Navigation Links
Bioanalytical Method Intraday Validation for the Quantitation of Paroxetine ,,, in Bovine Plasma using the TSQ Quantum Mass Spectrometer

Mark Churchill and Mark Harrison, Thermo Finnigan, Hemel Hempstead, UK

Bioanalytical Method Validation

The data presented here can be acquired using a Thermo Finnigan TSQ Quantum mass spectrometer Introduction Guidelines on Bioanalytical Method Validation acceptance criteria were established by the US Food and Drug Administration (FDA) from a conference on Analytical Methods Validation Bioavailability, Bioequivalence and Pharmacokinetic Studies held in Arlington,VA, December 3-5, 1990.1 These acceptance criteria were reviewed ten years on at Bioanalytical Methods Validation A Revisit with a Decade of Progress, Arlington,VA, January 12-14, 2000,2 a meeting sponsored by the American Association of Pharmaceutical Scientists and the FDA. This led to the issue of the FDA Guidance for Industry document on Bioanalytical Method Validation in May 20013. A full method validation should assess accuracy, precision, selectivity, sensitivity, reproducibility and stability of an analyte in matrix samples.

Paroxetine is an orally administered antidepressant which acts as a selective serotonin reuptake inhibitor (SSRI) but has only minimal effects on the neuronal reuptake of norepinephrine and dopamine. Paroxetine is administered for the treatment of depression, social anxiety disorder, obsessive compulsive disorder, panic disorder and generalized anxiety disorder. Goal This report describes the ease of use of the TSQ Quantum to validate bioanalytical methods in accordance with the guid elines issued by the US Food and Drug Administration.

The aim of this investigation was to demonstrate the sensitivity, linearity, and robustness of the TSQ Quantum to allow researchers to achieve excellent accuracy and precision for highthroughput quantitation of analytes in biological matrices. Paroxetine spiked into bovine plasma was injected as an intra-day batch consisting of blanks, calibration standards, and QCs at eight replicates. TSQ Quantum Tune View Optimization of Paroxetine Paroxetine (C19H20FNO3, molecular weight 329.37) was infused, 0.1 ng/L, into the ESI source and the four most abundant product ions for the MS/MS breakdown were determined using the automated compound optimization procedure in the TSQ Quantum (Figure 1).

The transition yielding the most abundant product ion (m/z 192.1) was used for the analysis of Paroxetine. Figure 1: Automated optimization of MS/MS parameters for Paroxetine Experimental Conditions Sample Preparation: Aliquots (1 milliliter) of Bovine Plasma (Sigma P4639) were spiked with Paroxetine (Figure 2) in the concentration range of 0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 200, 500 and 1000 pg/L for the calibration standards and at levels of 0.3, 25 and 750 pg/L for quality control samples. De-Fluoro-Paroxetine (Internal Standard, Figure 3) was spiked at a concentration of 25 pg/L. Blank Bovine plasma and plasma containing internal standard were also prepared. Eight replicate plasma aliquots of 100 L were taken from each spiked level and protein precipitated using 250 L of Acetonitrile, centrifuged at 13,000 rpm for 10 minutes and the supernatants decanted. The extracts were evaporated under a stream of nitrogen gas and reconstituted in 100 L of Water/Methanol/Acetic acid (80/20/0.1 v/v/v).

Sample Analysis: The plasma extracts were chromatographed (Thermo Finnigan Surveyor System) on a C18 Elite 100 mm 2.1 mm column (Thermo Hypersil-Keystone) at a flow rate of 250 L/min with a linear gradient of 10% Solvent B (methanol containing 0.1% acetic acid) to 95% B over five minutes. Solvent A was water containing 0.1% acetic acid. Injection volumes of 10 L were used. MS Conditions: Mass spectrometer: Thermo Finnigan TSQ Quantum
Ionization mode: Electrospray (ESI), positive ion
Resolution: 0.7 Dalton Full Width Half Maximum on Q1 and Q3
Selected Reaction Monitoring:
Paroxetine 330.20 > 192.1 +/- 0.3 Da, 22 V collision energy
De-Fluoro-Paroxetine 312.20 > 174.1 +/- 0.3 Da, 20 V collision energy
Argon collision gas pressure 1.5 mTorr

Figure 2: Paroxetine

Figure 3: De-Fluoro Paroxetine

LC/MS/MS Chromatograms of Bovine Plasma Spiked with Paroxetine and Internal Standard Blank Plasma,Plasma containing Internal Standard,Calibration Standards at 0.1 and 0.5 pg/L Figure 4a: Blank Plasma
Figure 4b: Plasma containing Int Std

Figure 4c: Plasma spiked at 0.1 pg/L
Figure 4d: Plasma spiked at 0.5 pg/L
LC/MS/MS Chromatograms of Bovine Plasma Spiked with Paroxetine and Internal Standard Quality Control samples at 0.3 and 25 pg/L Figure 5a: Quality Control at 0.3 pg/L

Figure 5b: Quality Control at 25 pg/L Results The chromatography (Figure 4a4d and 5a5b) was capable of resolving Paroxetine and De-Fluoro-Paroxetine from any matrix peaks and was suitable for the quantitation of unknowns.

A calibration curve was constructed from the first and eighth replicate set of calibration standards (Figure 6). The linear fit calibration curve, weighted 1/X, of area ratio plotted against concentration was used to calculate the concentrations of all calibration standards and quality control samples throughout the analytical batch.

A correlation coefficient of r2=0.9994 with an equation of Y=0.00346891 +0.0312025*x was obtained for the curve.

The internal standard peak area response was 3.1% cv for 121 injections (Figure 7). Figure 6: Paroxetine/Int Std area ratio

Figure 7: Internal Standard area response The calibration curve was used to calculate the values of the eight replicates at each level of quality control sample (Table 1) and of the remaining six replicates of standards (Table 2). Table 1: Intra-batch accuracy and precision of quality control data for Paroxetine in Bovine plasma

Table 2: Intra-batch accuracy and precision of calibration data for Paroxetine in Bovine Plasma
*Calibration data sets 1 and 8 used to construct curve Discussion The lowest calibration standard selected for this assay at 0.1 pg/L had a minimum signal-to-noise of greater than 5:1 and variability of 3% from eight determinations. This was well within the acceptance criteria set by the guidelines which defines that the Limit of Quantitation (LOQ) of an assay should have less than 20% variability.

The Intra-batch variability of back-calculated concentrations of Paroxetine in Bovine Plasma was less than 3% across the whole calibration range. This falls well within the expected method validation acceptance criteria as set by Shah et al of 20% variability at the LOQ and less than 15% variability at all other concentrations of the calibration standards.

The validated method accuracy and precision were determined from the backcalculated values of the Quality Control samples. The intra-batch accuracy was 103.6%, 104.0% and 99.7% at concentrations of 0.3, 25 and 750 pg/L respectively. The intra-batch precision was 2.2%, 0.6% and 0.6% at concentrations of 0.3, 25 and 750 pg/L respectively.

The validation data indicate that the TSQ Quantum is sensitive, robust and linear (four orders of dynamic range demonstrated here) and is an ideal instrument for the quantitation of analytical substances in Biological matrices. References 1 Shah, V.P., et al, Pharmaceutical Rese arch, Vol 9, No 4, 588-592, 1992
2 Shah, V.P., et al, Pharmaceutical Research, Vol 17, No 12, 1551-1557, 2000
3 Guidance for Industry, Bioanalytical Method Validation, U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER), May 2001


Page: All 1 2 3 4 5 6

Related biology technology :

1. A New PCR-based Mycoplasma Detection Method
2. DNA-free A NEW Method to Remove DNA
3. RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control
4. Comparison of Different Methods of mRNA Quantitation
5. Rapid Method Development With the BioLogic DuoFlow Chromatography System for the Purification of His-Tagged Proteins
6. A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF
7. Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A
8. Commercial Plasmid Preparation Methods Yield DNA With Different Transfection Capabilities
9. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A
10. A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF
11. A Robust LC/MS/MS Method for the Quantitation of Very Long Chain Fatty Acids for Research into the Characterization of Peroxisomal Disorders
Post Your Comments:

(Date:11/24/2015)... --> --> ... by Transparency Market Research, the global non-invasive prenatal testing ... 17.5% during the period between 2014 and 2022. The ... Analysis, Size, Volume, Share, Growth, Trends and Forecast 2014 ... to reach a valuation of US$2.38 bn by 2022. ...
(Date:11/24/2015)... ... November 24, 2015 , ... In harsh industrial ... points for in-line sensors can represent a weak spot where leaking process media ... of retractable sensor housings , which are designed to tolerate extreme process conditions. ...
(Date:11/24/2015)... LOS ANGELES , Nov. 24, 2015 ... a biotechnology company focused on the discovery, development and ... Marban , Ph.D., Chief Executive Officer, is scheduled to ... December 1, 2015 at 10:50 a.m. EST, at The ... York City . . ...
(Date:11/24/2015)... ... , ... Whitehouse Laboratories is pleased to announce that it has completed construction ... dedicated to basic USP 61, USP 62 and USP 51 testing specific to raw ... and micro testing performed by one supplier. Management has formally announced that ...
Breaking Biology Technology:
(Date:11/9/2015)... 09, 2015 ... the "Global Law Enforcement Biometrics Market ... --> ) has announced the ... Biometrics Market 2015-2019" report to their ... ( ) has announced the addition ...
(Date:10/29/2015)...   MedNet Solutions , an innovative SaaS-based eClinical ... research, is pleased to announce that it has been ... one of only three finalists for a 2015 ... Growing" category. The Tekne Awards honor Minnesota ... innovation and leadership. iMedNet™ eClinical ...
(Date:10/29/2015)... Oct. 29, 2015  Connected health pioneer, Joseph ... explosion of technology-enabled health and wellness, and the business ... The Internet of Healthy Things . ... smartphones even existed, Dr. Kvedar, vice president, Connected Health, ... care delivery, moving care from the hospital or doctor,s ...
Breaking Biology News(10 mins):