scholarly journals Rapid Quantification of Peptide Oxidation Isomers from Complex Mixtures

2019 ◽  
Author(s):  
Niloofar Abolhasani Khaje ◽  
Joshua S. Sharp
Keyword(s):  
Method Development ◽  
Electron Transfer Dissociation ◽  
Amino Acid Level ◽  
Reversed Phase ◽  
Residue Level ◽  
Oxidation Products ◽  
Peptide Sequence ◽  
Protein Footprinting ◽  
Hydroxyl Radical Protein Footprinting ◽  
Peptide Oxidation

AbstractHydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common, quantification at the residue level is challenging due to the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally co-elute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis at both the peptide and amino acid level.

scholarly journals Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

2017 ◽  
Author(s):  
Boer Xie ◽  
Amika Sood ◽  
Robert J. Woods ◽  
Joshua S. Sharp
Keyword(s):  
High Resolution ◽  
Hydroxyl Radical ◽  
Computational Models ◽  
Electron Transfer Dissociation ◽  
Molecular Model ◽  
Solvent Accessible Surface Area ◽  
Protein Footprinting ◽  
Backbone Rmsd ◽  
Hydroxyl Radical Protein Footprinting ◽  
Lower Accuracy

AbstractWe report an integrated workflow that allows mass spectrometry-based high-resolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area (<SASA>) of amino acid side chains. This approach is based on application of multi-point HR-HRPF, electron-transfer dissociation (ETD) tandem MS (MS/MS) acquisition, measurement of effective radical doses by radical dosimetry, and proper normalization of the inherent reactivity of the amino acids. The accuracy of the resulting <SASA> measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use <SASA> measurements from HR-HRPF to differentiate molecular models of high accuracy (< 3Å backbone RMSD) from models of lower accuracy (> 4Å backbone RMSD). The ability of <SASA> data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of <SASA> data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.

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 FOR THE ESTIMATION OF AMPRENAVIR IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–ELECTROSPRAY IONIZATION–TANDEM MASS SPECTROMETRY

2019 ◽  
pp. 137-141
Author(s):  
SUSMITHA K ◽  
MENAKA M
Keyword(s):  
Liquid Chromatography ◽  
Electrospray Ionization ◽  
Human Plasma ◽  
Method Development ◽  
Multiple Reaction Monitoring ◽  
Reversed Phase ◽  
Tandem Mass ◽  
Relative Standard ◽  
Tandem Mass Spectrometric ◽  
Chromatographic Peaks

Objective: The main aim of the present study was to develop a sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric technique for the quantitation of amprenavir in human plasma. Methods: Chromatographic separation was achieved on a reversed-phase Symmetry C18 (50 mm×4.6 mm, 3.5 μm) column with isocratic elution by acetonitrile and 0.1% v/v formic acid in the ratio of 90:10 v/v as mobile phase. Chromatographic peaks were resolved with 0.7 ml/min flow rate. Drug was extracted with ethyl acetate solvent by liquid–liquid extraction method. Monitoring of transition of m/z 506.2 and 71.0 for amprenavir and 628 and 421 for methyl-indinavir was made on multiple reaction monitoring. Results: Calibration curve of amprenavir was linear over 1–600 ng/ml concentration range with regression coefficient (r2) value of >0.99. The % relative standard deviation values were <8.5% for interday and intraday precision and accuracy. The method has excellent recovery, and the percentage recovery values of lower quality control (QC), median QC, and higher QC samples were 101.86%, 102.8%, and 99.28%, respectively. Conclusion: The drug was stable for more time at variable stability conditions, and method was successfully applicable to regular analysis of amprenavir in biological matrices.

Development and Validation of a Reversed-Phase Chiral HPLC Method to Determine the Chiral Purity of Bulk Batches of (S)-Enantiomer in Afoxolaner

2017 ◽  
Vol 100 (1) ◽  
pp. 65-73
Author(s):  
Nilusha Padivitage ◽  
Satish Kumar ◽  
Abu Rustum
Keyword(s):  
Method Development ◽  
Specific Rotation ◽  
Reversed Phase ◽  
Hplc Method ◽  
Racemic Mixture ◽  
Chiral Hplc ◽  
Development Tool ◽  
Chiral Purity ◽  
Development And Validation ◽  
First Time

Abstract Afoxolaner is a new antiparasitic molecule from the isoxazoline family that acts on insect acarine g-aminobutyric acid and glutamate receptors. Afoxolaner is a racemic mixture, which has a chiral center at the isoxazoline ring. A reversed-phase chiral HPLC method has been developed to determine the chiral purity of bulk batches of (S)-enantiomer in afoxolaner for the first time. This method can also be used to verifythat afoxolaner is a racemic mixture, which was demonstrated by specific rotation. ChromSword, an artificial intelligence method development tool, was used for initial method development. The column selected for the final method was CHIRALPAK AD-RH (150 × 4.6 mm, 5 μm particle size), maintained at 45°C, and isocratic elution using water–isopropanol–acetonitrile (40 + 50 + 10, v/v/v) as the mobile phasewith a detection wavelength of 312 nm. The run time for the method was 11 min. The resolution and selectivity factors of the two enantiomers were 2.3 and 1.24, respectively. LOQ and LOD of the method were 1.6 and 0.8 μg/mL, respectively. This method was appropriately validated according to International Conference on Harmonization guidelines for its intended use.

scholarly journals Reversed phase HPLC for strontium ranelate: Method development and validation applying experimental design

2018 ◽  
Vol 68 (2) ◽  
pp. 171-183
Author(s):  
Béla Kovács ◽  
Lajos Kristóf Kántor ◽  
Mircea Dumitru Croitoru ◽  
Éva Katalin Kelemen ◽  
Mona Obreja ◽  
...  
Keyword(s):  
Experimental Design ◽  
Strontium Ranelate ◽  
Method Development ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Reversed Phase ◽  
Hplc Method ◽  
Intermediate Precision ◽  
Limit Of Quantitation ◽  
Development And Validation

Abstract A reverse-phase HPLC (RP-HPLC) method was developed for strontium ranelate using a full factorial, screening experimental design. The analytical procedure was validated according to international guidelines for linearity, selectivity, sensitivity, accuracy and precision. A separate experimental design was used to demonstrate the robustness of the method. Strontium ranelate was eluted at 4.4 minutes and showed no interference with the excipients used in the formulation, at 321 nm. The method is linear in the range of 20–320 μg mL−1 (R2 = 0.99998). Recovery, tested in the range of 40–120 μg mL−1, was found to be 96.1–102.1 %. Intra-day and intermediate precision RSDs ranged from 1.0–1.4 and 1.2–1.4 %, resp. The limit of detection and limit of quantitation were 0.06 and 0.20 μg mL−1, resp. The proposed technique is fast, cost-effective, reliable and reproducible, and is proposed for the routine analysis of strontium ranelate.

Sign in / Sign up

Export Citation Format

Share Document