scholarly journals Separation and identification of permethylated glycan isomers by reversed phase nanoLC-NSI-MSn

2020 ◽  
Author(s):  
Simone Kurz ◽  
M. Osman Sheikh ◽  
Shan Lu ◽  
Lance Wells ◽  
Michael Tiemeyer
Keyword(s):  
High Performance ◽  
Method Development ◽  
Statistical Significance ◽  
Biological Significance ◽  
Structural Diversity ◽  
Structural Features ◽  
Reversed Phase ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Mobile Phase Optimization

SUMMARYHigh performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

scholarly journals Separation and identification of permethylated glycan isomers by reversed phase nanoLC-NSI-MSn

2020 ◽  
pp. mcp.RA120.002266
Author(s):  
Simone Kurz ◽  
M. Osman Sheikh ◽  
Shan Lu ◽  
Lance Wells ◽  
Michael Tiemeyer
Keyword(s):  
High Performance ◽  
Method Development ◽  
Statistical Significance ◽  
Biological Significance ◽  
Structural Diversity ◽  
Structural Features ◽  
Reversed Phase ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Mobile Phase Optimization

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

BIO ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULANEOUS ESTIMATION OF NITAZOXANIDE AND OFLOXACIN IN HUMAN PLASMA BY RP-HPLC

INDIAN DRUGS ◽  
2021 ◽  
Vol 57 (10) ◽  
pp. 47-57
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Method Development ◽  
Reversed Phase ◽  
Chromatography Method ◽  
Rp Hplc ◽  
Method Development And Validation ◽  
Liquid Chromatography Method ◽  
Development And Validation ◽  
Lower Limits

An isocratic Reversed-Phase High Performance Liquid Chromatography method has been developed for rapid and simultaneous separation and estimation of two antibiotics, namely, nitazoxanide and ofloxacin, in human plasma. Separation was carried out on Altima C8 (150 x 4.6 mm, 5µ) column using a mobile phase of 0.1% ortho phosphoric acid: acetonitrile (50:50, V/V) at 260 nm. The retention time of nitazoxanide and ofloxacin was noted to be 4.850 and 7.949 min, respectively. The average % recovery for nitazoxanide and ofloxacin were 98.012 % and 94.176 %, respectively and reproducibility was found to be satisfactory. The linearity was investigated in the concentration range of 0.02-2 µg/ml (r2=0.9996) for nitazoxanide and 0.008-0.8 µg/ml (r2=0.9998) for ofloxacin. The lower limits of quantification were 0.0196 µg/ml and 0.0079 µg/ml for nitazoxanide and ofloxacin, respectively, which reach the level of both drugs possibly found in human plasma. The proposed method can be applied for etermination of nitazoxanide and ofloxacin from dosage forms during pharmacokinetic study.

scholarly journals Method Development and Validation of Metoprolol and Felodipine by using RP-HPLC in Bulk and Pharmaceutical Dosage Form

2020 ◽  
Vol 11 (4) ◽  
pp. 8047-8053
Author(s):  
Potturi Ramadevi ◽  
Kantipudi Rambabu
Keyword(s):  
High Performance ◽  
Method Development ◽  
Pharmaceutical Preparations ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Concentration Limit ◽  
Uv Detector ◽  
Pharmaceutical Dosage Form ◽  
Pharmaceutical Dosage Forms ◽  
Rp Hplc

The main objective of this research is to develop and validate a simple, specific, precise, sensitive, cost effective and rapid Reversed-Phase High-Performance Liquid Chromatographic (RP-HPLC) method for simultaneous quantification of Felodipine and Metoprolol in bulk and pharmaceutical dosage forms. The separation of the analytes were carried out on a X-bridge phenyl column with a moving phase composed of 0.1 % Tri ethyl amine: acetonitrile (30:70 v/v) delivered at a stream of 1.0 ml/min, and separation has been observed by UV detector, at a detection wavelength of 235 nm. This method was proven to be linear over a concentration limit of 10-150 µg/ml for Metoprolol, 2-30 µg/ml for Felodipine with correlation coefficient of 0.999. The retention time of Metoprolol and Felodipine were 2.936, 4.535 minutes respectively. To separate Metoprolol and Felodipine peaks a run time of 8 min. was used. The validation results were in good agreement with acceptable limits. RSD values which are less than 2.0 % indicating the accuracy and precision of this method. Hence it was evident that the proposed method was said to be a suitable one for the regular analysis and quality control of pharmaceutical preparations which contain these active drugs either individually or in combination.

scholarly journals METHOD DEVELOPMENT AND VALIDATION OF LOPINAVIR IN TABLET DOSAGE FORM USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2018 ◽  
Vol 11 ◽  
Author(s):  
Sunkara Namratha ◽  
Vijayalakshmi A
Keyword(s):  
High Performance ◽  
Dosage Form ◽  
Method Development ◽  
Limit Of Detection ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Limit Of Quantification ◽  
Rp Hplc ◽  
Tablet Dosage ◽  
Liquid Chromatographic

Objective: Reversed-phase high-performance liquid chromatographic method (RP-HPLC) was developed for the assessment of lopinavir in the dosage form of tablet.Methods: Chromatogram was run through using Kromosil C18 4.5×150 mm using a mobile phase methanol: water of ratio 65:35% v/v with a rate of flow of 0.8 ml/min, measured by UV spectrometric detection at 265 nm. The method developed was validated in terms of precision, accuracy, linearity, and robustness parameters.Results: Retention time of lopinavir established at 2.482 min and percentage R.S.D of lopinavir found to be 1.0% and 0.5%, respectively. The method shows that good linearity range of 30–150 μg correlation coefficient of lopinavir was 0.997. The limit of detection was 2.97 and limit of quantification was 9.92, respectively. The percent purity of lopinavir was 99.87%.Conclusion: The suggested method (Rp-HPLC) for concurrent assay lopinavir was validated, which is appropriate method for the analysis oflopinavir quantitatively in tablet dosage forms and bulk.

scholarly journals A Novel Stress Indicating RP-HPLC Method Development and Validation for the Simultaneous Estimation of Velpatasvir and Sofosbuvir in Bulk and Its Tablet Dosage Form

2019 ◽  
pp. 1-10 ◽  
Author(s):  
D. Chinababu
Keyword(s):  
High Performance ◽  
Dosage Form ◽  
Method Development ◽  
Reversed Phase ◽  
Hplc Method ◽  
Simultaneous Estimation ◽  
Forced Degradation ◽  
Detection Range ◽  
Rp Hplc ◽  
Tablet Dosage

Aim: The objective of the study was simplest, accurate, precise and robust reversed phase high performance liquid chromatographic (RP-HPLC) method was developed for the estimation of Velpatasvir (VEL) and Sofosbuvir (SOF) in the bulk and its tablet dosage form. Study Design: The Quantitative and Qualitative estimation and designed forced degradation study of Velpatasvir & Sofosbuvir by RP-HPLC. Place and Duration of Study: The study was carried at Santhiram College of Pharmacy and time taken 4 months. Method: The method was attained by used Waters( 5µm, C18 250 x 4.6 mm) column with mobile phase consists of  0.5 mM disodium hydrogen phosphate buffer pH adjusted to 6.5, with Orthophosphoric acid and Methanol in the ratio of 78:22 v/v, a flow rate of 1.0 mL/min and ultraviolet detection at 285 nm. Results: The method was validated as per ICH guidelines with different parameters, the mean retention times of VEL and SOF were found to be 2.8 & 4.7 min respectively. The resolution between VEL and SOF was found to be 10.66. The Correlation coefficients for calibration curves within the detection range of 32.5 - 97.5 and 125 - 375 µg/mL were 0.999 for VEL and SOF respectively. The LOD and LOQ for VEL and SOF were found to be 0.0068-0.029 µg/mL and 0.104-0.342 µg/mL respectively. Conclusion: The results were indicated that the developed method was used for the routine analysis of VEL & SOF combined form in bulk and its commercial formulation. To the best of our knowledge, there was no method of RP-HPLC for the determination of VEL alone or in combination with SOF molecule.

scholarly journals RP-HPLC method development and validation for simultaneous estimation of efonidipine hydrochloride ethanolate and telmisartan in their synthetic mixture

2021 ◽  
pp. 190-195
Author(s):  
Grishma H Patel ◽  
Shreya D Adeshra ◽  
Dhananjay B Meshram
Keyword(s):  
Mobile Phase ◽  
High Performance ◽  
Method Development ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Hplc Method ◽  
Simultaneous Estimation ◽  
Quality Control Laboratory ◽  
Efficient Option ◽  
Liquid Chromatographic

A Novel, selective, accurate and rapid Reversed Phase High Performance Liquid Chromatographic (RPHPLC) method for the analysis of Efonidipine Hydrochloride Ethanolate and Telmisartan in binary mixture has been developed and validated. The chromatographic system consisted of a Phenomenex Kinetex ® 5µ C18 Size: 150 * 4.6mm column and the separation was achieved by using ambient temperature with a mobile phase containing mobile Phase Acetonitrile:25mM Phosphate Buffer pH 4.9 (45:55). The samples were monitored at 253 nm for detection at a flow rate of 1.0 mL/min and the retention time was about 7.77 and 4.10 mins for Efonidipine Hydrochloride Ehanolate and Telmisartan respectively. The calibration curve was linear over the concentration range 5-30 and 10-60 ?g/mL for Efonidipine Hydrochloride Ehanolate and Telmisartan respectively. The proposed method is accurate in the range of 99.75% - 100.10% recovery and precise (%RSD of intraday variation and % RSD of inter day variation were found to be within the acceptance criteria). Therefore, this method can be used as a more convenient and efficient option for the analysis of Efonidipine Hydrochloride Ehanolate and Telmisartan in Quality control laboratory.

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