Rapid, Sensitive and Simple LC-MS/MS Method Development and Validation for Estimation of Phenytoin in Human Plasma by using Deuterated Internal Standard

2021 ◽  
pp. 2937-2944
Author(s):  
Ajay I. Patel ◽  
Kishna Ram ◽  
Swati Guttikar ◽  
Amit Kumar J. Vyas ◽  
Ashok B. Patel ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Method Development ◽  
Dynamic Range ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Limit Of Quantification ◽  
Validation Parameters ◽  
Tandem Mass Spectrometric

A rapid, sensitive, accurate and precise high-performance liquid chromatography–tandem mass spectrometric (HPLC– MS/MS) method equipped with Electro Spray Ionization (ESI) source, operating in the Positive ion and Multiple Reaction Monitoring (MRM) mode was developed and validated for the estimation of Phenytoin in human plasma using Phenytoin D10 as an internal standard. Liquid-liquid extraction (LLE) technique was used to extract analyte and ISTDs from 0.2mL human plasma. The analytical separation was carried out in a reverse phase liquid chromatography by using C18 (150 x 4.6mm, 5µm) column, and 2mM Ammonium Acetate in water (pH 6.3): Methanol (30:70% v/v) mobile phase at 1mL/min in isocratic mode. Analytes were monitored in multiple reactions monitoring (MRM) mode using the respective [M+H]⁺ ions, m/z 253.10→ 182.30 for Phenytoin and m/z 263.30 → 192.20 for the internal standard, respectively. The Phenytoin and Phenytoin D10 retained at about 2.50 and 2.46 minutes respectively with total run time of 4 minute. The response of the LC-ESI-MS/MS method for both analyte and ISTD was linear over the dynamic range of 60-12000ng/mL with correlation coefficient r2 ≥ 0.9963. The Accuracy was well within the accepted limit of ± 20% at lower limit of quantification (LLOQ) and ± 15% at all the other concentrations in the linear range. Recovery of drug and ISTD was 87.52% and 86.42%, respectively. This method was fully validated for all the validation parameters and Stability studies. After optimization, the Bioanalytical method was validated according to USFDA, EMA and ANVISA guidelines for Bioanalysis.

scholarly journals Bioanalytical Method Development and Validation of Memantine in Human Plasma by High Performance Liquid Chromatography with Tandem Mass Spectrometry: Application to Bioequivalence Study

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Ravi Kumar Konda ◽  
B. R. Challa ◽  
Babu Rao Chandu ◽  
Kothapalli B. Chandrasekhar
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Method Development ◽  
Dynamic Range ◽  
Quantitative Estimation ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Bioequivalence Study ◽  
Healthy Human ◽  
Development And Validation

A simple, sensitive, and rapid HPLC-MS/MS method was developed and validated for quantitative estimation of memantine in human plasma. Chromatography was performed on Zorbax SB-C18(4.6×75 mm, 3.5 μm) column. Memantine (ME) and internal standard Memantine-d6(MED6) were extracted by using liquid-liquid extraction and analyzed by LC-ESI-MS/MS using multiple-reaction monitoring (MRM) mode. The assay exhibited a linear dynamic range of 50.00–50000.00 pg/ml for ME in human plasma. This method demonstrated an intra- and interday precision within the range of 2.1–3.7 and 1.4–7.8%, respectively. Further intra- and interday accuracy was within the range of 95.6–99.8 and 95.7–99.1% correspondingly. The mean recovery of ME and MED6 was86.07±6.87and80.31±5.70%, respectively. The described method was successfully employed in bioequivalence study of ME in Indian male healthy human volunteers under fasting conditions.

scholarly journals BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF DECITABINE AND CEDAZURIDINE IN HUMAN PLASMA USING LC-MS/MS

2021 ◽  
pp. 257-262
Author(s):  
SAI PRUDHVI N. ◽  
VENKATESWARLU B. S. ◽  
KUMUDHAVALLI M. V. ◽  
MURUGANANTHAM V.
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Human Plasma ◽  
Method Development ◽  
Limit Of Detection ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Simultaneous Estimation ◽  
Limit Of Quantification ◽  
Validation Parameters

Objective: The present work aimed to develop a novel, reliable and accurate Liquid Chromatography-Mass Spectrometry/Mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Decitabine and Cedazuridine a combined medication used for the treatment of chronic myelomonocytic leukemia in human plasma. Methods: Talazoparib drug is used as an internal standard in the study. Both the analytes and internal standard were isolated from 100 ml plasma samples by liquid-liquid extraction and then chromatographed on Zorbax SB-CN (4.6 mm×75 mm, 3.5 µm) column with a mobile phase consisting of 0.1 % ammonium formate and methanol in the ratio of 65:45 (v/v) pumped at 0.5 ml/min. The method had a chromatographic total run time of 5 min. Results: The developed method gave a symmetric peak at a retention time of 1.7 min for Decitabine, 2.2 min for Cedazuridine, 3.5 min for Talazoparib and satisfied all the peak properties as per USP guidelines. The mass spectral characterization of separated analytes in the LC method was performed using a mass detector operated at Multiple Reaction Monitoring mode with precursor-to-product ion transitions at m/z of 229 to m/z of 114 as MH+ion for Decitabine, m/z of 269 to m/z of 118 as MH+ion for Cedazuridine. A very sensitive limit of detection of 0.3 ng/ml was observed and showed a calibration curve linear over the concentration range of LLOQ (lower limit of quantification) to 500 ng/ml. The other validation parameters were found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction concentration was acceptable and very high for both the analytes in HQC (high-quality control concentration), MQC (medium quality control concentration) and LLOQ levels. The peak area response ratio of Decitabine and Cedazuridine with the internal standard in freeze-thaw, short term and long term stability studies was found to be acceptable confirms that the method is stable. Conclusion: It can be concluded that the proposed method is specific, accurate, and precise and could be used for the simultaneous estimation of Decitabine and Cedazuridine in human plasma.

scholarly journals NEW METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF FEBUXOSTAT IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY

2016 ◽  
Vol 8 (9) ◽  
pp. 61 ◽  
Author(s):  
Narottam Pal ◽  
Avanapu Srinivasa Rao ◽  
Pigilli Ravikumar
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
Method Development ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
New Method ◽  
Liquid Chromatography Mass Spectrometry ◽  
Chromatography Mass Spectrometry

<p><strong>Objective</strong>:<strong> </strong>To develop a new method and validate the same for the determination of Febuxostat (FBS) in human plasma by liquid chromatography–mass spectrometry (LCMS).</p><p><strong>Methods</strong>:<strong> </strong>The present method utilized reversed-phase high-performance liquid chromatography with tandem mass spectroscopy. Febuxostat D9 (FBS D9) was used as internal standard (IS). The analyte and internal standard were separated from human plasma by using solid phase extraction method. Zorbax Eclipse XDB, C<sub>8</sub>, 100 mm x 4.6 mm, 3.5 µm column was used and HPLC grade acetonitrile, 5 millimolar (mM) ammonium format (80: 20, v/v) as mobile phase, detected by mass spectrometry operating in positive ion and multiple reaction monitoring modes.</p><p><strong>Results</strong>:<strong> </strong>The parent and production transitions for FBS and internal standard were at m/z 317.1→261.0 and 326.1→262.0 respectively. The method was validated for system suitability, specificity, carryover effect, linearity, precision, accuracy, matrix effect, sensitivity and stability. The linearity range was from 20.131 ng/ml to10015. 534 ng/ml with a correlation coefficient of 0.999. Precision results (%CV) across six quality control samples were within the limit. The percentage recovery of FBS and internal standard from matrix samples was found to be 76.57% and 75.03% respectively.</p><p><strong>Conclusion</strong>:<strong> </strong>Present study describes new LC-MS method for the quantification of FBS in a pharmaceutical formulation. According to validation results, it was found to be a simple, sensitive, accurate and precise method and also free from any kind of interference. Therefore the proposed analytical method can be used for routine analysis for the estimation of FBS in its formulation.</p>

Rapid and Sensitive Bioanalytical Method Development and Validation for Quantification of Metoprolol Using LC-MS/MS in Human Plasma

2020 ◽  
Vol 42 (2) ◽  
pp. 171-171
Author(s):  
Beludari Mohammed Ishaq Beludari Mohammed Ishaq ◽  
Lingareddygari Siva Sanker Reddy Lingareddygari Siva Sanker Reddy ◽  
Gajula Mahaboob Basha Gajula Mahaboob Basha ◽  
Munna Sreenivasulu Munna Sreenivasulu ◽  
Challa Madhusudhana Chetty and Hindustan Abdul Ahad Challa Madhusudhana Chetty and Hindustan Abdul Ahad
Keyword(s):  
Human Plasma ◽  
Method Development ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Vascular Diseases ◽  
Storage Conditions ◽  
Spectrometric Analysis ◽  
Myocardial Infraction ◽  
Tandem Mass Spectrometric ◽  
Cardio Vascular

A novel, accurate, simple and selective LC-MS/MS method was developed and validated for the determination of metoprolol in human plasma. Due to structural resemblance Propranolol was selected as internal standard. Anti coagulant used was K2 EDTA. Metoprolol, used in the therapy and management of hypertension, myocardial infraction and other cardio vascular diseases. Liquid – liquid extraction technique with tert-butyl methyl ether was applied for the extraction of analyte from human plasma. Kromasil C18 column (5and#181;, 100 and#215; 4.6 mm) with an isocratic mobile phase of 5mM Ammonium Formate pH 3.5 and Acetonitrile (15:85 % V/V) was used for the resolution. Sample ionization was done with Electrospray ionization technique in positive ion mode. Selectivity was enhanced by tandem mass spectrometric analysis via two multiple reaction monitoring (MRM) transitions, m/z 268.15→115.90 for metoprolol and 260.17→115.90 for Propranolol respectively. The linearity of the method was established over a concentration range of 1.505 – 538.254 ng/mL, in human plasma, with the precision and accuracy ranging from 4.67 to 7.41% and 90.66 to 98.15% respectively. The stability of the analyte was evaluated in plasma under different storage conditions.

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.

scholarly journals A Simple High-Performance Liquid Chromatography Method for Simultaneous Determination of Three Triazole Antifungals in Human Plasma

2012 ◽  
Vol 57 (1) ◽  
pp. 484-489 ◽  
Author(s):  
Mei Zhang ◽  
Grant A. Moore ◽  
Murray L. Barclay ◽  
Evan J. Begg
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Simultaneous Determination ◽  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Gradient Elution ◽  
Limit Of Quantification ◽  
Chromatography Method

ABSTRACTA rapid and simple high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of three triazole antifungals (voriconazole, posaconazole, and itraconazole and the metabolite of itraconazole, hydroxyitraconazole) in human plasma. Sample preparation involved a simple one-step protein precipitation with 1.0 M perchloric acid and methanol. After centrifugation, the supernatant was injected directly into the HPLC system. Voriconazole, posaconazole, itraconazole, its metabolite hydroxyitraconazole, and the internal standard naproxen were resolved on a C6-phenyl column using gradient elution of 0.01 M phosphate buffer, pH 3.5, and acetonitrile and detected with UV detection at 262 nm. Standard curves were linear over the concentration range of 0.05 to 10 mg/liter (r2> 0.99). Bias was <8.0% from 0.05 to 10 mg/liter, intra- and interday coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/liter.

scholarly journals Enantioselective Determination Of S- And R-Isomers Of Ibuprofen In Plasma By Ultra-Performance Liquid Chromatography – Tandem Mass Spectrometry

2020 ◽  
Vol 15 (1) ◽  
pp. 40-46
Author(s):  
V.O. DOROSCHUK ◽  
V.Ye. Sabko ◽  
O.V. Ivashko ◽  
L.O. POPOVA ◽  
A.S. Shalamay
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Mass Spectrometric ◽  
Mass Spectrometric Detection ◽  
Tandem Mass ◽  
Ibuprofen Enantiomers ◽  
Tandem Mass Spectrometric

A new method of enantioselective determination of S- and R-isomers of ibuprofen in human plasma by ultraperformance liquid chromatography with tandem mass spectrometric detection using solid-phase extraction was developed. For enantioselective separation of ibuprofen isomers, a LUX Cellulose-3 chiral chromatographic column was used. Complete separation of the enathiomer peaks is achieved in the isocratic elution conditions with a mobile phase ratio of 0.05 % formic acid solution (%): methanol (%) = 30 : 70 (v/v) and a flow rate of 0.2 mL/min. The mass spectrometric detection was performed at negative ionization mode with multiple reaction monitoring, using the transitions at 205.13 > 161.14 Da and 208.09 > 164.03 Da for ibuprofen enantiomers and deuterated ibuprofen (internal standard), respectively. The method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within-run and between-run precision and accuracy. The LLOQ for the two enantiomers was 100 ng/mL in plasma. The calibration curves showed good linearity of each enantiomers in the ranges from 100 to 60000 ng/mL. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in human plasma.

scholarly journals A RAPID AND SENSITIVE LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY/MASS SPECTROMETRY METHOD FOR ESTIMATION OF PIOGLITAZONE, KETO PIOGLITAZONE AND HYDROXY PIOGLITAZONE IN HUMAN PLASMA

2017 ◽  
Vol 10 (12) ◽  
pp. 120
Author(s):  
Revathi Naga Lakshmi Ponnuri ◽  
Prahlad Pragallapati ◽  
Ravindra N ◽  
Venkata Basaveswara Rao Mandava
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Pharmacokinetic Parameters ◽  
Validation Parameters ◽  
Liquid Chromatography Tandem Mass ◽  
Multiple Reaction Monitoring Mode

  Objective: The main objective of the work was to develop a straightforward, fast and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay for determination of pioglitazone (PG), keto pioglitazone (KPG), and hydroxy pioglitazone (HPG) in human plasma and to validate as per recent guidelines.Methods: Analyte and the internal standard (IS) were extracted from plasma through liquid-liquid extraction and chromatographed on a Xterra RP18, 100×4.6, 5 μ column using methanol: acetonitrile mixture and 10 mM Ammonium formate buffer (70:30, v/v) as the mobile phase at a flow rate of 0.7 mL/min. The API-3200 Q Trap LC-MS/MS instrument in multiple reaction monitoring mode was used for detection. Diphenhydramine was utilized as IS.Results: The linearity was established in the concentration range of 20.15-1007.58 ng/mL for PG, 20.35-1017.58 ng/mL for KPG, and 19.68-491.22 ng/mL for HPG in human plasma. All the validation parameters were well within the acceptance limits.Conclusion: A new simple LC-MS/MS method was developed for the determination of PG, KPG, and HPG in human plasma. This method can be easily applied for the estimation of pharmacokinetic parameters of PG, KPG, and HPG.

scholarly journals Quantification of Amiridine in Human Plasma by High-Performance Liquid Chromatography Coupled with Electrospray Tandem Mass Spectrometry

2013 ◽  
Vol 2013 ◽  
pp. 1-5
Author(s):  
Igor I. Miroshnichenko ◽  
Angelina I. Platova
Keyword(s):  
Human Plasma ◽  
High Performance ◽  
Solid Phase ◽  
Internal Standard ◽  
Mass Spectrometric ◽  
Pharmacokinetic Studies ◽  
Tandem Mass ◽  
Ionization Source ◽  
Limit Of Quantification ◽  
Tandem Mass Spectrometric

The aim of this study was to develop and validate a high-performance liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for analysis of the amiridine in human plasma. The analyte and internal standard (IS), zolpidem, were extracted from human plasma by solid phase extraction (SPE with SOLA cartridges) and separated on a Zorbax SB-C18 column using methanol and 0.2% formic acid in water as mobile phase. Detection was performed using an electrospray ionization source and mass spectrometric positive multireaction monitoring mode (+MRM) at a voltage capillary of +2000 V. The assay was linear over the concentration range 0.5–200 ng/mL with the lowest limit of quantification (LLOQ) of 0.5 ng/mL. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and interday %), accuracy, recovery, and the stability of the analyte under various conditions. The method can be successfully applied to pharmacokinetic studies.

scholarly journals BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ENTRECTINIB IN RAT PLASMA BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

2020 ◽  
pp. 155-163
Author(s):  
PRAVALLIKA KE ◽  
PRAMEELA RANI A ◽  
RATNA KUMAR M
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Performance ◽  
Method Development ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Rat Plasma ◽  
Array Detector ◽  
Pharmaceutical Drugs ◽  
Relative Standard

Objective: The objective of the study was to develop and validate the bioanalytical liquid chromatography–mass spectrometry (LCMS/MS) method for the estimation of entrectinib in bulk and pharmaceutical drugs in rat plasma. Methods: Chromatographic separation of entrectinib with D4-entrectinib as internal standard (IS) was achieved using Waters Alliance high-performance liquid chromatography system, quaternary gradient pump of e2695, using Luna, 250×4.6 mm, 5 μm column and the mobile phase containing 0.1% formic acid and acetonitrile (ACN) within the ratio of 70:30% v/v. The flow was 1.0 ml/min; detection was carried out by absorption at 294 nm using a photodiode array detector at ambient temperature. Results: The peak of entrectinib was eluted at retention times of 5.225 min. The multiple reaction monitoring was 560.6/475.1 (m/z) for entrectinib and 580.6/496.3 (m/z) for IS entrectinib (D4). The linearity range was 1–20 ng/ml with a regression coefficient of 0.999. % relative standard deviation of peak areas of all measurements always <2.0. Conclusion: The method was successfully validated and it had been found to be within limits for accuracy, precision, and linearity and it is stable under analytical conditions used.

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