Reversed-phase Ultra-performance Liquid Chromatography Research Articles

Green and Sensitive Analysis of the Antihistaminic Drug Pheniramine Maleate and Its Main Toxic Impurity Using UPLC and TLC Methods, Blueness Assessment, and Greenness Assessments

For the first time, two direct and eco-friendly chromatographic approaches were adapted for the simultaneous estimation of pheniramine maleate (PAM) and its major toxic impurity, 2-benzyl pyridine (BNZ). Method A used reversed-phase ultra-performance liquid chromatography; separation was achieved within 4 min using a C18 column with a developing system of methanol/water (60:40 v/v) with a 0.1 mL/min flow rate. Photodiode array detection was adjusted at 215 nm. The method was linear in the ranges of 5.0–70.0 and 0.05–10.0 µg/mL for PAM and BNZ, correspondingly. Method B used thin-layer chromatography; separation was applied on silica gel TLC F254 using ethanol/ethyl acetate/liquid ammonia (8:2:0.1, in volumes) at room temperature, at 265 nm. Linearity was assured at concentration ranges 0.5–8.0 and 0.1–3.0 µg/band for the two components, respectively. Generally, the new UPLC and TLC methods outperform the old ones in terms of quickness, greenness, and sensitivity. Concisely, the greenness features were partially achieved using the Green Analytical Procedure Index (GAPI) and the Analytical Greenness (AGREE) pictograms. In contrast, the usefulness of the novel approaches was assured via the Blue Applicability Grade Index (BAGI) tool.

A-317 Development of a patient-focused volumetric absorptive microsampling strategy to assess urinary amino acid excretion

Abstract Background Biochemical determinations of urinary amino acid excretion patterns can diagnose specific inborn errors of metabolism (IEM). Clinical management of these genetic metabolic patients may require frequent urine amino acid tests. A simplified process for transporting home collected urine specimens would benefit the IEM community. Volumetric absorptive microsampling (VAMS) is an emergent alternative sampling technique for the collection of dried biological specimens. We describe the development and preliminary validation results of a VAMS strategy for the analysis of creatinine and amino acids (N=22) from microsampled dried urine. Methods Urine creatinine and amino acids were respectively measured in a clinical laboratory setting using established assays on a CobasPro (Roche) and a MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Two 30 µL VAMS tips from a Mitra device (Neoteryx) were used to sample each random urine specimen. An ultrasonication-based solvent extraction procedure was optimized to extract the amino acids and creatinine from the VAMS tips. Solvent extracts were subsequently dried by centrifugal evaporation. Extract from one tip was re-dissolved in water and tested for creatinine using an enzymatic method. The other extract was re-dissolved in 0.02 N sodium hydroxide and tested according to the MassTrak AAA procedure, which deploys pre-column AccQTag derivatization with reversed-phase UPLC on a C18 column (2.1 × 150 mm; 1.7 µm) and UV detection at 260 nm. Accuracy, within-batch precision and short-term stability of the optimized VAMS protocol were determined. Results Extraction solvent with a 60:40 (v/v) water:methanol mixture provided the highest average amino acid recovery. This extraction solvent produced higher average amino acid recoveries at pH 11 (89±13%), than pH 3 (82±10%) or pH 7 (72±11%). Creatinine measured from the VAMS tips (N=44) had a consistent negative bias of 20% relative to neat urine (R=0.9905, slope=0.802, intercept=0.02). The creatinine corrected urine amino acid results from the Mitra device were corrected for this observed concentration bias. The accuracy of the amino acid results derived from the VAMS tip vs neat urine was 104 ± 14%. The average within-batch precision (CV%) of the amino acids was 9.8% (range=6.7% (cystine) to 17.6% (arginine)) and 6.2% for creatinine. The average 1 week storage stability at ambient temperature was 91% (range=73% (ornithine) to 107% (glycine)) for the amino acids and 107% for creatinine. The devised VAMS strategy was applied to the analysis of microsampled dried urine from a patient with cystinuria (OMIM no. 220100). Marked elevations in arginine, cystine, lysine and ornithine were respectively observed and are consistent with the biochemical diagnosis of the patient. Conclusions Creatinine corrected amino acid results from the devised VAMS strategy exhibit good precision and accuracy when compared to tradition analyses of neat urine. The potential use of microsampled dried urine for the diagnosis and monitoring of selected IEM has been demonstrated. The analytical and clinical performance of this VAMS protocol must be further validated, including the use of microsampled dried urine from home specimen collections.

Characterization, quantitation, and natural variability of phenolics in Rhododendron arboreum leaves of western Himalayan origin using UPLC-PDA and UHPLC-ESI-Q/TOF-MS/MS

Rhododendron arboreum Sm. is very popular in the Indian Himalayan region due to its significant ethnomedicinal importance and potential commercial applications. Apart from the ornamental value derived from its vibrant red flowers, fresh flowers are used to prepare medicinal juices, and dried are utilized as spices. Despite being a rich source of phenolics, these lack identified marker chemicals and validated methods. The present work encompasses the chemical investigation of R. arboreum leaves using UHPLC-ESI-Q/TOF-MS/MS. The chemotypic variability was assessed through a reverse phase ultra-performance liquid chromatography method coupled with a photodiode array detector (RP-UPLC-PDA) chemical signatures of R. arboreum leaves from different altitudes combined with chemometric analysis. The chromatographic method was developed and validated for the quantitative analysis of three vital secondary metabolites, i.e., Isoquercetin or Quercetin-3-O-glucoside (Qg), Reynoutrin or Quercetin-3-O-xyloside (Qx), and Quercitrin or Quercetin-3-O-rhamnoside (Qr)The hyphenation of the method with ESI-Q/TOF-MS/MS has confirmed the distribution of 11 chemicals (nine known and two unknown) in R. arboreum leaves belonging to the phenolic and hydrolyzable tannins class. Samples from higher altitudes >3000 m (above mean sea level) were exceptionally rich in Quercitrin. The chemometric investigation, comprising hierarchical cluster analysis (HCA), principal component analysis (PCA), and partial least square method-discriminant analysis (PLS-DA), successfully categorized thirty-three samples from diverse geographic regions into two distinct categories. This current study provides a validated method for the chemotypic discrimination and quality assessment of the R. arboreum leaves from Uttarakhand, Western Himalayan region of India, and laid the foundation for the comprehensive utilization of these renewable and sustainable resources.

Innovative UPLC technique for concurrent quantification of etofenamate and benzyl nicotinate in the presence of methylparaben and benzyl alcohol in their topical cream: Greens, white, and Six Sigma methodologies.

The efficacious treatment of muscle and joint pain relies heavily on etofenamate (ETO) and benzyl nicotinate (BN), which possess robust anti-inflammatory and pain-relieving properties when paired with methylparaben (MP) or benzyl alcohol (BA). In this study, we have established and validated innovative RP-UPLC methods for assessing ETO and BN in the presence of MP or BA in their dosage forms, employing eight green tools to evaluate their eco-friendliness and effectiveness. Reversed phase-ultra-performance liquid chromatography (RP-UPLC) technique employs a flow rate of 0.3mL/min on Waters Acquity UPLC BEH Column (C18, 1.7μm, 100 mm × 2.1mm), detection at 254 nm using a photo diode array (PDA) detector and mobile phase of 0.05 M KH2PO4 buffer, acetonitrile, and methanol (50:15:35, v/v/v) adjusted pH6.0 with 0.2% triethylamine. For ETO, BN, MP, and BA, the calibration curves were linear and ranged from 0.005 to 1.0, from 0.001 to 0.2, from 0.002 to 0.08, and from 0.0001 to 0.1mg/mL, respectively. The correlation value was 0.9999, and the accuracy findings ranged from 98.81% to 100.56%. Consequently, the methodology has been successfully implemented in assay testing for the pharmaceuticals in the presence of the MP or BA, demonstrating the high selectivity of these approaches. The present study presents the Blue Applicability Grade Index (BAGI), an innovative approach that complements green metrics in practical white analytical chemistry. According to the International Council for Harmonisation (ICH) criteria, the procedures were effectively validated.

β-Aminobutyric acid promotes stress tolerance, physiological adjustments, as well as broad epigenetic changes at DNA and RNA nucleobases in field elms (Ulmus minor)

Backgroundβ-Aminobutyric acid (BABA) has been successfully used to prime stress resistance in numerous plant species; however, its effectiveness in forest trees has been poorly explored thus far. This study aimed to investigate the influence of BABA on morphological, physiological, and epigenetic parameters in field elms under various growth conditions. Epigenetic changes were assessed in both DNA and RNA through the use of reversed-phase ultra-performance liquid chromatography (UPLC) coupled with sensitive mass spectrometry.ResultsThe presented results confirm the influence of BABA on the development, physiology, and stress tolerance in field elms. However, the most important findings are related to the broad epigenetic changes promoted by this amino acid, which involve both DNA and RNA. Our findings confirm, for the first time, that BABA influences not only well-known epigenetic markers in plants, such as 5-methylcytosine, but also several other non-canonical nucleobases, such as 5-hydroxymethyluracil, 5-formylcytosine, 5-hydroxymethylcytosine, N6-methyladenine, uracil (in DNA) and thymine (in RNA). The significant effect on the levels of N6-methyladenine, the main bacterial epigenetic marker, is particularly noteworthy. In this case, the question arises as to whether this effect is due to epigenetic changes in the microbiome, the plant genome, or both.ConclusionsThe plant phenotype is the result of complex interactions between the plant’s DNA, the microbiome, and the environment. We propose that different types of epigenetic changes in the plant and microbiome may play important roles in the largely unknown memory process that enables plants to adapt faster to changing environmental conditions.

Formation of Micelles by Nonionic Detergent Molecules Leads to the Breakthrough Peak in Reversed-Phase Ultraperformance Liquid Chromatography (UPLC).

A peculiar phenomenon known as "breakthrough" occurs under reversed-phase ultraperformance liquid chromatography (UPLC) conditions and has been under scrutiny for decades. This effect takes place when a large volume of analyte solution, prepared in a solvent with an eluotropic strength significantly higher than that of the initial mobile phase solvent, is injected. According to the literature, under specific experimental conditions, a substantial portion of solutes is carried by the mobile phase and detected near the dead time of the chromatographic system. This phenomenon is typically observed when the injected volume of a particular analyte is sufficiently large. However, the underlying physicochemical principles governing this phenomenon have remained elusive. We present evidence demonstrating that breakthroughs can occur even when injecting a sample of a neat solvent devoid of any solute. By mass spectrometric analysis, we identified the breakthrough peak to represent the nonionic detergent Triton. When columns are equilibrated with water, Triton molecules, present as impurities in filtered water, accumulate on the nonpolar stationary phase. Upon the introduction of a solvent with a stronger elution strength, Triton molecules retained on the stationary phase are removed. As detergents, these Triton molecules aggregate into micelles featuring a hydrophobic inner core and a hydrophilic outer shell. These hydrophilic micelles are carried by the polar mobile phase and detected as the breakthrough peak at the dead time of the chromatographic system. When analytes are present, a portion of the injected solutes is captured by the micelles and transported with the breakthrough plug. This assertion was verified and confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis of a methanolic solution of perfluorooctanoic acid (PFOA). The mass spectra corresponding to the breakthrough plug featured a peak for the PFOA anion (m/z 413) in addition to those for Triton.

Analytical quality by design (AQbD) based optimization of RP-UPLC method for determination of nivolumab and relatlimab in bulk and pharmaceutical dosage forms

BackgroundThe Analytical Quality by Design (AQbD) methodology extends the application of Quality by Design (QbD) principles to the management of the analytical procedure life cycle, encompassing method creation, optimization, validation, and continuous improvement. AQbD assists in creating analytical procedures that are robust, reliable, precise, and cost-efficient. Opdualag™ is a combination of Nivolumab and Relatlimab, which are antibodies that block programmed death receptor-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) receptors, used to treat advanced melanoma. This work aims to develop and validate a reversed-phase ultra-performance liquid chromatography (RP-UPLC) method using AQbD principles to determine NLB and RTB in pharmaceutical products.ResultsA central composite design (CCD) comprising three factors arranged in five distinct levels was implemented via Design-expert® software to optimize the chromatographic conditions. A mathematical model was constructed and the effects of three independent factors namely flow rate (X1), percentage of methanol in the mobile phase (X2), and temperature (X3) on responses including retention time (Y1–2), resolution factor (Y3), theoretical plates (Y4–5), and tailing factor (Y6–7) were investigated. The software determined the optimal chromatographic conditions for the separation of NLB and RTB, which were as follows: 32.80% methanol in the mobile phase, 0.272 mL/min flow rate, 29.42 °C column temperature, and 260 nm UV detection. The retention time for NLB and RTB were 1.46 and 1.88 min, respectively. The method exhibited linearity across the concentration ranges of 4–24 µg/mL for RTB and 12–72 µg/mL for NLB. The limits of detection (LOD) and limit of quantification (LOQ) for NLB and RTB, respectively, were 0.89 µg/mL, 2.69 µg/mL and 0.15 µg/mL and 0.46 µg/mL. The percentage relative standard deviation (%RSD) of intraday and interday precision for NLB and RTB was below 2. The recovery percentages for NLB and RTB were determined to be 99.57–100.43% and 99.59–100.61%, respectively. Both drugs were found to be susceptible to oxidative and photolytic degradation in forced degradation studies.ConclusionsEmploying the AQbD-based methodology, a straightforward, fast, accurate, precise, specific, and stability-indicating RP-UPLC method has been established for the quantitative analysis of NLB and its RTB in pharmaceutical formulations.

Spray drying and ultrasonication processing of camel whey protein concentrate: Characterization and impact on bioactive properties

The production of whey protein concentrates (WPCs) from camel milk whey represents an effective approach to valorize this processing by-product. These concentrates harbor active ingredients with significant bioactive properties. Camel WPCs were spray-dried (SD) at inlet temperature of 170, 185 and 200°C, or Ultrasonicated (US) for 5, 10 and 15 min, then freeze-dried to obtain fine powder. The impact of both treatments on protein degradation was studied by sodium dodecyl sulfate-PAGE and reverse-phase ultraperformance liquid chromatography (RP-UPLC) techniques. Significantly enhanced protein degradation was observed after US treatment when compared with SD. Both SD and US treatments slightly enhanced the WPCs samples' antioxidant activities. The US exposure for 15 min exhibited highest 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (12.12 mmol TE/g). Moreover, US treatment for 10 min exhibited the highest in vitro anti-diabetic properties (α-amylase and α-glucosidase inhibition), and dipeptidyl-peptidase-IV inhibitory activity among all samples. In addition, the ultrasonication for 10 min and SD at 170°C showed the lowest IC50 values for in vitro anti-hypercholesterolemic activities in terms of pancreatic lipase and cholesteryl esterase inhibition. Conclusively, these green techniques can be adapted in the preservation and processing of camel milk whey into active ingredients with high bioactive properties.

Uncommon opsin’s retinal isomer is involved in mammalian sperm thermotaxis

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts—tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

A NEW RP-UPLC METHOD FOR THE SEPARATION AND SIMULTANEOUS QUANTIFICATION OF DORZOLAMIDE HCl AND TIMOLOL MALEATE

Objective: This investigation demonstrates a stability-indicating and reliable “reverse-phase ultra-performance liquid chromatography” method to simultaneously quantify timolol maleate and dorzolamide HCl in the pharmaceutical dosage form. Methods: Successful separation was accomplished using Phenyl column (100 mm x 2.1 mm, 1.7μm) with isocratic type of elution using mobile phase containing Acetonitrile+Ammonium Formate buffer (30:70), respectively with 0.2 ml/min flow rate. The wavelength sensor was attuned at 266 nm to quantify timolol maleate and dorzolamide HCl. Results: Dorzolamide HCl and timolol maleate peaks were eluted with fine resolution at retention times 0.7 min and 1.5 min, respectively. In the 55.75-334.5 μg/ml and 6.25-37.5 μg/ml concentration ranges for dorzolamide HCl and timolol maleate, the calibration graphs were linear, with regression coefficients of 0.99997 and 0.99991, respectively. The suggested ultra-performance liquid chromatography approach has been shown as sensitive, precise, robust, accurate, specific and stability, indicating through the resolution of dorzolamide HCl and timolol maleate from its degradation-based compounds. Conclusion: The established ultra-performance liquid chromatography technique was effectively extended to the evaluation of dorzolamide HCl and timolol Maleate in the pharmaceutical dosage form and the test results appeared satisfactory.

Development and validation of a stability-indicating analytical method for simultaneous determination of sodium phenylbutyrate and taurursodiol in bulk and formulation using reverse phase ultra-performance liquid chromatography

Background Amyotrophic Lateral Sclerosis, often known as Lou Gehrig’s disease, is typically treated with a combination therapy that includes both sodium phenylbutyrate (SPB) and taurursodiol (TRS). Objectives To assess both SPB and TRS in bulk and their dosage form concurrently, a stability-indicating analytical method was developed and validated using Ultra-Performance Liquid Chromatography. Patients and methods The chromatographic separation was carried out on a Waters C18 Column, with dimensions of 150×4.6 mm i.d. and a particle size of 2 μm. A mobile phase consisting of a phosphate buffer pH 2.5 with methanol in the ratio of 45 : 65 v/v was, then delivered at a flow rate of 1 ml/min. Detection of the analytes occurred at 285 nm using a photo diode array detector. An auto sampler injected a 10 μl sample into the column, and the column was maintained at a temperature of 30°C. Results and conclusion SPB and TRS were eluted at 1.483 and 2.492 nm, respectively. Linearity was established in the range of 567–1701 μg/ml for SPB and 189–567 μg/ml for TRS. The robustness of the method was assessed by intentionally modifying parameters such as flow rate, detection wavelength, and column temperature. Furthermore, studies on forced degradation under various stress conditions, including acid, base, peroxide, heat, and ultra violet exposure, indicated the method’s capability to identify stable materials. In summary, the developed analytical approach for simultaneously determining SPB and TRS in bulk and their formulation was found to be specific, accurate, precise, and reliable.

Plasma metabolites distinguish dementia with Lewy bodies from Alzheimer's disease: a cross-sectional metabolomic analysis.

In multifactorial diseases, alterations in the concentration of metabolites can identify novel pathological mechanisms at the intersection between genetic and environmental influences. This study aimed to profile the plasma metabolome of patients with dementia with Lewy bodies (DLB) and Alzheimer's disease (AD), two neurodegenerative disorders for which our understanding of the pathophysiology is incomplete. In the clinical setting, DLB is often mistaken for AD, highlighting a need for accurate diagnostic biomarkers. We therefore also aimed to determine the overlapping and differentiating metabolite patterns associated with each and establish whether identification of these patterns could be leveraged as biomarkers to support clinical diagnosis. A panel of 630 metabolites (Biocrates MxP Quant 500) and a further 232 metabolism indicators (biologically informative sums and ratios calculated from measured metabolites, each indicative for a specific pathway or synthesis; MetaboINDICATOR) were analyzed in plasma from patients with probable DLB (n = 15; age 77.6 ± 8.2 years), probable AD (n = 15; 76.1 ± 6.4 years), and age-matched cognitively healthy controls (HC; n = 15; 75.2 ± 6.9 years). Metabolites were quantified using a reversed-phase ultra-performance liquid chromatography column and triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, or by using flow injection analysis in MRM mode. Data underwent multivariate (PCA analysis), univariate and receiving operator characteristic (ROC) analysis. Metabolite data were also correlated (Spearman r) with the collected clinical neuroimaging and protein biomarker data. The PCA plot separated DLB, AD and HC groups (R2 = 0.518, Q2 = 0.348). Significant alterations in 17 detected metabolite parameters were identified (q ≤ 0.05), including neurotransmitters, amino acids and glycerophospholipids. Glutamine (Glu; q = 0.045) concentrations and indicators of sphingomyelin hydroxylation (q = 0.039) distinguished AD and DLB, and these significantly correlated with semi-quantitative measurement of cardiac sympathetic denervation. The most promising biomarker differentiating AD from DLB was Glu:lysophosphatidylcholine (lysoPC a 24:0) ratio (AUC = 0.92; 95%CI 0.809-0.996; sensitivity = 0.90; specificity = 0.90). Several plasma metabolomic aberrations are shared by both DLB and AD, but a rise in plasma glutamine was specific to DLB. When measured against plasma lysoPC a C24:0, glutamine could differentiate DLB from AD, and the reproducibility of this biomarker should be investigated in larger cohorts.

Biochemical assessment of spironolactone oral suspension in human plasma using ultra‐performance liquid chromatography‐tandem mass spectrometry: Application toward pharmacokinetic study

AbstractSpironolactone is one of the non‐selective mineralocorticoid receptor antagonists acting as anti‐diuretic drugs. Spironolactone is reported to possess mild to severe hyperkalemia which could often result in fatal conditions. Although several analytical methods for evaluating spironolactone as tablet dosage forms have been established in human biological systems, it is crucial to highlight that these approaches are still limited to the suspension dosage form. In this study, we have quantified spironolactone suspension in human plasma with an efficient, sensitive, and optimized ultra‐performance liquid chromatography (UPLC)‐tandem mass spectrometry‐based bioanalytical method using a deuterated internal standard method. A reverse phase UPLC analysis and mass spectrometric detection was performed using electrospray ionization in positive ion mode as an interface, and multiple reaction monitoring as a mode of acquisition. The retention of Spironolactone (SL) and Spironolactone d7 (SL‐d7) was found to be around 6.15 and 6.07 min, respectively. The linearity range was 1.007–100.224 ng/mL for SL with 0.1 mL sample volume. The method was successfully applied to the pharmacokinetic study of spironolactone in healthy male volunteers by administrating 25 mg/5 mL oral suspension and is in accordance with bioanalytical guidelines.

The development and validation of a stability indicating Rp-UPLC method for the simultaneous estimation of clarithromycin, amoxicillin, and vonoprazan in a physical mixture

A novel technique was developed for simultaneous quantification of clarithromycin (CLA) amoxicillin (AMO), and vonoprazan (VON) in a mixture using the reverse phase ultra-performance liquid chromatography (RP-UPLC) technique and validated as per International Council for Harmonization (ICH) guidelines as there was no literature published for its estimation by UPLC. The method was developed using an acquity UPLC system from waters corporation with Hibar Bis phosphonate C18 column (100 × 2.1 mm, 2 μm) at 35°C and tunable ultra-violet detector (TUV) with detection wavelength at 210 nm, has a run time of below 3 minutes. The mobile phase proportion of 60:40 of 0.1 N monobasic potassium phosphate buffer (pH 3.8) and acetonitrile at a flow velocity of 0.2 ml/ minute was utilized. Linearity was observed for CLA, AMO, and VON between the concentration ranges of 25–150, 25–150, and 1–6 μg/ml, respectively, and R2 was 0.999 for CLA, AMO, and VON. Accuracy and precision were within 2% of the coefficient of variation (RSD) for all drugs. The observed mean percentage recoveries for the CLA, AMO, and VON were determined to be 99.74%, 99.07%, and 99.8%, respectively. The stability of the approach was assessed using degradation studies by exposing it to acid, alkali, oxidizing agent, heat, Ultra Violet (UV) light, and water as per ICH guidelines.

Characterisation of the forest cobra (Naja melanoleuca) venom using a multifaceted mass spectrometric-based approach

Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated ‘omics’ and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches.

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of >75%.

Simultaneous UPLC Assay for Oxitropium Bromide and Formoterol Fumarate Dihydrate in Pressurized Metered Dose Inhaler Products for Chronic Obstructive Pulmonary Disease.

Oxitropium bromide (OB) and formoterol fumarate dihydrate (FFD) are inhaler molecules that are widely used in the treatment of chronic lung diseases. The goal of this work was to create a reversed phase-ultra performance liquid chromatography (RP-UPLC) technique for assay and identification of OB and FFD, as well as identification and estimate of its associated compounds in pressurized metered dose inhaler product (pMDI). Separation of oxitropium and formoterol peaks were enhanced on a C18 (50 × 2.1 mm × 1.7 μm) UPLC column with ethylene-bridged-hybrid technology, The mobile phase consists of buffer (0.07 M KH2PO4) and acetonitrile (80:20, v/v). The detector wavelength of 210 nm, flow rate of pump 0.6 mL/min, and oven temperature for column were set at 25°C. The injection volume was 10 μL. The method run time was 2 min. The mobile phase was used as the solvent. Retention times (RTs) were 0.5 min for OB and 1.0 min for FFD. The assay analysis was linear range for all analytes within the range for concentrations 0.03-14.8 µg/mL of OB, 0.01-0.88 µg/mL of FFD. LOD values and LOQ values 0.009 and 0.026 µg/mL for OB and 0.003 and 0.009 µg/mL for FFD, respectively. Recoveries were obtained at 96.3% for OB and 97.2% for FFD. Precisions values were (as RSD, %) ≤1.5%. With the UPLC method developed and validated according to the current ICH guidelines, it is possible to simultaneously detect OB and FFD of assay analysis in pMDI products accurately, precisely and selectively, independent of the matrix effect. The present method is the first method in the literature based on the UPLC method for this purpose. The UPLC method is a time-saving method, it provides a faster and cheaper technique than the high performance liquid chromatography (HPLC) method.

Determination of Clorsulon and its related substances in commercial bulk drug substance batches by a rapid ion-pair UPLC method

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H2O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific.

Isolation and Structural Characterization of a major degradation product of Ambrisentan using Q-TOF-MS, NMR and FTIR

Ambrisentan, an endothelin receptor antagonist, is subjected to forced degradation under neutral hydrolytic conditions, leading to a potential unknown impurity. This neutral degradation product (DP) has been detected using reverse-phase ultra-performance liquid chromatography (UPLC), where it has eluted at 1.24 relative retention time to the Ambrisentan peak. DP was isolated using semi-preparative HPLC from the degradation mixture. Later, the structure of DP was elucidated using high-resolution mass spectrometry, 2D Nuclear magnetic resonance spectroscopy (NMR), and Fourier transformed infrared spectroscopy (FTIR) spectroscopic techniques and characterized as 2-(2,2- diphenylvinyloxy)-4,6-dimethylpyrimidine.

Characterization of pneumococcal serotype 7F in vaccine conjugation

Streptococcus pneumoniae is a highly invasive bacterial pathogen that can cause a range of illnesses. Pneumococcal capsular polysaccharides (CPS) are the main virulence factors that causes invasive pneumococcal disease (IPD). Pneumococcal CPS serotype 7F along with a few other serotypes is more invasive and likely to cause IPD. Therefore, 7F is a target for pneumococcal vaccine development, and is included in the two recently approved multi-valent pneumococcal conjugated vaccines, i.e. VAXNEUVANCE and PREVNAR 20.To support process and development of our 15-valent pneumococcal conjugated vaccine (PCV15), chromatographic methods have been developed for 7F polysaccharide and conjugate characterization. A size-exclusion chromatography (SEC) method with UV, light scattering and refractive index detections was employed for concentration, size and conformation analysis. A reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was used for analysis of conjugate monosaccharide composition and degree of conjugation. The collective information obtained by these chromatographic analysis provided insights into the pneumococcal conjugate and conjugation process.Graphical