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

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.

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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.

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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.

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

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i9.34533 ◽

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.

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

Journal of Analytical Methods in Chemistry ◽

10.1155/2012/101249 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 10

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.

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

METHODS AND OBJECTS OF CHEMICAL ANALYSIS ◽

10.17721/moca.2020.40-46 ◽

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.

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SENSITIVE BIO ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF VENETOCLAX IN HUMAN PLASMA BY LC-ESI-MS/MS

INDIAN DRUGS ◽

10.53879/id.57.06.11999 ◽

2020 ◽

Vol 57 (06) ◽

pp. 32-38

Author(s):

H. Potluri ◽

Keyword(s):

Human Plasma ◽

Method Development ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Liquid Liquid Extraction ◽

Liquid Chromatography Tandem Mass ◽

Positive Mode ◽

Method Development And Validation ◽

Development And Validation

A specific and sensitive method of liquid chromatography–tandem mass spectrometry was demonstrated for the experimental determination of venetoclax in human plasma utilising venetoclax-D8 as an internal standard. The column Xbridge C18, 50 × 4.6mm, 5 µm was used for attaining chromatographic separation by utilising 10mM ammonium formate and methanol as isocratic mobile phase in the composition ratio of 20:80 (V/V). The flow-rate selected was 0.7ml/min. Venetoclax and venetoclax-D8 are identified in multiple reaction monitoring (MRM) positive mode with proton adducts at m/z 869.53 →553.21 and m/z 877.14 → 553.23, respectively. For the successful extraction of drug as well as internal standard, liquid-liquid extraction technique was efficiently utilised. The developed technique was established in a linear concentration range of 5.0-5000.0 pg/ml along with correlation coefficient (r2) of 0.9994. Intra and inter-day precisions were found to be 0.7 to 1.90% and 0.7 to 2.0 % for venetoclax and venetoclax-D8, respectively. Accuracy was found to be within 98.6 to 101.99% and 99.17 to 101.14 % for venetoclax and venetoclax-D8, respectively. It was observed that throughout the bench top studies, post-operative stability studies and freeze-thawing cycles, venetoclax retained stability.

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

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2021v13i5.41744 ◽

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.

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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.

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Validation of a Liquid Chromatographic/Tandem Mass Spectrometric Method for the Determination of Scopolamine Butylbromide in Human Plasma: Application of the Method to a Bioequivalence Study

Journal of AOAC International ◽

10.1093/jaoac/92.5.1366 ◽

2009 ◽

Vol 92 (5) ◽

pp. 1366-1372 ◽

Cited By ~ 9

Author(s):

Josélia Larger Manfio ◽

Mauricio Bedim dos Santos ◽

Wagner Alex Jann Favreto ◽

Fabiane Ines Hoffmann ◽

Adriana Cristina Mertin

Keyword(s):

Human Plasma ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Spectrometric Method ◽

Mass Spectrometric Method ◽

Healthy Human ◽

Scopolamine Butylbromide ◽

Validation Parameters ◽

Tandem Mass Spectrometric

Abstract A sensitive and specific LC/MS/MS method was developed and validated for the determination of scopolamine butylbromide in human plasma. Scopolamine butylbromide and propanolol (internal standard) were extracted from the plasma by liquidliquid extraction with dichloromethane as the extraction solvent and separated on a C18 analytical column (50 4.6 mm id) maintained at 40C. The analytes were eluted at a constant flow rate of 0.45 mL/min; the mobile phase consisted of acetonitrile and a buffer of 5 mM ammonium acetate and 0.1 formic acid (60 + 40, v/v). The mass spectrometer, equipped with an electrospray source in the positive ionization mode, was set up in the multiple-reaction monitoring mode to monitor the transitions m/z 360.6 > 102.5 (scopolamine butylbromide) and m/z 259.7 > 115.6 (propanolol). The chromatographic separation was obtained within 2.0 min, and the responses were linear over the concentration range of 0.1040.00 ng/mL. The mean extraction recoveries of scopolamine butylbromide and propanolol from plasma were 69.00 and 80.76, respectively. Method validation parameters, such as specificity, linearity, precision, accuracy, and stability, were within the acceptable range. Moreover, when the proposed method was successfully applied to a pharmacokinetic study of healthy human volunteers, the results showed that the two scopolamine butylbromide formulations tested are not bioequivalent in rate and extent of absorption.

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A Validated High-Performance Liquid Chromatography-Tandem Mass Spectrometric (Lc-Ms/Ms) Method for Simultaneous Determination of R(+)-Ketorolac and S(−)-Ketorolac in Human Plasma and Its Application to a Bioequivalence Study

Chromatography Research International ◽

10.4061/2011/214793 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Sabyasachi Patri ◽

Anil K. Patni ◽

Sunil S. Iyer ◽

Arshad H. Khuroo ◽

Tausif Monif ◽

...

Keyword(s):

Liquid Chromatography ◽

Human Plasma ◽

Solid Phase ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Bioequivalence Study ◽

Mass Spectrometric ◽

Tandem Mass ◽

Liquid Chromatography Tandem Mass ◽

Tandem Mass Spectrometric

We report a selective, accurate, and reproducible liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that employs solid phase extraction for quantification of ketorolac enantiomers in human plasma. Resolution of R(+)-ketorolac and S(−)-ketorolac was achieved using a Chiral-AGP column and a mobile phase of ammonium formate buffer (10 mM, pH ):acetonitrile (85 : 15, v/v and 70 : 30, v/v) in a gradient time program. S(+)-etodolac was used as the internal standard (IS). Quantification was achieved using a positive electrospray ionization (ESI+) interface under multiple reaction monitoring (MRM) condition. The method was validated over the concentration range of 9.36–1198.69 ng/ml for R(+)-ketorolac and 6.07–776.74 ng/ml for S(−)-ketorolac. Matrix effect was found negligible and the method showed good performances in terms of accuracy (89.6–102.7%) and precision (1.7–6.7%) for both enantiomers. Extraction recoveries of R(+)-ketorolac, S(−)-ketorolac, and S(+)-etodolac were 82.04, 70.94, and 93.90%, respectively. Results of all stability exercises in human plasma were within acceptable limits. The method was successfully applied to a single dose cross over bioequivalence study in healthy human male volunteers. Incurred Sample Reanalysis (ISR) was performed by randomly selecting 10% of total subject samples of the study using Statistical Analysis Software (SAS). Values of 91.1% for R (+)-ketorolac and 83.5% for S(−)-ketorolac indicated good acceptance for ISR.

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