Fundamentals: Bioanalytical LC/MS Method Validation -fit .
Fundamentals: Bioanalytical LC/MSmethod validation - fit for purposeSUMS SEMINAR SERIESJULY 2, 2020Ludmila AlexandrovaStaff Scientist, Stanford University Mass Spectrometry (SUMS)ludmilaa@stanford.edu
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Related Seminars in SUMS SeriesTHURSDAY APRIL 2, 2020 - SUMS SEMINAR SERIESFundamentals: Measuring concentrations of small molecules using mass spectrometry theory and practice, Part ITHURSDAY APRIL 23, 2020 - SUMS SEMINAR SERIESFundamentals: Measuring concentrations of small molecules using mass spectrometry theory and practice, Part IITHURSDAY May 21, 2020 - SUMS SEMINAR SERIESFundamentals: Applications of LC/MS in small molecule drug discoveryRecording and slides are available at https://mass-spec.stanford.edu/events3
Presentation OutlineIntroductionØ Bioanalysis in Drug Discovery and DevelopmentØ Fit For Purpose Concept in BioanalysisPart 1Ø Method Validation: Bioanalytical Assay Performance Attributes andRequirementsPart 2Ø Case study 1 – Melphalan LC-MS/MS Method Development andValidationØ Case study 2 –Determination of Global DNA Methylation Levels inClinical Samples by LC-MS/MSConclusion4
Introduction5
LILOCCSCLSP1P2P3Market LC/MS bioanalytical methodsParentMetaboliteNon-GLP /GCP method Pharmacokinetic/ADME studies Toxicology studies Clinical studies Biomarker researchIt is critical to use well characterized and appropriately validated LC/MSbioanalytical methods to ensure data integrity6GLP: Good Laboratory PracticeGCP: Good Clinical Practice
What Is Bioanalysis7qBioanalytical Method (BA) performs quantitative analysis of drugs andmetabolites in biological matrices (e.g. plasma, blood, serum or urine)in support of pharmacokinetic (PK), toxicokinetic (TK) and clinicalstudies, pharmacology evaluation, biomarker research, formulationdevelopment, etc. Small molecule platform – LC/MS/MSqFully validated BA methods support pivotal TK/PK and clinical studiesthat are used in regulatory submissionsqFor non regulatory studies, extent of BA method validation may bedefined by the purpose of the study.
Why is Bioanalytical Method Validation Important?q High data qualityØØØReproducible and reliable dataSensitivity establishedStability investigatedq Data used for safety and efficacy assessmentq Data integrity aids to results interpretationq Increasing demand of peer-reviewed scientific journalsWhy Not Done Routinely?qTime consuming and labor intensiveqNot always required e.g. screening studies, exploratory studies, endogenous/biomarkersquantitationFit for purpose approach is a useful tool in conserving resources andtime to generate reliable data8
Fit For Purpose (FFP)q “The fit for purpose (FFP) concept states that the level of validation should beappropriate for the intended purpose of the study”Ø FFP applies to drugs, their metabolites, endogenous compounds andbiomarkers and is used extensively during exploratory stages of drugdevelopmentØ It is important to evaluate study goals and objectives as well asquantification approaches and create validation strategyØ Extent of validation and key parameters should be specified and justified invalidation plan: e.g. accuracy, precision, stability etc.Ø Specific validation requirements and acceptance criteria may need to beestablished for each analyteFood and Drug administration. Bioanalytical method validation Guidance for od-validation-guidance-industry9
Method Validation: Bioanalytical Assay PerformanceAttributes and Requirements10
BA Method Validation Workflowo Method development Instrument sensitivity: optimize column chemistry, mobile phase, mode of ionization, SRM, etc. Sample preparation: PP simple, L-L less matrix impact, SPE more manipulations Determine curve fitting/weighting; assess accuracy/precision Key indicators: ion suppression, lot to lot variabilityo Validation (guidance driven) Precision and Accuracy: 20% at LLOQ)3 runs, each a curve 4 QC levels (n 6) à %CV and % Bias 15% (orStability in biological matrix (room temperature, Freeze-Thaw, frozen storage, auto-sampler) andstock solution (storage)Specificity (endogenous interference), matrix effect, selectivity, recovery, carryovero In-study (routine) analysis Curve, QC (3 levels, n 2), blank, contamination and carryover, IS, peak shape, RT11
Method Validation: Key ParametersoooSmall molecule platform – LC-MS/MS in SRM modeMethod Validation planBioanalytical assay performance attributes and requirementsoooooooooo12Reference Standards/Internal StandardsCalibration curve, range, regression modelSensitivityQC samplesAccuracyPrecisionDilution effectSelectivity, SpecificityRecoveryStability/Incurred sample reanalysisoooDocumentationInstrumentationSoftware
Reference Standards/ Internal Standards13oCertified reference standards with known identity and purity should be used toprepare spiking solutions of known concentrations (certificate of analysis, CoA)oInternal standard does not require CoA if it demonstrates that it does notinterfere with quantification of analyteoAnalyte stock and working solution stability should be tested to justify durationand storage conditionsoIsotopic exchange stability of IS (analyte specific)
Calibration Curveo14Calibration curve is a relationship between the instrument response andconcentration of analyte within selected concentration rangeØ Calibration curve should be prepared in the same matrix as the samplesand extracted side by side with the same procedureØ Choose concentration range of method on the basis of concentration rangeexpected in particular study: Lower Limit of Quantification (LLOQ) andUpper Limit of Quantification (ULOQ)Ø LLOQ defines the method sensitivity and is assessed during methodvalidationØ Use simplest regression model that describes the concentration – responserelationship; appropriate weighting schemeØ If more than one analyte, calibration curve should be generated for allanalytes
Quality Control SamplesoQuality control sample (QC) is a sample spiked with the known amount ofanalyteØ QCs are prepared in the same matrix as samplesØ QC samples are used to assess precision, accuracy of the assay andstability of analyte(s)Ø QCs prepared from a separate stock solutionØ It is recommended that blank matrix is screened before QC preparationØ To assess a dilution effect, QCs above Upper Limit of Quantification maybe prepared and analyzed in diluted form and monitored during validation15
Precision and AccuracyooPrecision is a repeatability of the series of measurements which isexpressed as % CVAccuracy is the ability to measure a true value based on nominal value which isexpressed as % deviation from nominal value (% Bias)ØØØ16Precision and Accuracy of the method is determined based on QC replicate analysisat the LLOQ, Low, Mid and High QC levels over full calibration range conducted overseveral days (at least 3 independent runs).Precision and accuracy is calculated within-run (Intra-assay) and between-run (Interassay)Each run includes a calibration curve and multiple QC concentrations that areanalyzed in replicates ( 5 replicates per each level).
Selectivity, Specificity and Matrix EffectoSelectivity is the ability to differentiate and measure the analyte of interest in the presence of endogenouscompoundsØØ17Selectivity demonstrated by analyzing blank samples of appropriate matrix from multiplesources (at least 6) to determine and minimize interferenceSelectivity should demonstrate minimal or no response related to interfering component at the RT ofanalyte in blank sample ( 20% LLOQ; 5% of the IS response)oSpecificity is the ability to unequivocally differentiate the analyte of interest in the presence of otheranalytesØ Potential interferences: compounds structurally similar to analyte, unstable metabolites (e.g.esters, unstable glucuronides, N-oxides etc.), isomers, impurities, decomposition products,concomitant medications and other xenobioticsØ Internal standard evaluated for interference on analyte quantitationoMatrix effect is a change in the analyte response due to unspecified components in the matrixØ Evaluated in relevant patients or special populations; hemolyzed or lipemic samples or ifstabilizer or enzyme inhibitor is used during sample collectionØ Determine ion matrix suppression or enhancement
Recovery, Carryoverq Recovery is the extraction efficiency of analytical process reported as a percentage of theknown amount of analyte carried out through the processing methodoRecovery needs to be determined and optimized to insure it is efficient andreproducible It does not need to be 100%, but consistent and reproducible over calibrationrange (Low, Mid, High) Applies to both, analyte and internal standardRecovery performed by comparing analytical results of extracted samples withcorresponding spiked post-extraction samples, representing 100% recoveryq Carryover is a change in measured concentration due to residual analyte interference frompreceding sampleoo18Carryover should be evaluated and eliminatedIf not possible, study samples should not be randomized
Stability of Analyte(s) in Biological Matrixq QCs are used for stability assessment 19Autosampler (re-injection reproducibility)Ø Stability of processed samples at autosampler temperatureBench-top (short-term) matrix stabilityØLaboratory handling conditions. Kept on the bench top at the same temperatureand for the same duration as study samplesExtraction/injection (sample processing stability)ØIn process sample stability to be used during sample analysis Freeze-Thaw matrix stabilityØ Stability assessment after multiple cycles of freezing and thawing according tothe same procedure applied to the study samples Long-term matrix stabilityØ Stability of analyte should be demonstrated under the same storage conditionsand duration as study samples
Incurred Sample Reanalysis (ISR)qqqqq20ISR is a repeat analysis of subset of samples which had been analyzedpreviously in a separate runISR confirms reliability of reported dataIt is used to support precision, accuracy and stability data establishedwith QCs.Ø E.g. presence of unstable metabolites which could degrade back toparent compoundISR acceptance criteria measured by % difference of the resultsbetween original value (1) and repeat value (2) divided by mean of 1and 2ISR required for regulatory studies
Case Study 1Development and Validation of LC-MS/MS Bioanalytical Method forDetermination of Melphalan in Human PlasmaKim JW, Alexandrova L, DeMarchis E, Lee D, Chien A. (2012) “LC-MS/MS Quantification Of Melphalan Plasma Levels In ChildrenUndergoing Selective Intra-arterial Infusion Of Chemotherapy For Retinoblastoma,” Investigative Ophthalmology & Visual Science. March2012, 53, 468.21
Background22qMelphalan is an alkylating agent with effective tumoricidal propertiesbut also severe systemic side effects.qA minority of subjects developed neutropenia, which suggests thatsystemic diffusion of the drug does occur.qDeveloping a reliable LC-MS/MS assay to determine plasma levels ofmelphalan following ophthalmic artery infusion is critical in optimizingthe benefits of this treatment.
Melphalan LC-MS/MS Method DevelopmentSUMS seminar seriesFundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practice, Part IFundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practice, Part IIFundamentals: Applications of LC/MS in small molecule drug discovery23
StructuresMelphalan24Internal Standard: Melphalan-D8
LC-MS/MS MethodooooooooTSQ Vantage (Thermo Fisher)CompoundNameSRM TransitionCollision Energy(v)Accela 1250CTC PAL AutosamplerMelphalan305 288305 246305 194122234Melphalan-D8313 254313 2932312ESI Column: Polaris A 5 µm C18 50x2.1 mmRun time 5.6 minInjection volume – 10 µLWash solutionso Strong – methanolo25Weak – 5% methanol/ 95%waterTime (min)%A (0.1%FAin water)%B (0.1%FA 55950.253.680200.255.680200.25
Extraction ProcedureKimJ 120402 35370 46 - TIC - SM: 5 RT: 0.62 - 4.62 NL: 4.19E7F: p ESI SRM ms2 305.032 [ 194.054-194.059, 246.042-246.047, 288.060-288.065]RT: 2.62100959085807570ooooooo100 µL plasma10 µL melphalan spiking solutions10 µL IS (500 ng/mL Melphalan-D8)400 µL ice cold methanolVortex, centrifugeTransfer to the injection vial10 µL injectedRelative 011.261.01.53 1.711.971.52.192.03.032.442.5Time (min)3.263.03.47 3.68 3.863.54.084.424.04.5KimJ 120402 35370 46 - TIC - SM: 5 RT: 0.60 - 4.60 NL: 1.22E7F: p ESI SRM ms2 313.145 [ 254.098-254.102, 296.118-296.123]RT: 2.601009590Melphalan-D885807570Relative 311.541.51.842.062.02.422.5Time (min)2.97 3.163.03.463.53.693.934.04.254.524.5
Melphalan LC-MS/MS Method Validation27
Melphalan LC-MS/MS Method Validation PlanExperimentWithin-run precision and accuracyQC level (ng/mL)LLOQ2LQC6MQC100HQC3200n 6n 6n 6n 6n 6n 6n 6Between-run precision and Accuracy(3 runs)Calibration Curve(10 points – 1 per run)Acceptance criteriaSelectivity and SpecificityRecoveryAcceptance criteria28ExperimentShort-term stability (bench) 20% 15% 15%3/3NA3/6NA3/6NALQCMQCHQC261003200n 1 for all calibration points 20% 20%Post-preparative Stability (reinjection)n 6n 6n 6Acceptance criteria 15% 15% 15%Freeze-Thaw Cycles (n 3)n 6n 6n 6Acceptance criteria 20% 20% 20% 15%6 different lots ( 20% LLOQ)LLOQ 20%Acceptance criteriaBack calculated concentrationsQC level (ng/mL) 20%
SelectivityLot #Measured concentration(ng/mL)% of LLOQ950.199.4960.115.6971.3970.31002003006 different lots of blank human plasma weretestedSome endogenous interference at the retentiontimes of the analyte was observed.Blank plasma from lot#97 was not used for themethod validation.endogenousKimJ 120329 35370 38 - TIC - SM: 5 RT: 0.57 - 4.57 NL: 1.59E5F: p ESI SRM ms2 305.032 [ 194.054-194.059, 246.042-246.047, 288.060-288.065]KimJ 120329 35370 38 - TIC - SM: 5 RT: 0.58 - 4.58 NL: 1.76E7F: p ESI SRM ms2 313.145 [ 254.098-254.102, 296.118-296.123]1.5410095903.67859085RT: lative IntensityRelative .202025201515101050.92001.029Acceptance criteria: 20% LLOQ6035305RT: 2.58100951.52.02.5Time (min)3.03.54.04.50.781.131.01.391.59 1.771.52.03 2.232.02.972.5Time (min)3.03.183.533.53.844.10 4.29 4.484.04.5It is important to screen blank plasma
Representative Calibration Curve Data (3 runs)Y 0.0171088 0.00829092*X R 2 0.9991 W: 1/X 2NominalConcentrations(ng/mL)MeanCV (%)Bias .17-2.403.02.82.6Area 01001011.990.732001980.291-0.83Calibration 08Y-int0.0167.13r20.9980.17Calibration curve concentration range: 2 ng/ml – 400 ng/mLLower Limit of Quantification 2 ng/mLAcceptance criteria: LLOQ 20%; Other levels: 15%ng/mL400
QC Precision and Accuracy Data31LLOQLQCMQCHQCConc. (ng/mL)261003200Aliquot (mL)0.10.10.050.01Dilution factor11210Mean% CV% Bias1.9510.3-2.50Mean% CV% etween-run6.421045.796.416.954.04Calibration curve range 2 – 400 ng/mLLLOQ 2 ng/mLMethod ULOQ 4000 ng/mL29898.63-6.5932898.932.78Within-run and between-run precision andaccuracy were assessed by calculating dailyand overall concentration values for sixreplicates of the LLOQ, Low-, Mid- and Highlevel validation QC samples extracted andanalyzed on the same day and in threeseparate runsAcceptance criteria:Ø LLOQ - %CV and % Bias 20%Ø%CV and % Bias 15%
Melphalan Extraction RecoveryQC% RecoveryLow97.1Mid100High92.2Recovery sampleso Extracted QC samples (Extracted )oControl samples: Extract blank plasma spiked with IS and analyte added after extraction(Post-Extracted)Absolute recovery determined based on analyte/IS area ratio32
Short-Term StabilityBench-top (short-term) matrix stabilityMelphalan stored in human plasma on the bench at ambient temperature for 4 hoursSamples at 2ng/mL and 200ng/mL were outside acceptance criteria ( 20%)Study plasma samples should be collected and frozen within 4 hours of the samplecollection time pointAutosampler (re-injection reproducibility)Melphalan is stable in human plasma extracts held at 10 C for 24 hoursEither an entire run containing analytical standards or individual samples may bereinjected as needed during this time period without compromising the accuracy ofthe determinationFreeze-Thaw CyclesNo significant changes in the measured melphalan concentrations outside the expectedaccuracy for the method (% Bias 20%)Melphalan is stable for at least 3 Freeze-Thaw cycles in human plasma when stored at-80 10 C.33
Melphalan LC-MS/MS Method Validation SummaryStandards-QCs (Low, Mid and High)7.71 – 9.6LLOQ10.3Within-run Accuracy (% Bias)-(-6.59) – 6.53(-2.50)Between-run Precision (%CV)0.29 – 4.05.79 – 8.939.03(-4.67) – 2.252.78 – 6.95(-0.94)Within-run Precision (%CV)Between-run Accuracy (% Bias)Analyte Recovery %Selectivity: Matrix BlanksStabilityPost PreparativeShort Term (Bench): 4 hours at room temperature inplasmaFreeze-Thaw CyclesIntermediate Solution Stability (Analyte)3492.2 - 100Low interference ( 20% of LLOQ)24 hours at 10 C in autosampler 4 hours at room temperature in plasma3 cycles 7 months at -80 C
Results35üBioanalytical method for determination of melphalan in human plasmawas shown to be accurate, precise and selective over the concentrationrange from 2 to 4000 ng/mLüMelphalan is stable in human plasma under conditions testedüThis method is suitable for human plasma samples analysis
Endogenous/Biomarker Method ValidationRecommendations36
Endogenous/Biomarkers: Method Development andValidation Recommendations37oNo consensus or guidelines for the assay validation approachesoUtilize the fit for purpose strategy to identify the most important elements ofmethod development and validation studiesoSame workflow as for drug method validationØ Method development, exploratory or definitive method validation, instudy sample analysisoCritical to ensure integrity of data when determining biomarker concentrationsoNeed to understand what is being measured, biological relevance, and datalimitation in a given assay
Endogenous/ Biomarkers: Calibration Curveq Calibration curve selection and preparation are central for the method development,validation and its applications Evaluate suitability of biological matrixØØØo38Matrix may contain residual levels of endogenous analytes which will effectaccuracyLevels may vary because of age, gender, disease etc.Screening for biological matrix with low levels of endogenous analyte which willnot interfere with quantification of analyte ( 20% LLOQ)Evaluate surrogate matrix (neat, artificial, stripped matrices)ØØStable isotope internal standards can be usefulInvestigate if concentration-response relationship of the analyte in the studysample matrix is similar as that in a substitute matrix
Endogenous/Biomarkers: QC Samples/StabilityoQC SamplesØØØØØoTotal QC concentrations validated before running samplesAlternate QC samples may be used for validationStability (case by case)Ø Short-term and bench-topØØ39The concentrations of QCs should represent expected study concentrationsThe endogenous concentration of the analyte in biological matrix evaluatedbefore QC preparationValidation QCs include both: un-spiked and spiked with known amount ofauthentic analyteAutosamplerLong-term stability
Case Study 2Global DNA Methylation as a Risk Marker for Cancer and NutritionalHealth.Yajing Hu1, Ludmila Alexandrova2, Allis S. Chien2, Megan P. Hitchins1; (1)Departments of Medicine, Stanford CancerInstitute, (2)Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University, Stanford, CA40
BackgroundMdCDNA methylation in mammals occurs at the 5-carbon site of the cytosine pyrimidine ring withinCpG dinucleotidesMethylation states can be modified by environmental or life-style factors and nutrientsEpigenetic modifications including DNA methylation are potentially reversible, however, itremains unknown whether dietary interventions resulting in significant weight loss willreverse pathological global methylation states to normal levelsThe goal of the study is to develop, optimize and validate liquid chromatography massspectrometry (LC-MS/MS) method to accurately and reproducibly quantify 5-methyl-2’deoxycytidine (MdC), 2’-deoxycytidine (dC) and 2’-deoxyguanosine (dG) in hydrolyzed DNAto determine global DNA methylation levels in clinical samples41
Development and Validation of LC-MS/MS Methodfor Quantification of MdC, dC and dG inHydrolyzed DNA42
LC-MS/MS Method Development (Fit For Purpose)SUMS seminar seriesFundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practice, Part IFundamentals: Measuring concentrations of small molecules using mass spectrometry - theory and practice, Part IIFundamentals: Applications of LC/MS in small molecule drug discovery43
Nucleoside StructuresMdC (5 Methyl 2’-deoxycytidine)15N -dC3dG (2’-deoxyguanosine)dC (2’-deoxycytidine)15N -dG544
LC-MS/MS MethodooooooShimadzu LC/MS 8030ESI Shimadzu LC-20ADXR ProminenceColumn: Synergy Fusion-RP 80Å, 150x2.0 mm;4µm particles (Phenomenex)Column T 40 CFlow rate: 0.2 mL/min SolventsA: 0.1% formic acid water ooo45B: 0.1% formic acid acetonitrileGradient: 0% to 80% B in 9 minutesRun time 13 minInjection volume – 10 µLCompoundNameSRMTransitionCollisionenergy (V)Dwell time (ms)MdC241.80 126.10-12100dC228.00 111.95-1250dG267.80 151.95-155015 N -dC3231.00 115.10-105015 N -dG5275.00 157.10-1050
DNA Extraction and Hydrolysis ProcedureDNA amount and hydrolysis protocol optimized during LC-MS/MS methoddevelopmentDNA extraction and HydrolysisØ Buffy coat DNA was extracted using standardphenol chloroform methodØ 0.5 µg of DNA was then hydrolyzed by a 3step enzyme digestionCalibration curveØ Calibration curve was prepared by spikingstandard solutions into DNA buffer mixtureand was treated in the same way as DNAsamples including enzymatic hydrolysis.q Internal standards 15N3-dC and 15N5-dG were added tothe hydrolysates.q DNA digests and calibration standards were diluted withwater and filtered to remove large proteins.q Prepared samples were injected into LC-MS46
Calibration Curve Ranges and QC SamplesAnalyteCalibration rangesMdC0.746 – 299 ng/mL3.1 nM - 1.24 µMdC18.7 – 1490 ng/mL82.4 nM – 6.56 µMdG18.7 – 1490 ng/mL70.0 nM – 5.58 µMQC DNAMethylation LevelQC1-LLOQHuman non methylated DNAQC2-MidPooled human PBL DNAQC3-High-1Human fully methylated DNA from HCT 116QC4-High-2Human fully methylated DNA from MilliporeNon methylated, fully methylated human DNA standards and pooled human PBLDNA ( baseline) were used for the validation47PBL: peripheral blood lymphocyte DNA
Calculation of Global DNA Methylation LevelAssay # 1%MdC MdC/(MdC dC)%MdC MdC/dGQC1MeanCV (%)2.642.922.782.55QC2MeanCV (%)4.202.744.433.49QC3MeanCV (%)4.991.305.381.03QC4MeanCV (%)5.614.326.045.68 DNA methylation (%) MdC/(MdC dC) DNA methylation (%) MdC/dG48
LC-MS/MS Method Validation (Fit For Purpose)49
Method Validation PlanExperimentExperimentWithin-run precisionQC for DNA methylationQC1QC2QC3QC4LLOQMidHigh-1High-2n 6n 6n cceptance criteriaNANANAn 6Selectivity/Specificity/Matrix effectBetween-run precision(3 runs)Calibration Curve(10 points – 1 per run)Acceptance criterian 4n 4n 4n 4Back calculated concentrations 20% 15% 15% 15%Ensure chromatographic separation forDNA and RNA nucleosides for potentialinterferencePost-preparative Stability (reinjection72hr)n 6Acceptance criteria 15%Freeze-Thaw Cycles (n 3)Acceptance criteria50QC for DNA methylationn 4 20%
Selectivity, Specificity and Matrix effect(Fit For Purpose)LC-MS nucleoside separationEnsure chromatographic separation for DNA and RNA nucleosides for potential interference51
Representative MdC Calibration Curve Data: Precisionand Accuracy (3 runs)Y (0.00420620)X (-7.91350e-005) R 2 0.996 W: 1/X 2MeanCV (%)Bias libration curve concentration range: 3.11 nM – 1.24 µMLower Limit of Quantification 3.11 nM52Acceptance criteria: LLOQ 20%; Other levels: 15%1.25MdC1.000.75Area 0200Concentration (ng/mL).250Calibration CurveparametersMeanCV (%)slope0.00415.53y-int.0.00057.47r20.99400.14
QC Precision DataDNA Methylation %MdC MdC/(MdC dC)QC1QC2QC3QC4nonmethylatedpooled bloodFullymethylatedHCT116FullymethylatedMPAliquot (ug)0.50.50.50.5Dilution factor1111Within-runMean2.975.006.116.31% % CV7.212.113.37.7Within-run precision was assessed by calculatingCV values for six replicates of the validationQC samples extracted and analyzed on thesame dayBetween-run precision was determined bycalculating CV values for 4 replicates ofvalidation QC samples extracted andanalyzed on 3 different daysAcceptance criteria:Ø LLOQ - %CV 20%Ø%CV 15%
Extraction Recovery: MdC, dC and dGnMMdCLow (15.5)Mid (125)High (622)dCLow (81.9)Mid (656)High (3290)dGLow (69.7)Mid (558)High (2790)54% Recovery10010311010093.310710396.3107Recovery samplesoAdd analyte and internal standard prior tohydrolysis and extraction (Extracted )oControl samples: Extract sample spiked withIS and analyte added after hydrolysis andextraction (Post-Extracted)Absolute recovery determined based onanalyte/IS area ratio
Freeze-Thaw StabilityDNA Methylation%MdC MdC/(MdC dC)QC2DNA methylation values are stable whenstored at -80 C for at least 3 freeze/thawcyclespooled bloodAliquot (µg)0.5Dilution factor1Freeze-Thaw 155Mean4.20% CV2.74Freeze-Thaw 3Mean4.27% CV2.96No significant changes in the measured DNAmethylation values outside the expectedprecision and accuracy of the method werefound, indicating that 3 Freeze-Thaw cyclesdid not affect the sample integrityAcceptance criteria:Ø %CV and % Bias 20%
Global DNA Methylation: Method Validation SummaryStandards-QCs (Low, Mid and High)2.6 - 4.58LLOQ5.1Within-run Accuracy (% Bias)---Between-run Precision (%CV)0.7 – 10.87.7 – 13.37.2(-5.24) – 13--Within-run Precision (%CV)Between-run Accuracy (% Bias)Analyte Recovery %Selectivity, Specificity, Matrix effectStabilityPost PreparativeFreeze-Thaw CyclesIntermediate Solution Stability (Analytes)5693.3 - 110Low interference72 hours at RT in autosampler3 cycles4 months at -80 C
Pilot Study: Effect of Low Fat vs. Low-Carbohydrate Dieton Global DNA MethylationDNA methylation (%) MdC/(MdC dC)57Diet TypeBaseline12 monthsDifferenceLow Carb4.314.05-0.26Low Carb4.183.94-0.24Low Carb4.63.8-0.80Low Carb4.063.92-0.14Low fat4.23.9-0.30Low fat3.964.070.11Low fat4.064.05-0.01Low fat3.773.820.05ØDNA methylation levels were determined in 16 paired clinical samples collected during atrial of obese individuals prior to, and following the dietary intervention (Prof. C. Gardner)ØResults show trend of reduced methylation level 12 months after intervention (pvalue 0.069, Wilcoxon signed ranks test).
ResultsLC-MS/MS method to accurately and reproducibly measures global DNA methylationlevels developed and validated.Fit for purpose approach was used to validate and monitor assay performance. DNA buffer matrix was evaluated and used for calibration curve preparation. Non-methylated and fully methylated human DNA commercial standards wereutilized as quality control samplesLC-MS/MS method was successfully applied to a pilot diet intervention study toinvestigate global DNA methylation levels as a potential marker to monitorhealth/disease statusPreliminary results showed trend of reduced methylation levels 12 months after dietaryintervention (p-value 0.069, Wilcoxon signed ranks test)58
Conclusion59ØMethod validation is an important tool in drug discovery and developmentØAssay performance parameters by their design may give an idea of methodcapabilities and limitations that can be experienced during routine sampleanalysisØFit for purpose validation is faster and less labor intensive and used extensivelyin drug discovery and biomarker researchØA practical fit for purpose validation delivers quality results to make informedproject decisions
ReferencesLiterature: Bioanalytical Method Validation; Guidance for Industry https://www.fda.gov/media/70858/download Handbook of LC-MS Bioanalysis. Best Practices, Experimental Protocols andRegulations by Li W., Zhang J., and Tse F.L.S. Biomarkers in Drug Discovery and Development: A Handbook of Practice,Application, and Strategy, Second Edition by Rahbari R., Van Neiwaal J. andBleavins M. R.Standards: Cambridge Isotope Labs - www.isotope.com Cerriliant - www.cerilliant.com Anaspec – www.anaspec.com Sigma-Aldrich – www.sigmaaldrich.com60
AcknowledgementsSUMS staffStanford Dean of ResearchVincent and Stella CoatesFoundation61
SUMS GroupProteomicsSmall MoleculeOpen AccessPeopleRyan Leib, PhDFang Liu, PhDRowan Matney, BAKratika Singhal, MSLudmila Alexandrova, PhDKarolina Krasinska, MSBeryl Xia, PhD(Theresa McLaughlin, MS)Theresa McLaughlin, MS(Beryl Xia, PhD)ApplicationsDirector – Allis Chien, Ph.D.Protein IDQuantitative – PRM, TMTProtein interactionsProtein structurePhosphoproteomics, PTMsAbsolute quantitationTargeted metabolomicsPK, Stability studiesMetabolite IDIntact proteinGC/MSLC/MSHRMSMALDI-TOF62Accounting Associate – William Lee, BS
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ØExtent of validation and key parameters should be specified and justified in validation plan: e.g. accuracy, precision, stability etc. ØSpecific validation requirements and acceptance criteria may need to be established for each analyte Food and Drug administration. Bioanalytical method validation Guidance for industry.
Bioanalytical Guidances 6 MHLW Draft Guideline on Bioanalytical Method Validation (Ligand Binding Assay) Validation in Pharmaceutical Development-2014 FDA Guidance for Industry: Bioanalytical Method validation-2013 MHLW Guideline on Bioanalytical Method Validation in Pharmaceutical Development-2013 ANVISA-Bioanalytical guidance-2012
“Bioanalytical Method Validation, A revisit 17: 1551-1557 (Jan,) AAPS Workshop on“Bioanalytical Methods Validation for Macromolecules” (Mar) al., “Workshop on Bioanalytical Methods Validation for Macromolecules: Summary Report” Pharm Res. 2001; 18: 1373-1383 FDA, Guidance for Industry (May) - Bioanalytical method validation - Post
Bioanalytical Method Validation 05/24/18 Bioanalytical Method Validation Guidance for Industry . U.S. Department of Health and Human Services Food and Drug Administration
Bioanalytical Method Validation 05/21/18 Bioanalytical Method Validation Guidance for Industry . U.S. Department of Health and Human Services Food and Drug Administration . Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM) May 2018 . Biopharmaceutics
National Health Surveillance Agency of Brazil (ANVISA) and the FDA Guidance for Industry Bioanalytical Method Validation (2013). The average of recovery was 98.71% in a linear range from 0.1 to 20 μg/mL. The intraday and interday precision and accuracy were within specified limits to bioanalytical methods.
Bioanalytical laboratories are required to comply with regulatory guidance documents such as, Guidelines for Bioanalytical Method Validation (EMA and FDA), 21CFR58, and 21CFR11, from method development through study archival. Procedural elements for data integrity in a bioanalytical laboratory can be broken down into three
1 Guidance for Industry, Bioanalytical Method Validation (2001) and Workshop/Conference Report – Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays (Viswanathan et al., AAPS J. 2007; 9 (1) Article 4).
EPA Test Method 1: EPA Test Method 2 EPA Test Method 3A. EPA Test Method 4 . Method 3A Oxygen & Carbon Dioxide . EPA Test Method 3A. Method 6C SO. 2. EPA Test Method 6C . Method 7E NOx . EPA Test Method 7E. Method 10 CO . EPA Test Method 10 . Method 25A Hydrocarbons (THC) EPA Test Method 25A. Method 30B Mercury (sorbent trap) EPA Test Method .
