk and Injectable Dosage Form

Developed and validated a simple, rapid, new mass compatible reverse phase ultra-performance liquid chromatography (UPLC) method for the simultaneous estimation of assay and related substance of remdesivir in bulk and injectable dosage form. The developed method is capable of separating all the potential process and degradation impurities within 15 min. Chromatography was carried out using Waters Acquity BEH C18 column (50 mm length, 2.1 mm internal diameter and 1.7 μ particle size) with gradient elution. Ghost buster column (30 mm length, 2.1 mm internal diameter) was used as inline filter to suppress the response of peaks originating from mobile phase. Mobile phase A composed of 10 mM ammonium acetate buffer pH 3.5 and acetonitrile in the ratio of 90:10 v/v and mobile phase B composed of 10 mM ammonium acetate buffer pH 3.5 and acetonitrile in the ratio of 10:90 v/v. The flow rate of the mobile phase was set at 0.35 mL/min and the column temperature was maintained at 35 ºC. The detection was carried out using UV detector at 245 and 270 nm. Method validation was performed according to ICH guidelines. The validated method is simple, rapid, mass compatible, economical, specific, accurate, linear, precise, rugged, sensitive, robust and stability indicating. This method can be employed for the simultaneous estimation of assay and related substance of remdesivir in bulk and injectable dosage forms at quality control laboratories of pharmaceutical industries.

A Novel Ultra Performance Liquid Chromatographic Method for the Estimation of Lercanidipine in Bulk and Tablet Dosage Form

Aim: In this present study an accurate reverse phase ultra-performance liquid chromatography (RP-UPLC) method has been developed, validated and applied to stability indicating studies to determine Lercanidipine HCL in bulk and marketed dosage form.&#x0D; Methods: Optimized chromatographic conditions were achieved by using Waters Acquity BEH C18 (2.1 x 50mm, 1.7m) UPLC column. Empower 2 is a software, dihydrogen Orthophosphate Buffer : Methanol (40 : 60) as eluent at flow rate 0.3 ml/min. PDA detection was performed at 254nm.&#x0D; Results: The developed method was validated and stability study was conducted as per ICH guidelines. The retention time was found at 0.503 min. The method shows linearity over a range of 1 μg/ml to 60 μg /ml with the obtained correlation coefficient is 0.999. The LOD and LOQ values were found 0.025 and 0.05 μg /ml. The acidic and peroxide stressed study shows more degradation of 6.23% and 3.03%.&#x0D; Conclusion: The present developed method was found stability indicating, reliable, validated method was applied for the routine analysis of lercanidipine in bulk drug and the pharmaceutical formulations.

Production, characterization, and bioactivity of novel camel milk-based infant formula in comparison to bovine and commercial sources

The study reports formulation, characterization and in-vitro digestion of novel camel milk-based infant formula (IF) in comparison to bovine and commercial IF. Camel milk IF was further fortified with camel casein or whey at different levels to study the effect of different protein supplementations. A simulated infant gastrointestinal digestion (SIGID) model was used to compare the digestibility of different IFs. Protein digestibility of various IFs were studied through degree of hydrolysis (DH), sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and reverse-phase ultra-performance liquid chromatography (RP-UPLC). Upon SIGID, camel milk IF fortified with 10% whey showed the highest DH (3092.2 μg NH3 g−1) followed by camel milk IF (1308.16 μg NH3 g−1) indicating significant hydrolysis of the protein fraction. The fate of proteins and fats after SIGID was also studied using fluorescence microscopy that reflected homogenous breakdown of the proteins and lipid fractions. Furthermore, the effect of SIGID on bioactive properties was also analyzed via antioxidant (ABTS and DPPH radical scavenging activities) and anti-inflammatory activities. After SIGID, camel IF samples fortified with whey and casein proteins showed superior radical scavenging and anti-inflammatory activities. The study concludes that camel milk can be a potential alternative for the development of infant formula with additional health benefits.

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles.

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Multidimensional Mass Spectrometry of Multicomponent Nonionic Surfactant Blends.

Ultraperformance liquid chromatography (UPLC) and ion mobility (IM) spectrometry were interfaced with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize a complex nonionic surfactant mixture. The surfactant was composed of a glycerol core, functionalized with poly(ethylene oxide) units (PEOn) that were partially esterified by caprylic and/or capric acid. Reversed-phase UPLC classified the blend based on polarity into four groups of eluates, corresponding to compounds with zero, one, two, or three fatty acid residues. Additional separation within each eluate group was achieved according to the length of the fatty acid chains. Coeluting molecules of similar polarity were dispersed in the gas phase by their collision cross section in the IM dimension. Performed in series, UPLC and IM allowed for the separation and detection of several isomeric and isobaric blend constituents, thereby enabling their isolation for conclusive MS/MS analysis to confirm or elucidate their primary structures and architectures (overall four-dimensional, 4D, characterization).

Analytical Method Development and Validation for the Analysis of Nepafenac and its related substances using Ultra Performance Liquid Chromatography

A novel, economic and time-efficient reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method has been developed for the analysis of Nepafenac in the presence of both impurities and degradation products generated by forced degradation. When Nepafenac was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and thermal stress, observed degradation only in oxidative and base hydrolysis. The drug was found to be stable to other stress conditions. Various method development trails were performed for the separation of drug from impurities. However, best chromatographic separation was achieved on a Waters Acquity CSHC18, 100mm x 2.1mm, 1.7µ particle size column, UV detection at 245nm, a gradient elution of Ammonium formate (pH 4.0), mixture of organic solvents (Acetonitrile, Methanol) as mobile phase for drug, its impurities and it was captured. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of Nepafenac. Total elution time was about 6.5 min and equilibration time of about 1.5 min which allowed analysis of more than 100 samples per day. The analytical method discussed in United states Pharmacopoeia was pH sensitive and compatible to LC-MS analysis.

First Determination of Glycidyl Ester Species in Edible Oils by Reverse-Phase Ultra-Performance Liquid Chromatography Coupled with an Evaporative Light-Scattering Detector.

In this work, a new ultra-performance liquid chromatograph-evaporative light-scattering detector (UPLC-ELSD) method for quantitation of glycidyl esters (GE) contents in edible oils is presented. The method features complete separation of five GE species within 20 min by a C18 column and gradient elution with a mobile phase consisting of 85% and 2.5% methanol aqueous solutions. The coefficients of regression (R2) were all ≥0.9999 for the linear-quadratic regression curves of GE species in a concentration range of 5~80 μg/mL. The intraday and interday recoveries (%) of GE species in solvent were in a range of 81.3~107.3%, and the intraday and interday coefficients of variation (CVs, %) were all ≤8.6%. The average recovery (%) of GE species spiked in extra-virgin olive oil samples ranged from 88.3~107.8% and the intermediate precision (CV, %) of ≤14% indicated acceptable accuracy and precision. The method exhibited limit of quantification (LOQ) for each GE species (0.6 μg glycidol equivalents/g oil). The method was applied to determine GE concentrations of six commercial oil samples, and total glycidol equivalents were consistent with data obtained by GC-MS method. This UPLC-ELSD method could be adopted for precursory screening and research purposes to improve food safety when MS detectors are unavailable.

Development of new stability indicating UPLC-UV method for the extraction and quantification of perindopril and indapamide from human plasma

BackgroundThe hypertension and cardiovascular ailments are the leading cause of deaths worldwide. The combination therapy was found to be effective on the cardiovascular illness by reducing the blood pressure. The indapamide and perindopril combination therapy showed excellent results on reducing high blood pressure. With this in mind, the stability indicating reverse phase UPLC method was developed for the simultaneous identification and quantification of indapamide and perindopril from human plasma. In this work, we developed a new solid phase extraction method for the extraction of indapamide and perindopril in human plasma. It is a simple, accurate, and selective method for the extraction of these two drugs from human plasma with elution time of 2 min. The extracted drugs were identified and quantified by using stability indicating UPLC method. The method showed high recovery rate as well as low detection and quantification limits of two drugs.ResultsA novel, simple, highly accurate, and precise stability indicating ultra-performance liquid chromatography (UPLC) method was developed for the identification and quantification of perindopril (PP) (brand name Coversyl) and indapamide (IP) (brand name Lorvas) from human plasma. In this UPLC method, HSS C18 column (100 × 2.1 mm, 1.8 μm) and mobile phase acetonitrile (ACN), 10 mM KH2PO4 buffer solution (pH 3.0) mixture was used in the ratio of 65:35. Colum temperature of 30 °C, flow rate of 1.0 mL per minute and UV wave length of 254 nm were used. PP and IP were eluted below 2 min runtime with high resolution. Solid phase extraction (SPE) method was used for the extraction of PP and IP from human plasma. Different solvents were used to extract the analyte from SPE such as ACN, methanol, acetone, tertiary butyl diethyl ether (TBDE), chloroform (CHCl3), and ethanol (EtOH). Among these, ACN gave good recovery percentages (94.56 to 101.58%). From the linearity graph, good correlation coefficient values of 0.9996 for PP and 0.9997 for IP were achieved. The coefficient variance values for intra and inter day precision is in between 1.08 and 12.5%. The LOD and LOQ values were determined by the signal to noise ratio method. LOD and LOQ values for IP and PP were found to be 8.6 and 33.5 ng/mL and 28.33 and 110.5 ng/mL respectively. The developed method was statistically validated as per ICH guidelines.ConclusionIn summary, a novel stability indicating UPLC-UV method was developed and validated for the simultaneous identification and quantification of perindopril and indapamide drugs in human plasma and tested the stability as per ICH guidelines. It is a simple, accurate, and specific method for the extraction of these two drugs from human plasma and eluted within 2 min runtime. The method showed high recovery rate as well as low detection and quantification limits of two drugs. The developed method is suitable for routine analysis as well as in bioanalytical and clinical studies.Graphical abstract

HA1 (Hemagglutinin) quantitation for influenza A H1N1 and H3N2 high yield reassortant vaccine candidate seed viruses by RP-UPLC

The only effective measure to decrease morbidity and mortality caused by the influenza virus in the human population is worldwide vaccination. Vaccination produces neutralizing antibodies that target the HA1 subunit of the HA (hemagglutinin) protein and are strain specific. The effectiveness of new influenza vaccines are linked to two factors, the correct prediction of the circulating strains in the population in a particular season and the concentration of the HA1 protein in the vaccine formulation. With the advent of the licensing of quadrivalent vaccines, pharmaceutical manufacturers are under considerable pressure due to time constraints and dedicated resources to deliver 194–198 million doses (2020–2021 U.S. market) of vaccine. Considering the valuable resources needed to produce the influenza vaccine in a timely manner, the efficient quantitation of the HA1 protein (the main component in the influenza vaccine) is required. Currently the only method approved by regulatory agencies for quantitation of the HA antigen in vaccines is the single radial immunodiffusion assay (SRID), an antibody dependent assay that is not time efficient. Time efficient methods that are antibody independent e.g. reverse phase-high performance liquid chromatography (RP-HPLC) or size exclusion-HPLC (SE-HPLC) are available. An improved method implementing reverse phase-ultra performance liquid chromatography (RP-UPLC) has been developed to quantitate the HA1 protein antigen present in the high yield reassortant vaccine seed viruses from influenza A H1N1 and H3N2 subtypes harvested from inoculated embryonated chicken eggs. This method differentiates between high yield and lower yielding reassortants in order to select the best vaccine candidate seed virus with the highest growth ‘in ovo’. This direct capability to monitor the HA1 concentration of potential reassortant seed viruses and to choose the best yielding HA influenza reassortant when faced with multiple viral seed candidates provides a major advantage on the industrial scale to the influenza vaccine process.

Stability indicating analytical method development and validation of eltrombopag olamine in tablet dosage form by RP-UPLC

A simple, precise and accurate stability indicating isocratic Reverse Phase Ultra-Performance Liquid Chromatographic (RP-UPLC) method was developed for quantitative determination of Eltrombopag olamine in the presence of degradant products. The method was developed using Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) column with mobile phase containing a mixture of water (pH 3 with 30 % formic acid) and acetonitrile in ratio 30:70. The eluted compound was monitored at 244 nm and run time was within 2 min. Eltrombopag olamine was subjected to the stress condition of oxidative, acid, base, thermal and photolytic degradation. The drug was found to degrade significantly in acid, base and oxidative stress condition and stable in thermal and photolytic degradation condition. The degradation products were well resolved from main peak, providing stability indicating power of the method. The developed method was validated as per ICH Q2(R1) guideline with Linearity range was between 25-75 μg/ml, % Recovery range was found as 99.8-101.9 and Precision was found as RSD less than 2.

Ultra-performance liquid chromatography (UPLC) for the determination of organic acids – The intermediates of branched-chain amino acid biosynthesis in Escherichia coli strains

Simple and rapid reverse-phase ultra-performance liquid chromatography with UV-detection (RP-UPLC-UV) was developed and validated for the simultaneous analysis of 2-isopropylmalate (2-IPM), 3-isopropylmalate (3-IPM), ketoisovalerate (KIV), ketoisocaproate (KIC), and α-keto-β-methylvalerate (KMV) in the culture fluid (CF) of branched-chain amino acid (BCAA) Escherichia coli strain producers. The RP-UPLC-UV method was developed using a Waters Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with a mobile phase consisting of phosphate monobasic salt adjusted to a pH of 3.5 and methanol solution. The flow rate of the mobile phase was 0.5 mL min−1 with gradient elution. The RP-UPLC-UV method was compared with reverse-phase high-performance liquid chromatography with UV-detection (RP-HPLC-UV), ion-exchange high-performance liquid chromatography with UV-detection (IE-HPLC-UV), and ion-exchange high-performance liquid chromatography with electrochemical detection (IE-HPLC-ECD) and a postcolumn buffer for organic acid analysis. The proposed RP-UPLC-UV method was highly sensitive (LOD for KIV was 2.5 × 10−3 mg mL−1, for 2-IPM – 4.5 × 10−3 mg mL−1, for 3-IPM – 4.4 × 10−3 mg mL−1, for KIC – 3.4 × 10−3 mg mL−1, and for KMV – 2.8 × 10−3 mg mL−1) and successfully applied for the analysis of organic acids in the CF of E. coli strain producers.

Stability-indicating Reversed Phase-Ultra Performance Liquid Chromatography Method Development and Validation for Simultaneous Determination of Encorafenib and Binimetinib in Formulation

A simple, accurate and precise stability indicating method was developed for the simultaneous estimation of the encorafenib (ECRB) and binimetinib (BMTB) in a dosage form by UPLC. Chromatographic elution was processed through a HSS C18 (100 x 2.1 mm, 1.8m) reverse phase column and the mobile phase composition of 0.01N KH2PO4 buffer (3.5 pH) and acetonitrile in the proportion of 55:45 was processed thru a column at a flow rate of 1.0 ml/min. Temperature of the column oven was kept at 30.0°C and the wavelength maximum of detection system was set to 294 nm. Retention times of ECRB and BMTB were found to be 0.767 min and 1.130 min respectively. Repeatability of the method was determined in the form of %RSD and findings were 0.3 and 0.6 for ECRB and BMTB respectively. The percentage recovery of the method was found to be 99.59% and 99.70% for ECRB and BMTB respectively. LOD, LOQ values obtained from regression equations of ECRB and BMTB were 0.51, 1.55mg/ml and 1.47, 4.44 mg/ml respectively. Regression equation of ECRB was y = 6684.x + 18102 and BMTB was y = 13118x + 2159. Two analytes were subjected for acid, peroxide, photolytic, alkali, neutral and thermal degradation studies and the results shown that the percentage of degradation was found between 0.76% and 6.88%. Retention times and total run time of two drugs were decreased and the developed method was simple and economical. So, the developed method can be adopted in industries as a regular quality control test for the quantification of ECRB and BMTB.

Lipidomic UPLC-MS/MS Profiles of Normal-Appearing White Matter Differentiate Primary and Secondary Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a neurodegenerative inflammatory disease where an autoimmune response to components of the central nervous system leads to a loss of myelin and subsequent neurological deterioration. People with MS can develop primary or secondary progressive disease (PPMS, SPMS) and differentiation of the specific differences in the pathogenesis of these two courses, at the molecular level, is currently unclear. Recently, lipidomics studies using human biofluids, mainly plasma and cerebrospinal fluid, have highlighted a possible role for lipids in the initiation and progression of MS. However, there is a lack of lipidomics studies in MS on CNS tissues, such as normal-appearing white matter (NAWM), where local inflammation initially occurs. Herein, we developed an untargeted reverse phase ultra-performance liquid chromatography time of flight tandem mass spectrometry (RP-UPLC-TOF MSE)-based workflow, in combination with multivariate and univariate statistical analysis, to assess significant differences in lipid profiles in brain NAWM from post-mortem cases of PPMS, SPMS and controls. Groups of eight control, nine PPMS and seven SPMS NAWM samples were used. Correlation analysis of the identified lipids by RP-UPLC-TOF MSE was undertaken to remove those lipids that correlated with age, gender and post-mortem interval as confounding factors. We demonstrate that there is a significantly altered lipid profile of control cases compared with MS cases and that progressive disease, PPMS and SPMS, can be differentiated on the basis of the lipidome of NAWM with good sensitivity, specificity and prediction accuracy based on receiver operating characteristic (ROC) curve analysis. Metabolic pathway analysis revealed that the most altered lipid pathways between PPMS and SPMS were glycerophospholipid metabolism, glycerophosphatidyl inositol (GPI) anchor synthesis and linoleic acid metabolism. Further understanding of the impact of these lipid alterations described herein associated with progression will provide an increased understanding of the mechanisms underpinning progression and highlight possible new therapeutic targets.

Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation.

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a KD of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichiacoli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.