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

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.

Download Full-text

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

Research Journal of Pharmacy and Technology ◽

10.52711/0974-360x.2021.00515 ◽

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.

Download Full-text

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

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016v8i9.11968 ◽

2016 ◽

Vol 8 (9) ◽

pp. 61 ◽

Cited By ~ 6

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

Objective: To develop a new method and validate the same for the determination of Febuxostat (FBS) in human plasma by liquid chromatography–mass spectrometry (LCMS).Methods: 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, C8, 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.Results: 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.Conclusion: 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.

Download Full-text

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

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2017.v10i12.20284 ◽

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.

Download Full-text

Application of a Reliable LC-MS/MS Method for Determination of Rizatriptan in Healthy Subject Samples: Demonstration of Assay Reproducibility by Incurred Sample Reanalysis

ISRN Chromatography ◽

10.5402/2012/761679 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Dinesh S. Patel ◽

Naveen Sharma ◽

Mukesh C. Patel ◽

Bhavin N. Patel ◽

Pranav S. Shrivastav ◽

...

Keyword(s):

Mass Spectrometry ◽

Quality Control ◽

Tandem Mass Spectrometry ◽

Human Plasma ◽

Dynamic Range ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Tandem Mass ◽

Limit Of Quantitation

A reliable, rapid, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been proposed for the determination of rizatriptan in human plasma using sumatriptan as internal standard (IS). The analyte and IS were extracted from 300 μL human plasma via liquid-liquid extraction and the chromatography was achieved on Hypurity C18 (50 mm × 4.6 mm, 5 μm) column under isocratic conditions. Detection of rizatriptan and IS was done by tandem mass spectrometry, operating in positive ionization and multiple-reaction monitoring mode. The limit of detection and lower limit of quantitation of the method were 0.04 and 0.20 ng/mL, respectively, with a linear dynamic range of 0.20–60.0 ng/mL. The intrabatch and interbatch precision (% CV) was ≤8.4% while the mean extraction recovery was >78% across quality control levels. Bench top stability, freeze and thaw stability, processed sample stability, and long-term stability in plasma were evaluated at two quality control levels. It was successfully applied to a bioequivalence study of 10 mg rizatriptan orally disintegrating tablet formulation in 40 and 32 healthy Indian male subjects under fasting and fed conditions, respectively. The reproducibility in the measurement of study data was demonstrated by reanalysis of 254 incurred samples.

Download Full-text

SIMULTANEOUS ESTIMATION OF PROPAFENONE AND ITS TWO METABOLITES IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY LC-MS/MS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2017v9i8.18978 ◽

2017 ◽

Vol 9 (8) ◽

pp. 192

Author(s):

Rajeev Kumar Gupta ◽

Anand Chaurasia ◽

Brijeshkunvar Mishra

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Human Plasma ◽

Dynamic Range ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Simultaneous Estimation ◽

Tandem Mass ◽

Plasma Samples

Objective: A simple, sensitive and rapid performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was to be developed and validated for quantification of propafenone (PPF) and its two major metabolite 5-hydroxy propafenone (5-OHP) and N-depropyl propafenone (NDP) in human plasma.Methods: Liquid-liquid extraction (LLE) with ethyl acetate was used of extraction of plasma samples. The analytes were separated using an isocratic mixture of 0.1% formic acid/acetonitrile (20:80 v/v) on a reversed-phase column Hypurity Advance C18 50 x2.1 mm, 5µ and analysed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H] Ions. The m/z was 342.20/116.10 for propafenone, m/z 299.80/74.10 for N depropyl propafenone and m/z 358.30/98.10 for 5-hydroxy propfenone along with m/z 409.2/238.0 for Amlodipine as internal standard respectively.Results: The method had a short chromatographic run time of 1.5 min. The method exhibited a linear dynamic range over 5.11 to 1000.73 ng/ml for propafenone, 0.51 to 100.06 ng/ml for N-depropyl propafenone and 5.11 to 1001.64 ng/ml for 5-hydroxy propafenone respectively, in human plasma.Conclusion: The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetics, bioavailability and bioequivalence studies.

Download Full-text

Quick and Sensitive UPLC-ESI-MS/MS Method for Simultaneous Estimation of Sofosbuvir and Its Metabolite in Human Plasma

Molecules ◽

10.3390/molecules24071302 ◽

2019 ◽

Vol 24 (7) ◽

pp. 1302 ◽

Cited By ~ 1

Author(s):

Mohammad Semreen ◽

Hasan Alniss ◽

Muath Mousa ◽

Hassan Aboul-Enein

Keyword(s):

Human Plasma ◽

Matrix Effects ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Simultaneous Estimation ◽

High Throughput Analysis ◽

Elution Time ◽

Validation Parameters ◽

Multiple Reaction Monitoring Mode ◽

Acquity Uplc

A simple, fast and highly sensitive RP-UPLC-MS/MS method was developed and validated for the simultaneous determination of sofosbuvir (SR) and its metabolite GS331007 in human plasma using ketotifen as an internal standard (IS). The separation was achieved on Acquity UPLC BEH C18 (50 × 2.1 mm, i.d. 1.7 µm, Waters, USA) column using acetonitrile:5 mM ammonium formate:0.1% formic acid (85:15:0.1% v/v/v) as a mobile phase at a flow rate of 0.35 mL/min in an isocratic elution. The Xevo TQD UPLC-MS/MS was operated under the multiple-reaction monitoring mode using positive electrospray ionization. Extraction with dichloromethane was used in the sample preparation. Method validation was performed as per the Food and Drug Administration (FDA) guidelines and the calibration curves of the proposed method were found to be linear in the range of 1–1000 ng/mL for SR and in the range of 10–1500 ng/mL for its metabolite (GS331007) with an elution time of 1.83 min. All validation parameters were within the acceptable range according to the bioanalytical methods validation guidelines. Furthermore, the obtained results of matrix effects indicate that ion suppression or enhancement from human plasma components was negligible under the optimized conditions. The proposed method can be applied in high-throughput analysis required for pharmacokinetic and bioequivalence studies in human samples.

Download Full-text

BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF CANAGLIFLOZIN IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i18.33228 ◽

2019 ◽

pp. 46-51 ◽

Cited By ~ 1

Author(s):

DEEPAN T ◽

BASAVESWARA RAO MV ◽

DHANARAJU MD

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Human Plasma ◽

Method Development ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

Objective: A validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for canagliflozin in human plasma along with stability studies. Methods: The chromatographic separation of canagliflozin was performed on Zorbax XDB phenyl (75 × 4.6 mm, 3.5 mm) using methanol:acetate buffer (80:20 v/v) at a flow rate of 1.0 ml/min. The LC–MS/MS system consists of API 4000 triple quadrupole mass spectrometer equipped with turbospray ionization and an AS8020 automatic sample injector. Results: The retention time of canagliflozin was 1.15 min and total runtime was 2 min. The multiple reaction monitoring was 462.5/267.1 (m/z) for canagliflozin and 466.4/267.2 (m/z) for internal standard (canagliflozin D4), respectively. The method was linear over the range of 10–7505 ng/ml. The calculated slope ranged from 0.0451 to 0.0502 and intercepts from 0.0102 to 0.0456 with coefficients of the determination of 0.9970. The overall mean recovery of internal standard and canagliflozin was 76.66 and 79.77, respectively. Conclusion: The method was successfully validated and it was found to be within the limits for accuracy, precision, and linearity and it is stable under analytical conditions used.

Download Full-text

A Novel Simultaneous Estimation of Sofosbuvir and Velpatasvir in Human Plasma by Liquid Chromatography Tandem-Mass Spectrometry after Protein Precipitation Method

Current Pharmaceutical Analysis ◽

10.2174/1573412914666180910102353 ◽

2019 ◽

Vol 15 (7) ◽

pp. 710-715

Author(s):

S.T. Narenderan ◽

Basuvan Babu ◽

T. Gokul ◽

Subramania Nainar Meyyanathan

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Human Plasma ◽

Multiple Reaction Monitoring ◽

Simultaneous Estimation ◽

Pharmacokinetic Studies ◽

Tandem Mass ◽

Mass Spectrometric Method ◽

Highly Sensitive ◽

Tandem Mass Spectrometric

Objective: The aim of the present work is to achieve a novel highly sensitive chromatographic method for the simultaneous determination of hepatitis C agents, sofosbuvir and velpatasvir from human plasma using ritonavir as an internal standard. Methods: Chromatographic separation was achieved using Hypersil C18 column (50mm x 4.6mm, 3μm) with an isocratic elution mode using the mobile phase composition 10 mM ammonium formate buffer (pH 5.0): acetonitrile (20:80 v/v) pumped at a flow rate of 0.5 ml/min. The detection was carried out by tandem mass spectrometry using Multiple Reaction Monitoring (MRM) positive Electrospray Ionization (ESI) with proton adducts at m/z 530.10 > 243.10, 883.40 > 114.0 and 721.25 > 197.0. Results: The method validated as per USFDA guidelines with respect to linearity, accuracy, and precision was found to be acceptable over the concentration range of 0.2–2000 ng/ml and 5-2000 ng/ml for sofosbuvir and velpatasvir respectively and the method was found to be highly sensitive and selective. Conclusion: The developed tandem mass spectrometric method is robust and can be applied for the monitoring of plasma levels of the analyzed drug in preclinical and clinical pharmacokinetic studies.

Download Full-text

Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Biomedicines ◽

10.3390/biomedicines9060621 ◽

2021 ◽

Vol 9 (6) ◽

pp. 621

Author(s):

Aurélien Millet ◽

Nihel Khoudour ◽

Jérôme Guitton ◽

Dorothée Lebert ◽

François Goldwasser ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Plasma ◽

High Resolution Mass Spectrometry ◽

Internal Standard ◽

Therapeutic Drug ◽

Limit Of Quantification ◽

Immunoglobulin G4 ◽

Positive Mode ◽

Surrogate Peptide ◽

First Time

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

Download Full-text

Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0612 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1461-1468 ◽

Cited By ~ 2

Author(s):

Jean-Claude Alvarez ◽

Pierre Moine ◽

Isabelle Etting ◽

Djillali Annane ◽

Islam Amine Larabi

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Half Life ◽

Limit Of Detection ◽

Treated Patient ◽

Internal Standard ◽

Therapeutic Drug ◽

Active Metabolites ◽

Limit Of Quantification ◽

Positive Mode

AbstractObjectivesA method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524.MethodsA simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.ResultsCalibration curves were linear in the 1–5000 μg/L range for remdesivir and 5–2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6–110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.ConclusionsThis method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

Download Full-text