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A Novel Solution for an Endogenous Biomarker:
QUANTITATION OF 4ß-HYDROXYCHOLESTEROL USING A SURROGATE ANALYTE LC-MS/MS APPROACH
Stephanie Cape, Ph.D.Associate Director | Bioanalytical Chemistry
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Overview
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► Viability of 4-Hydroxycholesterol (4-HC) as a biomarker
► Strategies for absolute quantitation of endogenous molecules
► LC-MS/MS method for the quantitation of 4-HC in human plasma
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VIABILITY OF 4-HYDROXYCHOLESTEROLAS A BIOMARKER
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Cytochrome P450 3A Metabolism
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► 4-HC has been proposed as a biomarker to indicate CYP450 3A activity
► CYP subfamily members: • Essential for production of key biological molecules
• Cholesterol, steroids, prostacyclins, etc. • Critical for detoxification of foreign chemicals• Mediate metabolism of ~50% of marketed drugs.
► Shift in CYP450 metabolic capacity may result in changes in therapeutic response or intensity of adverse effects
Cytochrome P450
Image from: ScientificAmerican.com
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Cytochrome P450 3A Mediated DDIs
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► Many drug-drug interactions (DDIs) are the result of an alteration of CYP450 metabolism
► Multiple drugs have been pulled from the market due to CYP450 metabolism disruption (Lynch 2007).
cisapride (Propulsid), astemizole (Hismanal), & terfenadine (Seldane)– Terfenadine pulled from market due to cardiotoxic effects caused
by DDI with CYP3A4 inhibitors, including grapefruit
► Conventional approach to assess includes probe substrates for in vivo metabolic activity assessment:
• Current standard
• Studies are complex
• Challenges in certain patient populations
• Patient safety concerns
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4-Hydroxycholesterol as a CYP450 3A biomarker
► Benefits Eliminate the need for probe drug
The subject can serve as own-control
Evidence 4-HC levels reflect P450 3A activity specifically
• Not impacted by cholesterol auto-oxidation (Breuer 1996)
• Not impacted by activity of other hepatic enzymes (Bodin 2001)
Quantitation of 4-Hydroxycholesterol December 10, 2014
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4-Hydroxycholesterol as a Biomarker
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CYTOCHROME P450 3A Inducer
CYTOCHROME P450 3A Inhibitor
CYP3A4/5Auto-oxidation
H
HH
H
HO
cholesterol
H
HH
H
HO
4- -hydroxycholesterolOH
Figure adapted from: Goodenough 2011
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STRATEGIES FOR QUANTITATION OF ENDOGENOUS COMPOUNDS VIA LC-MS/MS
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Quantitation of Endogenous Compounds
► Endogenous presence of analytes in the native matrix► Measuring small changes within larger concentrations► Interfering native structurally similar species► Lack of regulatory clarity for validation
CHALLENGES
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STRATEGIES► Mathematical Correction► Standard Addition► Surrogate Matrix► Surrogate Analyte
(Jones 2012, van de Merbel 2008)
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Quantitation of Endogenous Compounds
► Background subtraction technique during data processing to correct for endogenous concentration
• “Quick and dirty” estimate of changes in concentration.
• Impractical if background levels are significantly higher than the change being measured.
MATHEMATICAL CORRECTION
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► Aliquots of native matrix are fortified with increasing concentrations of the analyte to create a calibration curve.
• Determination of the x-intercept yields endogenous concentration
• Can suffer from a limited linear range
• Curve weighting factor can contribute toimprecision in endogenous measurement.
STANDARD ADDITION
y = mx + b
x: concentration
y: s
igna
l
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Quantitation of Endogenous Compounds
An analyte-free “surrogate matrix” is used for the preparation of calibration standards.
► Common examples of surrogate matrices:• Analyte depleted matrix
• Activated Carbon (Charcoal) stripped• Immuno-depletion
• Commercially available matrix substitutes• SeraSum or UriSub (CST Technologies)
• Same matrix from alternate gender or species• Buffers or other solvents
• BSA in PBS• Methanol
► Appropriate surrogate matrix is not always available► Stability, matrix effects, solubility, and recovery can differ.
SURROGATE MATRIX APPROACH
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Quantitation of Endogenous Compounds
A “surrogate analyte” is used to create calibration standards in native matrix.
► Assumes properties of authentic and surrogate analytes are similar
• Surrogate analyte is generally a stable isotope labeled (SIL) analog of the target molecule
• Response factor • Ratio of response of surrogate analyte versus authentic analyte• Must be balanced
• Multiple stable labeled versions of the target molecule are required
SURROGATE ANALYTE APPROACH
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LC-MS/MS METHOD FOR THE QUANTITATION OF 4-HYDROXYCHOLESTEROLIN HUMAN PLASMA
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Quantitation of 4-HC: Surrogate Analyte
CD3
H
HH
H
HO
CH3
4- -hydroxycholesterol-d4
OH
D
Quantitation of 4-Hydroxycholesterol December 10, 2014
H
HH
H
HO
4- -hydroxycholesterolOH
EndogenousMS/MS Transition: 385 97
Surrogate Analyte: MS/MS Transition: 389 97
Internal StandardMS/MS Transition: 392 97
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Quantitative LC-MS/MS Analysis of 4-HC
► Derivatization is not required. • Previously published LC-MS/MS method used picolinyl ester
derivative (Goodenough 2011)
► Specialized source is not required• Such as atmospheric pressure photo-ionisation (APPI) (van de
Merbel 2011)
ADVANTAGES OVER PREVIOUS METHODS
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Quantitative LC-MS/MS Analysis of 4-HC
► Calibration Curve: 4 to 100 ng/mL
► Aliquot Volume: 400 µL Human Plasma (K2EDTA)
► Alkaline Hydrolysis:
• Treatment with sodium methoxide to obtain the ‘free’ HC from the long-chain fatty acid esters
► Extraction:
• Liquid/liquid and SPE (Isolute Diol Cartridges)• Avoid exposure to light and heat
► Separation and detection:
• LC-MS/MS with positive ion Atmospheric Pressure Chemical Ionization (APCI)
OUTLINE OF METHOD
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LC-MS/MS Method
HPLC Column: ► Waters, Acquity UPLC BEH C18, 50x2.1mm, 1.7 µm
Mobile Phases: ► A: 0.1% formic acid in water
► B: 0.1% formic acid in methanol : acetonitrile (20:80, v:v)
Gradient: ► Elution during 1 min. gradient from 85-95% B.
Mass Spectrometer: ► Sciex API 5500 positive APCI [(M-H2O) + H+]
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Validation Study Design
PREPARATION OF QUALITY CONTROLS
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• 8 concentrations of d4 4β-HC were prepared to be used as the calibration curve.
PREPARATION OF CALIBRATION STANDARDS
Sample Preparation
LLOQ, LQC, MQC, HQC, and DQC
Prepared by fortifying native matrix with d4 4β-HC to appropriate concentration
QC-END Measured endogenous 4β-HC
DQC-END Measured endogenous 4β-HC + 4β-HC
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Validation Study Design
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Validation Test Modification
Precision & Accuracy Determined using 4 levels of d4 4β-HC and 1 of 4β-HC
Sensitivity, Carryover, Recovery, and Matrix Factor
Determined using d4 4β-HC
Selectivity Fortified 4β-HC in addition to the endogenous 4β-HC
Dilution Integrity Determined using both d4 4β-HC and 4β-HC. (Samples diluted with PBS.)
Hemolysis Hemolyzed sample compared against non-hemolyzed reference generated from the same whole blood.
Hyperlipemic Testing completed using d4 4β-HC
Stability Designed as fit for purpose
Response Factor Unique to this type of experimental design
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SYSTEM EVALUATION AND ADJUSTMENT
Response Factor
► Prior to each run: • Compare response generated from mixture of equal concentrations of d4
4β-HC against 4β-HC• Adjust appropriate source parameters to achieve RF ~1• Evaluation is acceptable when responses compare within +/- 10%
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CHROMATOGRAPHIC SELECTIVITY
Chromatogram Showing a Mixture of 4-HC and 4-HC
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4-HC
4-HC
Quantitative LC-MS/MS Analysis of 4-HC
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BlanksCHROMATOGRAMS OF PLASMA BLANK SAMPLE
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Endogenous 4β-HC transition
Surrogate Analyte: D4 4β-HC transition
Internal Standard: D7 4β-HC transition
Internal Standard : D7 4β-HC transition
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SensitivityCHROMATOGRAM OF CALIBRATION SAMPLE AT LLOQ
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Endogenous 4β-HC
Surrogate Analyte LLOQ (4 ng/mL): D4 4β-HC
Internal Standard: D7 4β-HC
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Validation Data: Precision & AccuracyINTER-RUN STATISTICS:
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LLOQ QC 4.00
ng/mL
LQC 10.0
ng/mL
MQC 30.0
ng/mL
HQC 80.0
ng/mLQC END
41.2 ng/mL
Mean Concentration Found (ng/mL) 3.99 10.4 30.5 82.2 41.8
Inter-run RSD (%) 17.3 7.9 7.7 5.2 5.7
Inter-run %Bias -0.3 4.0 1.7 2.8 1.5
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QUANTITATION IN INDIVIDUAL MATRIX LOTS
Validation Data: Selectivity
Individual Lots of Matrix
Lot 167.9
ng/mL
Lot 239.9
ng/mL
Lot 355.8
ng/mL
Lot 458.4
ng/mL
Lot 555.7
ng/mL
Lot 650.3
ng/mL
Mean (n=6) 61.6 38.0 50.9 54.6 51.0 46.9
RSD (%) 1.7 2.6 1.9 3.3 2.6 2.4
Accuracy (%) 90.7 95.2 91.2 93.5 91.6 93.2
% Bias -9.3 -4.8 -8.8 -6.5 -8.4 -6.8
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StabilityESTABLISHED STABILITY OF 4-HC
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• Frozen in plasma at -60º C to -80º C for 387 days • Frozen in plasma at -10º C to -30º C for 160 days• 4 freeze/thaw cycles
• Solution at -10º C to -30º C for 225 days• Solution at room temperature for 6 hours
• Plasma (on ice) for 4 hours • Whole blood at room temperature for 1 hour• Extracted/Processed samples at 2º C to 10º C for 96 hours
ESTABLISHED STABILITY OF D4 4Β-HC
• Sufficient stability established to support laboratory handling of compound in appropriate solutions and matrices.
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Cross Validation
BETWEEN TWO MASS SPECTROMETRICPLATFORMS AND TWO SITES
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Cross Validation
QC Concentration
Covance Site 1Result
(mean of n=6)
Covance Site 2Result
(mean of n=6) % BiasQC 1* 70.0 72.3 69.5 3.9QC 2* 45.0 47.1 44.3 6.1QC 3* 25.0 25.7 23.8 7.7QC 4* 15.0 15.3 14.9 2.6QC 5** 31.4 30.9 29.6 4.3
* Spiked D4 4-HC Control** Endogenous 4-HC Control% Bias = ( Site 1- Site 2) / (Mean) x 100%
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Clinical Sample Analysis
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► Supported multiple clinical studies using this method
Successful ISR testing
► Covance has developed and validated a robust method for the quantitation of 4-HC in human plasma
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Conclusions
► There is increasing interest in 4-HC as a potential biomarker for CYP450 activity
► Thoughtful strategies must be employed to overcome challenges with quantitation of endogenous molecules
► Covance has developed and validated a robust method for the quantitation of 4-HC in human plasma
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Acknowledgements
► Barbara Bell
► Stuart McDougall
► Janine McKnight
► Matt Byers
► Christopher Schmidt
► Yao Shi
► Jamie Farnham
► Aaron Ledvina
COVANCE TEAMS AT ALNWICK AND MADISON WHO DEVELOPED AND VALIDATED THE METHOD:
COVANCE ISOTOPE CHEMISTRY TEAM AT ALNWICK FOR SYNTHESIS OF THE STABLE LABELLED STANDARDS
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References
• Lynch, Tom ; Price, Amy. (2007) The effect of Cytochrome P450 Metabolism on Drug Response, Interactions, and Adverse Effects. Am. Fam Physician;76:391-6
• Bodin, K., Betillon, L., Aden, Y., Bertilsson, L., Broome, U., Einarsson, C., and Diczfalusy, U. (2001) Antiepileptic drugs increase plasma levels of 4-hydroxycholesterol in humans. Evidence for involvement of cytochrome P450 3A4. J. Biol. Chem.; 276, 38685-38689.
• Goodenough, A. K.; Onorato, J. M.; Ouyang, Z.; Chang, S.; Rodrigues, A. D.; Kasichayanula, S.; Huang, S.; Turley, W.; Burrell, R.; Bifano, M.; Jemal, M.; LaCreta, F.; Tymiak, A.; Wang-Iverson, D. (2011) Quantification of 4-beta-hydroxycholesterol in human plasma using automated sample preparation and LC-ESI-MS/MS analysis. Chem. Res. Toxicol. 24, 1575-1585
• Van de Merbel, N. C. (2008) Quantitative determination of endogenous compounds in biological samples using chromatographic techniques. Trends in Analytical Chemistry, 27(10), 924-933.
• Jones, B. R.; Schultz, G. A.; Eckstein, J. A.; Ackermann, B. L. (2012) Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules Bioanalysis, 4(19), 2343-2356.
• Van de Merbel, N. C., Bronsema, K. J., van Hout, M. W. J., Milsson, R., and Sillen, H. (2011) A validated liquid chromatography-tandem mass spectrometry method for the quantitative determination of 4-hydroxycholesterol in human plasma. J. Pharm. Biomed. Anal. 55, 1089-1095
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Thank You for Attending
Stephanie Cape, Ph.D.Associate Director, Bioanalytical Chemistry, Covance
Stephanie.Cape@Covance.com
A Novel Solution for an Endogenous Biomarker:
QUANTITATION OF 4ß-HYDROXYCHOLESTEROL USING A SURROGATE ANALYTE LC-MS/MS APPROACH
Quantitation of 4-Hydroxycholesterol December 10, 201432