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Copyright © 2014 Covance. All Rights Reserved

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

Quantitation of 4-Hydroxycholesterol December 10, 20142

► 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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Copyright © 2014 Covance. All Rights Reserved3

VIABILITY OF 4-HYDROXYCHOLESTEROLAS A BIOMARKER

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Cytochrome P450 3A Metabolism


► 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


► 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)


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4-Hydroxycholesterol as a Biomarker


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


STRATEGIES► Mathematical Correction► Standard Addition► Surrogate Matrix► Surrogate Analyte

(Jones 2012, van de Merbel 2008)

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► 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


► 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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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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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


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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► 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

18 Quantitation of 4-Hydroxycholesterol December 10, 2014

• 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

19 Quantitation of 4-Hydroxycholesterol December 10, 2014

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


4-HC

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:


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


• 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


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


► 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

[email protected]

A Novel Solution for an Endogenous Biomarker:

QUANTITATION OF 4ß-HYDROXYCHOLESTEROL USING A SURROGATE ANALYTE LC-MS/MS APPROACH


slides: a novel solution for an endogenous biomarker: quantitation of 4ß-hydroxycholesterol using a...

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