Reversed Phase Ultra Performance Research Articles

Allelic Analysis of the Gli-B1 Locus in Hexaploid Wheat Using Reverse-Phase–Ultra-Performance Liquid Chromatography

Wheat (Triticum aestivum L.) omega-5 gliadin, a major allergen responsible for wheat-dependent exercise-induced anaphylaxis in humans, is encoded by genes located at the Gli-B1 locus on chromosome 1B, which exhibits genetic polymorphism. Gli-B1 alleles have generally been identified based on the electrophoretic mobilities of the encoded gamma-, omega-1,2, and omega-5 gliadins in acid polyacrylamide gel electrophoresis. However, the similar mobilities of omega-5 gliadin variants make it difficult to distinguish them among different wheat varieties. In this study, we optimized reverse-phase–ultra-performance liquid chromatography (RP-UPLC) conditions to separate omega-5 gliadins in the reference wheat cultivar Chinese Spring and its nullisomic–tetrasomic lines for chromosome 1B. Five chromatographic peaks corresponded to omega-5 gliadin, and the average relative standard deviation to each peak retention time ranged from 0.31% to 0.93%, indicating that the method is accurate and reproducible for fractionating omega-5 gliadins in gliadin extracts from wheat flour. Using the optimized RP-UPLC method, we analyzed omega-5 gliadins in 24 wheat varieties with the Gli-B1f allele. The result showed that the wheat varieties were sorted into eight groups according to the composition of omega-5 gliadin, indicating that the classification of Gli-B1 alleles based on A-PAGE could not explain the composition of omega-5 gliadin in wheat. We reclassified 73 wheat varieties containing 16 unique Gli-B1 alleles into 31 groups based on the chromatographic patterns of their omega-5 gliadins. Our results provide information on the specific Gli-B1 alleles of wheat varieties belonging to each group and demonstrate the potential for RP-UPLC to facilitate genetic studies of wheat varieties.

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.

A validated stability‐indicating reversed‐phase‐UPLC method for simultaneous estimation of promethazine hydrochloride, methylparaben, propylparaben and sodium benzoate assay of cough suppressant and antihistamine liquid oral dosage forms

A quick, simple, sensitive, efficient and stability-indicating reverse-phase ultraperformance liquid chromatographic method for the estimation of propylparaben, methylparaben and sodium benzoate in a pharmaceutical liquid oral formulation was developed. A Waters Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 μm i.d. column was used to perform chromatographic separation with a 0.1% perchloric acid mobile phase used as solvent A and a mixture of 0.1 % perchloric acid and methanol in the ratio 20:80 (v/v), respectively, as solvent B. The experiments were carried out at a flow rate of 0.4 ml/min and the detection wavelength was 240 nm. The compartment temperature of the column was set at 40°C and the injection volume was set at 2 μl. The main aim of the research was to develop a single UPLC assay method for promethazine (active ingredient) and preservatives in the oral solution of promethazine HCl and dextromethorphan HBr that contains promethazine (active ingredient) and methylparaben, propylparaben and sodium benzoate (preservatives). An assay of dextromethorphan HBr was developed and validated by another HPLC method. The drug and preservatives were eluted at retention times of 19.3 min for promethazine HCl, 9.3 min for methylparaben, 18.9 min for propylparaben and 8.9 min for sodium benzoate. Validation of the developed method was carried out as stated by the International Conference on Harmonization guidelines ICH Q2B and under USP<1225>. The analytical parameters verified specificity/selectivity, linearity, accuracy, ruggedness and robustness. The linearity ranges of promethazine HCL, methylparaben, propylparaben and sodium benzoate were 10-100, 10-80, 1.0-8.0 and 10-80 μg/ml, respectively, with a correlation coefficient of active ingredients and preservatives of 1.00. Percentage recoveries of promethazine, propylparaben, methylparaben, and sodium benzoate were 100.0-100.2, 99.0-100.3, 99.5-98.0 and 99.0-100.0%. The validated analytical method proves that the method is specific, precise, linear, accurate, sensitive, rugged and stable, indicating the quantification of the active ingredient and all preservatives in liquid oral formulations.

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

The production of whey protein concentrates (WPC) from camel milk whey represents an effective approach to valorize this processing byproduct. These concentrates harbor active ingredients with significant bioactive properties. Camel WPC were spray-dried 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 effect of both treatments on protein degradation was studied by sodium dodecyl sulfate-PAGE and reverse-phase ultraperformance liquid chromatography techniques. Significantly Substantially enhanced protein degradation was observed after US treatment when compared with spray-drying (SPD). Both SPD and US treatments slightly enhanced the WPC samples' antioxidant activities. The US exposure for 15 min exhibited the highest 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (12.12 mmol Trolox equivalent per gram). Moreover, US treatment for 10 min exhibited the highest in vitro antidiabetic properties (α-amylase and α-glucosidase inhibition), and dipeptidyl peptidase-IV inhibitory activity among all samples. In addition, the US for 10 min and SPD at 170°C showed the lowest median inhibitory concentration (IC50) values for in vitro antihypercholesterolemic 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.

Improved Stability-Indicating RP-UPLC Method for the Levamisole Hydrochloride Assay and Estimation of Its Related Compounds.

Levamisole hydrochloride (LVM) is an anthelmintic drug substance with immunomodulatory and anticancer activities. LVM has also found usage as a cutting agent in street cocaine. This study was aimed to develop and validate an alternative and improved stability-indicating reversed-phase ultraperformance liquid chromatography (RP-UPLC) method for the determination of LVM and the estimation of its related compounds. The UPLC method for the assay was optimized in terms of organic solvents consumed, pH, column temperature, and flow rate. Determination of LVM and its related compounds was performed using a gradient elution on a Waters ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm i.d., 130 Å). The column temperature was maintained at 35°C. Mobile phase A was composed of aqueous 5 mM ammonium hydroxide, and mobile phase B was composed of acetonitrile. All the analytes were monitored by UV detection at 215 nm with a flow rate of 0.7 mL/min. The total runtime of the method with column equilibration is 4.0 min. The developed method met all the acceptance criteria of the current International Council for Harmonization [ICH Q2 (R1)] guidelines. The method was tested in terms of specificity, linearity (R2 > 0.999), limit of detection (LOD; 0.06 μg/mL), limit of quantitation (LOQ; 0.2 μg/mL), accuracy, precision, and robustness. With a short analysis time (<2.5 min) and reduced consumption of organic solvents, the proposed method is considered a greener alternative to conventional chromatographic methods. An alternative and improved UPLC method was successfully developed and validated in accordance with the ICH guidelines for the determination of LVM and the estimation of its related compounds. Due to its high degree of selectivity, speed, and accuracy, the developed method can significantly benefit the end-users with laboratory efficiency and throughput during routine analysis of production batches and stability monitoring of LVM-related drug products.

Determination of Haloacetic Acids, Disinfection Byproducts, in Tap Water with Reversed-Phase Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry

To establish a method for quantitative analysis of haloacetic acids (HAAs), disinfection byproducts, in tap water with reversed-phase ultra-performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry. Tap water samples were collected and 0.70 g/L ascorbic acid was added to eliminate residual chlorine. Then, the water samples were directly injected into the instrument for analysis after filtration. After separation on a pentafluorobenzene (PFP) column with an inner diameter of 1.0 mm at a higher linear velocity and a lower volume flow rate compared with those of a narrow-bore column, nine HAAs, namely, monochloroacetic acid (MCAA), monobromoacetic acid (MBAA), dichloroacetic acid (DCAA), bromochloroacetic acid (BCAA), dibromoacetic acid (DBAA), trichloroacetic acid (TCAA), bromodichloroacetic acid（BDCAA), chlorodibromoacetic acid (CDBAA) and tribromoacetic acid (TBAA), were examined by negative electrospray ionization and full MS/dd-MS 2 acquisition mode. In order to adjust for the matrix effect, matrix matching calibration curves were used to quantitate the nine HAAs. Good linearity was obtained for each of the nine HAAs within their respective linear ranges. The detection limits and quantification limits of the method were 0.020-1.0 μg/L and 0.060-3.0 μg/L. The recoveries were 69.8%-119%. The proposed method showed strengths in separation speed and qualitative accuracy. It did not require for complicated pretreatment procedures and can meet the need of tap water sample analysis.

A NEW RP-UPLC METHOD FOR SIMULTANEOUS QUANTIFICATION OF IVABRADINE AND METOPROLOL IN COMBINED DOSAGE FORMS

An accurate and yet fast, easy-to-use, affordable, sensitive, single method for measuring Ivabradine and Metoprolol in bulk and pharmaceutical products using Reversed-Phase Ultra-Performance Liquid Chromatography (RP-UPLC) was developed. A Hibar C18 column was used with a running phase composed of Water: ACN (60:40 v/v) at a flow rate of 0.5 ml/min. UV detection was used at a wavelength of 260 nm. The correlation parameters for Ivabradine and Metoprolol were found to be of the order of 99.9% and % MSD &lt;2. 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

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.

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.

Implementation of Two Chromatographic Methods for Simultaneous Quantitation of Thioctic Acid, Benfotiamine and Cyanocobalamin.

Two accurate and sensitive chromatographic methods have been introduced and validated for the simultaneous determination of thioctic acid, benfotiamine and cyanocobalamin in bulk powders and in their pharmaceutical formulation. Method A is reversed-phase ultra performance liquid chromatographic method with an isocratic elution, where a rapid separation was accomplished on a Zorbax C8 column using a mobile phase of acetonitrile:0.05 M phosphate buffer (pH 6 adjusted by o-phosphoric acid) (23:77, v/v). The retention times (tR) were 0.578, 0.852 and 1.376 for cyanocobalamin, benfotiamine and thioctic acid, respectively. The separated peaks were revealed at 210.0 nm. Method B is a thin-layer densitometric method where the separation of the studied drugs was carried out on silica gel plates using methanol-chloroform-heptane-1-sulphonic acid sodium salt (0.4%) (7:3:0.1, by volume) as a mobile phase, and scanning of the separated bands was done at 240.0 nm. The retardation factor (Rf) values were 0.17, 0.48 and 0.75 for cyanocobalamin, benfotiamine and thioctic acid, respectively. Validation of the methods was achieved following ICH guidelines and the applied methods succeeded to determine the cited drugs in their pure forms and capsules. Results were statistically compared to the manufacturer's method where no significant difference was observed.

Stability Indicating RP-UPLC Photo-Diode Array based Method for Determination of Edoxaban Tosylate

The objective of the current study was to develop a specific, precise, accurate and robust gradient stability indicating reversed-phase ultra performance liquid chromatography (RP-UPLC-PDA) assay method and validated for determination of edoxaban tosylate in API. Gradient separation was achieved on an acquity UPLC BEH C18 column (50 mm, 2.1 mm and 1.7 μm) column using mobile phase of acetoitrile:20 mM potassium dihydrogen phosphate, pH 3.0 ± 0.05 adjust with OPA at flow rate of 0.6 mL/min, the injection volume was 1 μL and the detection was carried out of 289 nm by using photo-diode array detector. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress condition. The method was linear in the drug concentration range of 100-300 μg/mL with correlation coefficient of 0.999. Degradation products produced as a result of stress studies did not interfere with detection of edoxaban tosylate and the assay, thus developed stability indicating method can be used for routine analysis in pharmaceutical industry.

Characterization of metabolite profiles of white and green spears of asparagus officinalis L. from Caoxian, East China

China is the largest planting country of asparagus (Asparagus officinalis L.) in the world. Caoxian, as the famous asparagus township in China, enjoys a reputation for producing asparagus with high yield and good quality, due to its unique geological characteristic. In this study, a method of reverse-phase ultraperformance liquid chromatography coupled with electrospray tandem mass spectrometry (RP-UPLC-ESI-MS/MS) was established for profiling metabolites from three segments (tip, mid, and base) of 'Caoxian white and green Asparagus'. A total of 114 metabolites were identified, among them, 43 were found for the first time in this vegetable. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) was applied to provide an overview of the metabolite profiles of Caoxian asparagus and to separate different segments of spears. The variables most decisive to discriminate among segments included 9 of the metabolites tentatively identified. This study will help to improve the protection of Caoxian asparagus geographical indication.

Characterisation of the Urinary Metabolic Profile of Liver Fluke-Associated Cholangiocarcinoma

BackgroundHuman infection with Opisthorchis viverrini, a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis.MethodsGlobal molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand.ResultsMultivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury.ConclusionsSeveral metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA.

들깨 유전자원의 지방산 변이 및 rosmarinic acid와 caffeic acid의 함량에 따른 항산화 활성 비교

Background: The aim of this study was to compare the fatty acid composition, antioxidant activity, and the content of two major phenolic acids, caffeic and rosmarinic acids, of 45 Perilla accessions collected from Russia.BR Methods and Results: A total of 45 accessions of the genus Perilla were used in this study. The antioxidant activities of these accessions were analyzed using a spectrophotometer, and their caffeic and rosmarinic acid contents were determined using a reversedphase ultraperformance liquid chromatography (UPLC) system. The Perilla seed oil was recovered using hexane in a soxhlet extraction method. The fatty acid compositions were analyzed using a Shimadzu QP2010 gas chromatography-mass spectoroscopy system. The results showed that accessions IT226732 and IT274300 had the highest content of caffeic and rosmarinic acid, respectively. A cluster analysis based on antioxidant assay results and concentration of phenolic acids led to the categorization of Perilla accessions into three major groups. The accessions in Group I were characterized by higher caffeic and rosmarinic acid content and antioxidant activity, compared to the accessions in the other two groups (p 0.05). The total oil content ranged between 28.39 and 46.89%. The compositions of oleic, linoleic, and linolenic acid ranged from 11.83 to 19.55%, 11.92 to 16.71%, and 59.19 to 67.28%, respectively.BR Conclusions: The results of this study indicated that accessions IT274300, IT226732, IT274293, IT235818, and IT235820 could be used as a source of functional materials.

Development and Validation of UPLC–PDA Method for Quality Control of Dikamali gum—A Natural Tablet Binder: Fast Simultaneous Quantitation of Six Polymethoxyflavones

Dikamali gum (Gardenia lucida Roxb., Family—Rubiaceae) in wet and dry form is used as one of the popular tablet binders in the Indian traditional herbal formulations. Recently, its application as a binder in modern drug delivery has also been reported. However, its quality specification is yet to be established. To the best of our knowledge, no validated method is reported so far for the quality assurance of Dikamali. A new reverse-phase ultraperformance liquid chromatography–photodiode array (UPLC–PDA) method was developed for the simultaneous quantification of six polymethoxyflavones (PMFs), viz., gardenin E, gardenin D, xanthomicrol, 5-desmethynobiletin, gardenin A, and gardenin B, in Dikamali gum using a Waters X-select C18 (2.5 µm, 2.1 × 100 mm) column. The influence of acid additive in mobile phase on peak response and column temperature on the peak resolution was also investigated. ICH guideline was followed for method validation of analytical procedures for the compliance of precision, accuracy, linearity, etc. The method is about three times faster than classical HPLC analysis and completed within 15 min. The limits of detection (LOD) and limit of quantitation (LOQ) were in the range 0.02–0.06 and 0.08–0.20 µg mL− 1, respectively. Calibration curves were linear from 0.18 to 1.78 µg mL− 1 with acceptable correlation coefficients ranged 0.9993–0.9999. The percent mean recoveries of the PMFs were ranged from 96.07 to 101.69%. Successful analysis of three G. lucida samples from different locations demonstrated the method to be reproducible and convenient. The present work also provides some references for quality specification of G. lucida based on polymethoxyflavones.

SIMULTANEOUS REVERSE-PHASE ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF GRAZOPREVIR AND ELBASVIR

Objective: The objective of this study was to develop and validate rapid, specific, sensitive, and precise reverse-phase ultra performance liquid chromatography (RP-UPLC) method for the quantitative determination of grazoprevir and elbasvir, as there are no official monograph and no analytical method by UPLC.Methods: Chromatographic separation was achieved on a Waters Acquity UPLC HSS C18 (2.1 mm × 100 mm, 1.8 micron) column with a 45:55 (v/v) mixture of 0.1% orthophosphoric acid (pH 2.8) and acetonitrile as a mobile phase, thermostated at 30°C with a short run time of 3.0 min.Results: The retention times were 0.73 and 1.29 min for grazoprevir and elbasvir, respectively. Quantification is achieved with TUV detection at 254 nm over the concentration range of 25–150 μg/ml for grazoprevir and for elbasvir 12.5–75 μg/ml, with a correlation coefficient of 0.999 and 0.999, respectively. The developed method was validated according to the International Conference on Harmonization (ICH) guidelines with respect to linearity, accuracy, precision, specificity, and robustness. Forced degradation study was extended out under acidic, alkaline, oxidative, photolytic, and thermal conditions to demonstrate the stability-indicating capability of the developed UPLC method. The degradation products were well resolved from the main peak, thus proved the stability-indicating power of the method. The results of the analysis were validated statistically.Conclusion: The method is precise, accurate, linear, robust, and fast. The short retention time allows the analysis of a large number of samples in a short period of time and, therefore, should be cost-effective for routine analysis in the pharmaceutical industry.

The Preparation and Physicochemical Characterization of Aluminum Hydroxide/TLR7a, a Novel Vaccine Adjuvant Comprising a Small Molecule Adsorbed to Aluminum Hydroxide

Adjuvants are necessary to enable vaccine development against a significant number of challenging pathogens for which effective vaccines are not available. We engineered a novel small-molecule immune potentiator, a benzonaphthyridine agonist targeting toll-like receptor 7 (TLR7), as a vaccine adjuvant. TLR7 agonist (TLR7a) was engineered to be adsorbed onto aluminum hydroxide (AlOH), and the resulting AlOH/TLR7a was evaluated as a vaccine adjuvant. AlOH/TLR7a exploits the flexibility of AlOH formulations, has an application in many vaccine candidates, and induced good efficacy and safety profiles against all tested antigens (bacterial- and viral-derived protein antigens, toxoids, glycoconjugates, and so forth) in many animal models, including nonhuman primates. In this article, we describe the outcome of the physicochemical characterization of AlOH/TLR7a. Reverse-phase ultra performance liquid chromatography, confocal microscopy, flow cytometry, zeta potential, and phosphophilicity assays were used as tools to demonstrate the association of TLR7a to AlOH and to characterize this novel formulation. Raman spectroscopy, nuclear magnetic resonance, and mass spectroscopy were also used to investigate the interaction between TLR7a and AlOH (data not shown). This pivotal work paved the way for AlOH/TLR7a to progress into the clinic for evaluation as an adjuvant platform for vaccines against challenging preventable diseases.

Simultaneous Determination of Impurities of Atazanavir and Ritonavir in Tablet Dosage Form by Using Reversed-Phase Ultra Performance Liquid Chromatographic Method.

A simple, rapid, selective and stability indicating reversed phase-ultra performance liquid chromatography method was developed and validated for the simultaneous quantification of process related and degradation impurities present in Atazanavir and Ritonavir tablets. The two proposed drug components and their respective impurities were separated using Acquity BEH C18 (100 mm × 2.1 mm), 1.7 μ column at a flow rate of 0.4 mL/min. Buffer used as Mobile phase-A which consists of 0.01 M monobasic potassium hydrogen phosphate adjusted the pH to 3.6 and acetonitrile is used as organic modifier (mobile phase-B). The detector wavelength of 240 nm was used for quantifying the impurities. Both the drug components along with their impurities were eluted within a runtime of 18 min. The performance of the developed method was checked by validating the method according to the requirements of International Conference on Harmonization for parameters such as specificity, precision, linearity, ruggedness, accuracy, sensitivity (limit of detection (LOD) and limit of quantitation (LOQ)) and robustness. Linearity and range were established from LOQ level to 150% level. Accuracy of the method was demonstrated from LOQ level to 150% level. The developed stability indicating method is capable for determination of impurities of Atazanavir and Ritonavir in combined tablet dosage form as well as individual dosage forms also. The reported method enables lesser solvent consumption and reduces time and cost of the analysis in quality control laboratory.