Comparison among validations of twoanalitical methods for the determinationof cidofovir in human plasma“Comparison of the ...
2Analitical issueCitofovir(CVD, HPMPC, VISTIDE®) is anucleotide analogue of cytosine with antiviralactivity against a broa...
3Analitical procedureI)“Comparison of the quantitativeperformances and measurementuncertainty estimates obtained duringmet...
4Analitical procedureI)“Comparison of the quantitativeperformances and measurementuncertainty estimates obtained duringmet...
5Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien,...
6Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien,...
7Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien,...
8Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien,...
9Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien,...
10Selectivity● 6 indipendent source of plasma wereanalysed;No endogenous source of interference wasobserved at the ritenti...
11Matrix effectHPLC-MS-MS Breddeman et al.To analize possible matrix effect on thequantitative essays, sample extracts wit...
12Reagents stabilityHPLC-MS-MS Breddeman et al.● Relevant data regarding the stability ofCDV and IS at different concentra...
13Precision● Precision of this analitical method wasdetermined by computing relative standarddeviationr(RSD%) for repeatab...
14PrecisionHPLC-MS-MS Breddeman et al.
15Trueness and accuracy● Trueness espressed in terms of relativebias(%)5 concentration levels of spiked plasmaranging from...
16Trueness and accuracy● Accuracy takes into account thetotal(systematic and random)error. Thisinterval defines a region w...
17Trueness and accuracy● Accuracy espressed as percentages(%)ofnominal concentrationsAccuracy and precision were assested ...
18Trueness and accuracyHPLC-MS-MS Breddeman et al.● Accuracy espressed as percentages(%)ofnominal concentrationsAccuracy a...
19Calibration● For the method calibration, calibration andvalidation standards were prepared spikingblank human plasma.Cal...
20Fitting● Response function coresponds to the assessment of therelationship between the chromatographic response andthe c...
21FittingHILIC-UV Lecomte et al.quadraticmodelweighted 1/xquadratic modelweighted 1/x^2quadratic modelweighted 1/xlinear m...
22Fitting● In order to demostrate the linearity of theresults, a regression line was fitted betweenthe back-calculated con...
23Fitting● The calibration curve was obtained by fitting the ratio of the integrated mass peakarea of CDV to the integrate...
24LOD e LOQ● The lower limit of quantification (LLOQ)is defined as the smallest quantity that canbe quantitatiely determin...
25● The lower limit of quantification (LLOQ)is defined as the smallest quantity that canbe quantitatiely determined uner t...
26HPLC-MS-MS Breddeman et al.According to FDA, Food and DrugAdministration:“the lowest standard on the calibration curvesh...
27HPLC-MS-MS Breddeman et al.According to FDA, Food and DrugAdministration:“the lowest standard on the calibration curvesh...
28HPLC-MS-MS Breddeman et al.According to FDA, Food and DrugAdministration:“the lowest standard on the calibration curvesh...
29Recovery● The recoveries of CDV were determinedusing blank plasma sample spiked at threedifferent concentrations ranging...
30Recovery● The recoveries of CDV were determinedusing blank plasma sample spiked at threedifferent concentrations ranging...
31Risk assessmentThe risk of having future measurements fallingoutside the specified acceptance limits wasevaluated using ...
32Routine performanceThe validated method was applied routinely to thequantitative determination of CDV in human plasmasam...
33For the first trial:● 10 routine runs (one run per day)wereperformed, leading to the analysis of– 40 QC samples (spikedp...
34As can be seen, for the two highest QC levels, all(100%) QC samples fell within the acceptancelimits, thereby confirming...
35Routine performanceIon chromatogram of a quality control extract (spiked with 1250 ng/ml CDV) forCDV and the internal st...
36ConclusionsSELECTIVITYMATRIX EFFECTREAGENTS STABILITYPRECISIONACCURACYTRUTHNESSLOQ E LODRECOVERYRISK ASSESSMENTCONTROL C...
37ConclusionsSELECTIVITYMATRIX EFFECTREAGENTS STABILITYPRECISIONACCURACYTRUTHNESSLOQ E LODRECOVERYRISK ASSESSMENTCONTROL C...
38Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.Both the proposed methods seem to satisfy the fund...
39Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.●Lecomte et al. writes:”As can be seen the LC–MS/M...
40Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.●Lecomte et al. Denounce :”As can be seen the LC–M...
41Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.● However, the adequate treatment often requires p...
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Method Validation: Comparison among two analitical methods for the determination of cidofovir in human plasma

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A comparison between the method validation of two analytical methods. I did this presentation for an exam during my master degree course at University of Pisa. Greta Dalle Luche

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Method Validation: Comparison among two analitical methods for the determination of cidofovir in human plasma

  1. 1. Comparison among validations of twoanalitical methods for the determinationof cidofovir in human plasma“Comparison of the quantitative performancesand measurement uncertainty estimatesobtained during method validation versusroutine applications of a novel hydrophilicinteraction chromatography method for thedetermination of cidofovir in human plasma”F. Lecomte, C. Hubert, S. Demarche, C. De Bleye, A. Dispas, M. Jost , F. Frankenne,Journal ofPharmaceutical and Biomedical AnalysisJournal of Pharmaceutical and Biomedical Analysis 57 (2012) 153– 165GRETA DALLE LUCHEChimica Analitica III - 2 Luglio 2012“Quantification of cidofovir in human serum byLC–MS/MS for children”Andr´e Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aerJournal of Chromatography B, 861 (2008) 1–9
  2. 2. 2Analitical issueCitofovir(CVD, HPMPC, VISTIDE®) is anucleotide analogue of cytosine with antiviralactivity against a broad spectrum of DNAviruses. It has been approved for the treatmentof Cytomegalovirus retinis in AIDS patients. Itstherapeutic use is still under investigation, as itcould be applied to a much greater number ofpatologyes. Information about thepharmacokinetic properties of CVD areurgently required to determine the individualdrug exposure for different metabolismpatients(children or patients affected by renalinsufficiency) or different topictreatments(papilloma virus infection)..2.157.004.57An accurate quantification of CDV in human plasma in needed to run pre-clinical trials and to avoidtoxic dosing. The analytical method should quantify CDV in human plasma over, at least aconcentration v range of 100-1000 ng/mL..
  3. 3. 3Analitical procedureI)“Comparison of the quantitativeperformances and measurementuncertainty estimates obtained duringmethod validation versus routineapplications of a novel hydrophilicinteraction chromatography method forthe determination of cidofovir in humanplasma”F. Lecomte, C. Hubert, S. Demarche, C. De Bleye, A. Dispas, M.Jost , F. Frankenne,Journal of Pharmaceutical and BiomedicalAnalysisJournal of Pharmaceutical and Biomedical Analysis 57 (2012) 153–165● Plasmatic protein precipitation850 μL plasma + 850 μL HPO3(5% w/v)● Centrifugation● SPE Plexa PCXcatridges packedwith mixed mode polimeric cationexchange sorbentconditionment:1mL AcN, 500 μL ammonium formate (Ph3;20mM)washing:250 μL AcN-water(85:15v/v)elution:1.0 mL AcN-ammonium carbonate(Ph 10.0;20 mM)● HP HILIC-UV(275 nm)
  4. 4. 4Analitical procedureI)“Comparison of the quantitativeperformances and measurementuncertainty estimates obtained duringmethod validation versus routineapplications of a novel hydrophilicinteraction chromatography method forthe determination of cidofovir in humanplasma”F. Lecomte, C. Hubert, S. Demarche, C. De Bleye, A. Dispas, M.Jost , F. Frankenne,Journal of Pharmaceutical and BiomedicalAnalysisJournal of Pharmaceutical and Biomedical Analysis 57 (2012) 153–165Alltima HP HILIC● Volume of injection 5 μL● Column 150mm x 2.1 mm i.d., 3 μm● Pre-column 7.5mm x 2.1 mm i.d., 5 μm● isocratic separationAcN-NH4HCO3(72:28)(pH7; 20mM)● 25°C● Flow rate 0.21 mL/min● UV(275 nm)
  5. 5. 5Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aerJournal of Chromatography B, 861 (2008) 1–9● IS:300 μL plasma + 30 μL PMEG (100μg/mL in H2O)->1000 μL● Vortex● SPE catridges strong anionexchange(SAX) Varian Bond Elutconditionment:2 mL MeOH, 2 mL H2Owashing:2 x1 mL H2Oelution:2 X1.0 mL MeOH containg 3%HCl● Dryness 40°C (N2)● ->300 μL MeOH-H2O(50:50, v/v)modified with 3%ammonia● Vortex● Centrifugated 20 min 3220 x g(4000 rpm)● HPLCMS/MS
  6. 6. 6Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aerJournal of Chromatography B, 861 (2008) 1–9● IS:300 μL plasma + 30 μL PMEG (100μg/mL in H2O)->1000 μL● Vortex● SPE catridges strong anionexchange(SAX) Varian Bond Elutconditionment:2 mL MeOH, 2 mL H2Owashing:2 x1 mL H2Oelution:2 X1.0 mL MeOH containg 3%HCl● Dryness 40°C (N2)● ->300 μL MeOH-H2O(50:50, v/v)modified with 3%ammonia● Vortex● Centrifugated 20 min 3220 x g(4000 rpm)● HPLCMS/MS9-(2-phosphonylmethoxyethyl)guanine
  7. 7. 7Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aerJournal of Chromatography B, 861 (2008) 1–9● IS:300 μL plasma + 30 μL PMEG (100μg/mL in H2O)->1000 μL● Vortex● SPE catridges strong anionexchange(SAX) Varian Bond Elutconditionment:2 mL MeOH, 2 mL H2Owashing:2 x1 mL H2Oelution:2 X1.0 mL MeOH containg 3%HCl● Dryness 40°C (N2)● ->300 μL MeOH-H2O(50:50, v/v)modified with 3%ammonia● Vortex● Centrifugated 20 min 3220 x g(4000 rpm)● HPLCMS/MS
  8. 8. 8Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aerJournal of Chromatography B, 861 (2008) 1–9● IS:300 μL plasma + 30 μL PMEG (100μg/mL in H2O)->1000 μL● Vortex● SPE catridges strong anionexchange(SAX) Varian Bond Elutconditionment:2 mL MeOH, 2 mL H2Owashing:2 x1 mL H2Oelution:2 X1.0 mL MeOH containg 3%HCl● Dryness 40°C (N2)● ->300 μL MeOH-H2O(50:50, v/v)modified with 3%ammoniaHPLCMS/MSShimazu controller SCL10Avp with 2 separateShimazu pumps LC10Avp● Triple quadrupole massspectrometer API 2000(AppliedBiosystems/SCIEX, Concord)● Column(tetraalkoxysilanecolumn): Purospher Star RP-18 (125mm x 2 mm, 5um, Merk KgaA,Darmstadt)● Pre-column: 4 mm x 4 mm, amefeatures● T= 30 °C● Isocraticelution:50:49:1(v/v)methanol,water,ammonia Ph=10.5● 0.3 mL/minMRM, m/z:● CDV278.1->234.9● IS288.1->133.1● Negative electronspray ionization
  9. 9. 9Analitical procedureII)““Quantification of cidofovir inhuman serum by LC–MS/MS forchildren”André Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aerJournal of Chromatography B, 861 (2008) 1–9● IS:300 μL plasma + 30 μL PMEG (100μg/mL in H2O)->1000 μL● Vortex● SPE catridges strong anionexchange(SAX) Varian Bond Elutconditionment:2 mL MeOH, 2 mL H2Owashing:2 x1 mL H2Oelution:2 X1.0 mL MeOH containg 3%HCl● Dryness 40°C (N2)● ->300 μL MeOH-H2O(50:50, v/v)modified with 3%ammoniaHPLCMS/MSShimazu controller SCL10Avp with 2 separateShimazu pumps LC10Avp● Triple quadrupole massspectrometer API 2000(AppliedBiosystems/SCIEX, Concord)● Column(tetraalkoxysilanecolumn): Purospher Star RP-18 (125mm x 2 mm, 5um, Merk KgaA,Darmstadt)● Pre-column: 4 mm x 4 mm, amefeatures● T= 30 °C● Ph=10.5● 0.3 mL/minMRM, m/z:● CDV278.1->234.9● IS288.1->133.1● Negative electronspray ionization
  10. 10. 10Selectivity● 6 indipendent source of plasma wereanalysed;No endogenous source of interference wasobserved at the ritention time of CVD.Chromatograms of (A) a blank plasma chromatogram, (B) a spiked plasma at 100ng/mL and (C) a spiked plasma at 1000 ng/mL of cidofovir. Peak identification:(1) cidofovir, (2) endogenous compound.HILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.● Interferring drug or endogenous compoundwas considered if there was a signal closeto 0.3 min of the retention time of theanalyte or the IS7 serum extracts from 7 different pediatriccancer patients not receiving CDV therapywere analysed;● Possible “cross-talk” between the MS/MSchannelsThis was achieved by separately injecting:CDV at the highest concentrtion of thecalibration line(10000 ng/mL) andmonitoring the response in IS channel; byinjecting a serum spiked only with internalstandard and monitoring the response withthe CDV channel.Interferences were not observed.
  11. 11. 11Matrix effectHPLC-MS-MS Breddeman et al.To analize possible matrix effect on thequantitative essays, sample extracts with theanalyte of interest added post extraction werecompared with pure solutions prepared inmobile phase, containing equivalents amountsof the analyte of interest.Plasma spiked after extraction - Pure solution x100%Pure solutionIon-suppressive matrix effect on the ionisationof the IS is consistent with the matrix effect ofthe analyte. Therefore, the technical necessityfor the reliable quantification may not beadversely affectedby this matrix effect..(b)Typical ion chromatogram of blank human serum extract. (b) Typical ionchromatogram of LLOQ-human serum extract (spiked with 78.125 ng/ml CDV)for CDV.78 ng/mL -72.94%1250 ng/mL -72.14%10000 ng/mL -74.96%IS 10000 ng/mL -69.68%
  12. 12. 12Reagents stabilityHPLC-MS-MS Breddeman et al.● Relevant data regarding the stability ofCDV and IS at different concentrationwere not avaiable. Fo this reaon, stabilitytests were conducted in the followingdifferent matrices:Conditions CDV ISCalibration solution (Ringerssolution)After 1 month storing at 4°C -1.91% -0.14%Extracted QC samples (4 conclevels; 5 replicates)After 24h stoing at room T +(11.51-0.92)%Human plasma (4 conc levels; 5replicates)After 1 month storing at -20°C 94.36-98.72% ofnominalconcentrationAfter 3 freeze-thaw cycles 106.15-110.15% ofnominalconcentration
  13. 13. 13Precision● Precision of this analitical method wasdetermined by computing relative standarddeviationr(RSD%) for repeatability andtime-different intermediate precision ateach concentration level.5 concentration levels of spiked plasmaranging from 50 to 1020 ng/mL4 days of analysis, 5 repetitions each day● Precision espressed as relative standarddeviation(CV%)Accuracy and precision were assested bydeterming quality control samples at the:4 concentration levels (lower limit ofquatification, low, mid, highconcentration); five samples eachconcentrationon 3 subsequent validation daysIntra- and inter-day precisions(CV%) wereless than 7.8%>>>HILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.
  14. 14. 14PrecisionHPLC-MS-MS Breddeman et al.
  15. 15. 15Trueness and accuracy● Trueness espressed in terms of relativebias(%)5 concentration levels of spiked plasmaranging from 50 to 1020 ng/mL4 days of analysis, 5 repetitions each day● Accuracy espressed as percentages(%)ofnominal concentrationsAccuracy and precision were assested bydeterming quality control samples at the:4 concentration levels (lower limit ofquatification, low, mid, highconcentration); five samples eachconcentrationon 3 subsequent validation daysHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.
  16. 16. 16Trueness and accuracy● Accuracy takes into account thetotal(systematic and random)error. Thisinterval defines a region where each futureresults generated by the bioanalyticalprocedure has 95% chance to fall.The accuracy profile is obtained by linkingon one hand the lower bounds and on theother hands the upper bounds of β-expectation tolerance limits calculated ateach concentration levels for the validationstandards.β-expectation tolerance limits:X ± ks σˆ“β-Expectation and β-Content Tolerance Limits for BalancedOne-Way ANOVA Random Model Robert” W. Mee,Technometrics, Vol. 26, No. 3 (Aug., 1984), pp. 251-254HILIC-UV Lecomte et al.
  17. 17. 17Trueness and accuracy● Accuracy espressed as percentages(%)ofnominal concentrationsAccuracy and precision were assested bydeterming quality control samples at the:4 concentration levels (lower limit ofquatification, low, mid, highconcentration); 5 samples eachconcentrationon 3 subsequent validation daysIntra- and inter-day accuracies(CV%) werewithin ± 12.1%>>>HILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.● Accuracy takes into account thetotal(systematic and random)error. Thisinterval defines a region where each futureresults generated by the bioanalyticalprocedure has 95% chance to fall.
  18. 18. 18Trueness and accuracyHPLC-MS-MS Breddeman et al.● Accuracy espressed as percentages(%)ofnominal concentrationsAccuracy and precision were assested bydeterming quality control samples at the:4 concentration levels (lower limit ofquatification, low, mid, highconcentration); five samples eachconcentrationon 3 subsequent validation days
  19. 19. 19Calibration● For the method calibration, calibration andvalidation standards were prepared spikingblank human plasma.Calibration stadards were prepared at 7concentration levels, ranging from 50 to1000 ng/mL.4 series were performed by injecting allthe calibration stadards in triplicate.● The CDVstandard solution werepreparedby dissolving the accuratelyweighed reference standard in Ringerssolution to obtain a final concentration of100 μg/mL. The standard solution was thenserially diliuted with Ringers solution toobtain 8 working solutions, ranging from781.25 ng/mL to100 μg/mL.The calibration standards were thenprepared by spiking 270 μL blank humanplasma with 30 μL of the workingsolutions. The 8 calibration solutionobtained range from 78.125 ng/mL to 10μg/mLHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.
  20. 20. 20Fitting● Response function coresponds to the assessment of therelationship between the chromatographic response andthe concentration of the analyte. The optimal regressionmodel should be the one that firstly allows toaccurately quantify CDV over the widest concetrationrange and secondly provides the smallest bias over thisconcentration range.From each response function tested, theconcentration of the spiked plasmavalidation standards were back calculatedin order to determine the upper and lowerexpectation limits at β=95%.Theacceptance limit were set at +30%.The only fuction that complied with thedefined criteria is the weighted quadraticmodel using the weight 1/X^2HILIC-UV Lecomte et al.
  21. 21. 21FittingHILIC-UV Lecomte et al.quadraticmodelweighted 1/xquadratic modelweighted 1/x^2quadratic modelweighted 1/xlinear modellinear model
  22. 22. 22Fitting● In order to demostrate the linearity of theresults, a regression line was fitted betweenthe back-calculated concentration ofvalidation standards versus the introducedconcentration applaying a linear regressionmodel.HILIC-UV Lecomte et al.
  23. 23. 23Fitting● The calibration curve was obtained by fitting the ratio of the integrated mass peakarea of CDV to the integrated mass peak of the IS (y-axis) against the range ofadded analyte concentration(x-axis) using 1/X linear regression plot.HPLC-MS-MS Breddeman et al.
  24. 24. 24LOD e LOQ● The lower limit of quantification (LLOQ)is defined as the smallest quantity that canbe quantitatiely determined uner theexperimental conditions with the welldefined accuracy.Using the accuracy profile, the LLOQ wasestimated as 92.7 ng/mL. The LLOQ wasobtained calculating the smallestconcentration for which the β-expectationtolerance limit cross the acceptance limits.HILIC-UV Lecomte et al.
  25. 25. 25● The lower limit of quantification (LLOQ)is defined as the smallest quantity that canbe quantitatiely determined uner theexperimental conditions with the welldefined accuracy.Using the accuracy profile, the LLOQ wasestimated as 92.7 ng/mL. The LLOQ wasobtained calculating the smallestconcentration for which the β-expectationtolerance limit cross the acceptance limits.The limit of detection(LOD) was estimatedusing the mean intercept of the calibrationmodel and the residual variance of theregression and was evaluated to be 28.1ng/mL.HILIC-UV Lecomte et al.LOD = Sb + 3 σbLOD e LOQ
  26. 26. 26HPLC-MS-MS Breddeman et al.According to FDA, Food and DrugAdministration:“the lowest standard on the calibration curveshould be accepted as the limit of quantificationif the following conditions are met:● The analyte response at the LLOQ shouldbe at least 5 times the response comparedto blank response.● Analyte peak (response) should beidentifiable, discrete, and reproduciblewith a precision of 20% and accuracy of80-120%”U.S. Department of Health and Human Services, Food and DrugAdministration,Center for Drug Evaluation and Research (CDER), 2001.http://www.fda.gov/cder/guidance/4252fnl.htm..(b)Typical ion chromatogram of blank human serum extract. (b) Typical ionchromatogram of LLOQ-human serum extract (spiked with 78.125 ng/ml CDV)for CDV.LOD e LOQ
  27. 27. 27HPLC-MS-MS Breddeman et al.According to FDA, Food and DrugAdministration:“the lowest standard on the calibration curveshould be accepted as the limit of quantificationif the following conditions are met:● The analyte response at the LLOQ shouldbe at least 5 times the response comparedto blank response.● Analyte peak (response) should beidentifiable, discrete, and reproduciblewith a precision of 20% and accuracy of80-120%”U.S. Department of Health and Human Services, Food and DrugAdministration,Center for Drug Evaluation and Research (CDER), 2001.http://www.fda.gov/cder/guidance/4252fnl.htm.LOD e LOQ
  28. 28. 28HPLC-MS-MS Breddeman et al.According to FDA, Food and DrugAdministration:“the lowest standard on the calibration curveshould be accepted as the limit of quantificationif the following conditions are met:● The analyte response at the LLOQ shouldbe at least 5 times the response comparedto blank response.● Analyte peak (response) should beidentifiable, discrete, and reproduciblewith a precision of 20% and accuracy of80-120%”U.S. Department of Health and Human Services, Food and DrugAdministration,Center for Drug Evaluation and Research (CDER), 2001.http://www.fda.gov/cder/guidance/4252fnl.htm....e il LOD?LOD e LOQ
  29. 29. 29Recovery● The recoveries of CDV were determinedusing blank plasma sample spiked at threedifferent concentrations ranging from 100to 1020 ng/mLRecoveries were found to be constantaround 85% over the entire range studied.● The absolute recovery was determined by acomparison of the peak areas derived fromserum samples(QC sample at 3concentrations with 5 samples for eachconcentration) spiked before extractionwith the peak areas from serum samplesspiked after the extraction procedure.Plasma spiked before extraction x100%Plasma spiked after extractionThe recovery of IS(PMEG) wasdetermined similarly78 ng/mL 62.69%1250 ng/mL 51.38%10000 ng/mL 52.45%IS 10000 ng/mL 60.94%HILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.
  30. 30. 30Recovery● The recoveries of CDV were determinedusing blank plasma sample spiked at threedifferent concentrations ranging from 100to 1020 ng/mLRecoveries were found to be constantaround 85% over the entire range studied.● The absolute recovery was determined by acomparison of the peak areas derived fromserum samples(QC sample at 3concentrations with 5 samples for eachconcentration) spiked before extractionwith the peak areas from serum samplesspiked after the extraction procedure.Plasma spiked before extraction x100%Plasma spiked after extractionThe recovery of IS(PMEG) wasdetermined similarly78 ng/mL 62.69%1250 ng/mL 51.38%10000 ng/mL 52.45%IS 10000 ng/mL 60.94%...e le dev standard?riproducibilità?HILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.
  31. 31. 31Risk assessmentThe risk of having future measurements fallingoutside the specified acceptance limits wasevaluated using the β-expectation tolerance intervalsobtained with the previously selected regressionmodel. This risk is computed for each concentrationlevel investigated, as the sum of the proportion ofresults effectively lying outside the upperacceptance limit on one hand and under the loweracceptance limit on the other hand. The maximumrisk tolerated was set to 5%, meaning that it isaccepted that at most each future result provided bythe developed method will have five chances out of100 to fall outside the acceptance limits of ±30%.The risk was clearly smaller than 5% over the validconcentration range. However, this risk was about36% for the smallest concentration level (50 ng/mL)thus confirmingthe inaccuracy of the resultsgenerated by the bioanalytical method at thisconcentration level.HILIC-UV Lecomte et al.Illustrates the risk profile for CDV results obtained at each validationstandardconcentration level.
  32. 32. 32Routine performanceThe validated method was applied routinely to thequantitative determination of CDV in human plasmasamples from a pre-clinical trial.The estimate concentration level, of a patient after adefined time T5, is equal to 184.9 ng/mL anddemonstrating the good selectivity of thecurrentmethod at low concentration levels (twotimes the LLOQ).In each trial apart from the calibration standards andthe real unknownsamples, each analytical runinvolved quality control (QC) samples prepared inblank plasma spiked with CDV in order to reachthree concentration levels: 150, 500 and 850 ng/mL.HILIC-UV Lecomte et al.Fig. 8 illustrates chromatograms obtained from incurred blank sample at T0 (pre-dose) and a real unknown sample from the same patient at T5
  33. 33. 33For the first trial:● 10 routine runs (one run per day)wereperformed, leading to the analysis of– 40 QC samples (spikedplasmasamples) at each of the threeconcentration levels– as well as 192 real samples.For the second trial:● 15 routine runs were realized.– The numbers of QC samples for thistrial add up to 172– the number of real samples to 252.Each level of concentration was analysed inquadruplicates.HILIC-UV Lecomte et al.Routine performance150 ng/mL500 ng/mL850 ng/mL
  34. 34. 34As can be seen, for the two highest QC levels, all(100%) QC samples fell within the acceptancelimits, thereby confirming the validation stepprediction.For the smallest QC level (150 ng/mL) only twosamples out of 106 at this level fell outside theacceptance limits of ±30% resulting in a proportionof 98.1% which is above the minimum requirementof95% defined during the method validation.The routine performance of thisbioanalyticalmethod demonstrates that this methodis clearly appropriate forits final use since at least95% of the QC samples are included in theroutineacceptance limits.HILIC-UV Lecomte et al.Routine performance150 ng/mL500 ng/mL850 ng/mL
  35. 35. 35Routine performanceIon chromatogram of a quality control extract (spiked with 1250 ng/ml CDV) forCDV and the internal standard PMEG. (b) Ion chromatogram of apae diatricpatient sample extract (blood withdrawal 1 h after infusion of 2.5 mg/kg CDV,the measured CDV concentration amounted to 5270 ng/ml) for CDV andtheinternal standard PMEG.HPLC-MS-MS Breddeman et al.To apply and test the new LC–MS/MS methodunder realistic application conditions, weanalysed blood samples of a paediatric cancerpatient who intravenously received 2.5 mg perkg bodyweight CDV. Blood samples werecollectedat 0 h (pre-treatment), 1 h, 2 h, 3 h, 4 hand 6 h after infusion....no quantitative data
  36. 36. 36ConclusionsSELECTIVITYMATRIX EFFECTREAGENTS STABILITYPRECISIONACCURACYTRUTHNESSLOQ E LODRECOVERYRISK ASSESSMENTCONTROL CHARTHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.✔--✔✔✔✔✔✔✔✔✔✔✔✔-✔X--
  37. 37. 37ConclusionsSELECTIVITYMATRIX EFFECTREAGENTS STABILITYPRECISIONACCURACYTRUTHNESSLOQ E LODRECOVERYRISK ASSESSMENTCONTROL CHARTHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.✔X-✔✔✔✔✔✔✔✔✔✔✔✔-✔X--3.4-9.7(RSD%) 3.0-4.0(RSD%)100.0 ng/mL e 28.09 78.125 ng7mL.. no LODRange 78-10.000 ng/mLRange 93-1020 ng/mL
  38. 38. 38Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.Both the proposed methods seem to satisfy the fundamental prerequisite for the investigationof the pharmacokinetic properties of CDV:● With a linear signal response in the concentration range from 78.125 ng/ml to 10000and from 100.0 to 1000 ng/ml, the developed methods covers the expectedconcentrations in patients at least up to four half-lives.● The required plasma volume for the analysis is restricted for both the method: 300 μL
  39. 39. 39Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.●Lecomte et al. writes:”As can be seen the LC–MS/MS method of Breddemann et al.is only able to provide accurate results 95 times out of 100 over the concentration range2000–4870 ng/mL of CDV. Indeed, for concentration levels of CDV smaller than 2000ng/mL, the risk to obtain results out of the±30% acceptance limits is greater than 5 %.This means that there are more than five chances out of 100 that future results will falloutside the acceptance limits recommended by the Food and Drug Administration(FDA).
  40. 40. 40Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.●Lecomte et al. Denounce :”As can be seen the LC–MS/MS method of Breddemannet al. is only able to provide accurate results 95 times out of 100 over the concentrationrange 2000–4870 ng/mL of CDV. Indeed, for concentration levels of CDV smaller than2000 ng/mL, the risk to obtain results out of the±30% acceptance limits is greater than 5%. This means that there are more than five chances out of 100 that future results willfall outside the acceptance limits recommended by the Food and Drug Administration(FDA).
  41. 41. 41Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.● However, the adequate treatment often requires polymedication(e.g. cancer patients)which is related to potential analytical interference. Therefore, analytical methods for thedetermination ofCDV in human serum necessitate a selective detection.●Moreover matrix effect has not been investigated in the HILIC-UV.●In the HPLC-MS-MS method, the conducted tests to investigate these effects clearlyindicatea pronounced ion-suppressive matrix effect. But this effect wasconsistent for boththe analyte and the internal standard. Therefore, the technical necessity for the reliablequantification may not be adversely affected by this matrix effect.

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