# Presentation: 750033

## Chemical Assay of Drugs and Drug Metabolites

- Sanford P. Markey
- Laboratory of Neurotoxicology
- NIMH

- November 30, 2006

## correction

- On notes from lecture Nov 16
- Change "percent homology" to "percent sequence identity" in reference to Cytochrome P450s family relationships

## Lecture Outline

- Quantification principles
- Analytical PK lab tasks
- Chromatography
- Detection - spectroscopies
- Optical
- Mass
- Examples
- Resveratrol
- FddA
- CYP450 Assays
- Cyclosporin A
- References

## Definition of Analytical Terms

- Limits of detection (LOD)
- Sensitivity is the minimum detectable concentration change that can be observed at a specified concentration
- LOD is the minimum mass or concentration of analyte that can be detected at an acceptable signal to noise (S/N) ratio
- Limits of quantification (LOQ)
- Analyte mass or concentration required to give an acceptable level of confidence in the measured analyte quantity
- Always greater (usually 3x) than the minimum LOD

## Accuracy vs. Precision

- Good accuracy
- Poor precision

- Poor accuracy
- Good precision

- Good accuracy
- Good precision

## Pharmaceutical Industry PK Lab Analytical Assays (1)

- Parent drug usually the target analyte for Phase 1 dose response and safety determinations
- Scale of runs: 30-50 samples/patient, plus 10-15 standards, procedural blanks, plus 10-15 QC pools or previously analyzed samples
- Several patients per run - effort to optimize patient/(standards + QC) ratio. Result is >100 samples/run
- Analytical runs require automation & rugged instrumentation, continuous operation for  assay cycle time X number of samples
- Develop assays on 96 well or 384 well devices

**Notes:**
15 min/sample for 150 samples = 37 hrs

## Pharmaceutical Industry PK Lab Analytical Assays (2)

- Speed of assay development principal determinant of methodology choice
- Avoid derivatization chemistry
- Use solid phase extraction or simple methanol/acetonitrile protein precipitation
- Time is money (5 min LC/MS/MS assay vs. 40 min HPLC)
- Use automated LC/MS/MS methods with high sensitivity and specificity

**Notes:**
15 min/sample for 150 samples = 37 hrs

## Assay Issues

- What to assay (what is important?)
- Species -
- man, non-human primate, rat, mouse (transgenic)
- Tissue/Fluid
- liver, target organ, plasma, excreta
- Isolated organ/tissue fluids
- liver slices, human liver microsomes, CYPs, other enzymes

## Assay Issues

- Commercial Aides
- Drug metabolizing preparations
- Human liver tissue or hepatocytes – all enzymes present in fresh (not frozen) tissue – single use only
- Microsomes from frozen liver; easily stored
- Recombinant CYPs and other enzymes - widely available (yeast, baculovirus, bacteria) and some mammalian cells with NADPH CYP reductase
- CYP substrates, antibodies, inhibitors, inducers
- Computer software - predict metabolites, pKa, pLogD, logP
- Contract Research Organizations

## Separation Methods 

- Liquid _[chromatography ](slide11.xml)_
- _[High Performance](slide12.xml)_ (HPLC)
- Separation Principles of Columns
- Immunoaffinity. Reverse Phase, Cation & Anion Exchange
- Gas chromatography
- Capillary electrophoresis

## Liquid Chromatography

- stationary phase

- column

- mixture

- solvent
- (eluent)

## HPLC Apparatus

## Detection Principles

- _[Ultraviolet ](slide14.xml)_ or _[Fluorescence](slide15.xml)_ Spectroscopy
- chromophore in drug or derivatized drug
- most useful for known target analytes
- Nuclear Magnetic Resonance Spectrometry
- most useful for totally unknown chemical structure characterization
- least sensitive
- _[Mass Spectrometry](slide16.xml)_
- versatile ionization modes for liquids and gases
- electron, chemical, _[electrospray](slide17.xml)_,desorption
- versatile _[mass analyzers ](slide18.xml)_with varying capabilities
- magnetic, ion trap, quadrupole, time-of-flight
- very sensitive and structurally informative –example: _[air, acetaminophen](slide21.xml)_

## UV Absorption Spectrophotometer

## Emission Spectrophotometer

## Mass Spectrometer Component Overview

## Mass Spectrometer Ionizers

- Electron
- Ionization
- (in vacuo)

- Electrospray
- Ionization
- (external)

## Mass Analyzers

- Time-of-flight
- (TOF)

- Quadrupole
- (q)

## Quadrupole Ion Trap

## Electrospray-Ion Trap Mass Spectometer

## Mass Spectrum of Air

- m/z

- Relative
- Intensity

- 18

- 28

- 40

- 44

- 32

- H2O

- N2

- O2

- Ar

- CO2

## Mass spectrum of acetaminophen

## Multidimensional Analyses

- time

- response

- chromatogram

- m/z

- m/z

- m/z

## Tandem Mass Spectrometry (MS/MS or MSn)

- Mass Spectrometer

- Tandem Mass Analysis (MS/MS)

- Tandem MS permits selection and isolation of specific ions for subsequent analysis.

- Tandem Mass Spectrometry (MS/MS or MSn)

- D. Sheeley, NCRR, NIH

## Pharmaceutical Industry PK Lab Analytical Assay Work Load for New Chemical Entities

| Method | 1990 | 1998 | 2000 | 2006 |
| --- | --- | --- | --- | --- |
| HPLC | 75% | 50-60% | 20% | 2% |
| GC/MS | 12% | 3% | 2% | 0 |
| LC/MS/MS | 3% | 40-50% | 60-75% | 98% |
| RIA | 10% | 10% | 10% | 0 |
| Preliminary lead profile time | 18 m | 4 m | 0 | 0 |

- Conclusion: requirement for speed (not instrumentation cost)
-  dictates choice of analytical methods

**Notes:**
table

## Popular Methods for Qualitative & Quantitative Assays in Clinical Pharmacology

- LC/MS/MS
- High speed, reduced requirement for sample preparation
- HPLC/UV or Fluorescence
- Very robust, routine assay technology – F-dAA
-  Enzyme Linked Immunoassay (ELISA)
- Many 96 well formatted colorimetric or radiometric commercial assay kits for specific compounds
- Florescence polarization immunoassay (FPIA)
- Measures difference in florescence between bound and free antigen
- Important in therapeutic drug monitoring - CsA

## Examples of Analytical MethodsApplied in Drug Analyses

- 1. Resveratrol - bioavailability
- 2. FddA
- HPLC/UV vs HPLC/MS/MS for PK study
- 3. CYP450 Assays - LC/MS/MS
- 4. Cyclosporin - FPIA, HPLC/UV, LC/MS/MS

## Example 1 -Where Do Drugs Go?

- Radiochemical tracers (14C, 3H)
- requires availability of labeled drug
- useful for bioavailability, kinetics - **Reservatrol**
- detection of protein adducts/localization (autoradiography)
- Non-radiochemical methods
- Unique drug elements (fluorine, etc.) or structural property (fluorescence)
- Specific atom or isotope detectors
- Accelerator mass spectrometry (AMS) - detection of 14C at near natural background levels (K. Turteltaub et al.)
- Combustion reaction isotope mass spectrometry (CRIMS)  (F. Abramson et al.)

## Resveratrol

- Washington Post,** **November 2, 2006
**A Compound in Red Wine Makes Fat Mice Healthy**
*By *Rob Stein

- A substance found in red wine protected mice from the ill effects
- of obesity and extended their life spans, raising the tantalizing
- prospect that the compound could do the same for humans and
- may also help people live longer, healthier lives, researchers
- reported yesterday...
- "We've been looking for something like this for the last 100,000 years, and maybe it's right around the corner -- a molecule that could be taken in a single pill to delay the diseases of aging and keep you healthier as you grow old," said David A. Sinclair, a Harvard Medical School molecular biologist who led the study.

## Resveratrol

- JA Baur, et al...DA Sinclair
*Nature* **444**, 337-342 (16 November 2006)
- Resveratrol improves health and survival of mice on a high-calorie diet

- 22.4±0.4 mg/kg-1/day-1 in food

***SD***
***HC***
***HCR***

## Resveratrol

- Widely sold at health food stores as antioxidant
- Proposed chemopreventive for cardiac diseases, cancer
- based on _*in vitro *_evidence
- Absorption?
- Bioavailability?
- Metabolism?

- HIGH ABSORPTION BUT VERY LOW BIOAVAILABILITY OF ORAL RESVERATROL IN HUMANS *T.Walle et al., Drug Metab Disp 32:1377-1382 (2004)*

## Resveratrol plasma concentration-time curves (total radioactivity)

*T.Walle et al., Drug Metab Disp 32:1377-1382 (2004)*

## Resveratrol Urine Metabolites (0-12 hr)

*LC/MS*
*(selected ions)*
*M1,2=RV-Glu*
*M3= RV-H**2**-Glu*
*M4= RV-SO4*
*M5 = RV-H**2**-SO4*

*LC/UV*
*305 nm*
*(note absence of M3,M5)*

*T.Walle et al., Drug Metab Disp 32:1377-1382 (2004)*

## Resveratrol Recovery of Radioactivity

|  | 25 mg Oral |  | 0.2 mg i.v. |  |
| --- | --- | --- | --- | --- |
|  | Urine | Feces | Urine | Feces |
| N=6 | 70.5 ± 4.3 | 12.7 ± 6.1 | 64.1 ± 7.7 | 10.4 ± 3.7 |

*T.Walle et al., Drug Metab Disp 32:1377-1382 (2004)*

## HPLC Radiochromatogram0-12 hr urine extract

*Glucuronidase shifts M1-M3 to Reservatrol r.t.*

*T.Walle et al., Drug Metab Disp 32:1377-1382 (2004)*

## Resveratrol Study Conclusions

- Unmetabolized resveratrol not detectable in plasma
- Absorption of resveratrol is at least 70%
- No evidence for further oxidation - only conjugation ± reduction
- Bioavailability of resveratrol limited
- Highly accumulated in intestinal epithelial cells
- Target sites of breast and prostate unlikely unless RV-SO4 is active species or reservoir of parent

- Oral bioavailability of resveratrol is almost zero
- Potential biologic activity of resveratrol conjugates should be considered in future investigations

*T.Walle et al., Drug Metab Disp 32:1377-1382 (2004)*

*Wenzel E, Somoza V. Mol Nutr Food Res. 2005 May;49(5):472-81*.

## Example 2.  Analytical studies for pharmacokinetic measurements  in the development of laudanosine

-     James A. Kelley  (DCT, NCI)

**Example 2.  Analytical studies for pharmacokinetic measurements  in the development of laudanosine**

- _LC/MS/MS_
- Jian Wang
- Jeri S. Roth
- _PHASE I F-ddA_
- Harry Ford, Jr.
- Nancy M. Malinowski
- Robert Yarchoan (HAMB,DCS, NCI)
- Richard F. Little (HAMB, DCS, NCI)

- _F-ddATP Analysis_
- Harry Ford, Jr.
- Christine Dai
- Heping Zhang
- Mark F. Kavlick (MB, DCS, NCI)
- _Fddl Products_
- John S. Driscoll
- Victor E. Marquez
- Robert J. Lutz (BEIP)
- Robert L. Dedrick (BEIP)

## Synthetic dideoxynucleoside
Acid stable (pH 1.0, 37 °C)
More lipophilic than inosine analogue 
(Log P = -0.183 versus -1.210)
More resistant to metabolic degradation than Didanosine (ddI)
Inhibitor of HIV reverse transcriptase

- Acid stable (pH 1.0, 37 °C)
- More lipophilic than inosine analogue
**(Log P = -0.183 versus -1.210)**
- More resistant to metabolic degradation than Didanosine (ddI)
- Inhibitor of HIV reverse transcriptase

## Slide 39

## HPLC of Plasma

- Pre-dose plasma
- plus internal std.

- Plasma at min 85 of 100
- min 1.6 mg/kg infusion
- (internal std. added)

- JS Roth et al (1998)
- J. Chrom B _712_:199

## Comparison of Methods for Analysis of F-ddA and F-ddI

- JS Roth et al (2000) J. Mass Spectrom 35:1313-9

- LC/MS/MS

- HPLC-UV

*F-ddI*

*F-ddA*

## F-ddA/F-ddI Assay Summary

- HPLC-UV
- Measures both F-ddA and F-ddI
- Primary assay for clinical samples
- Adequate sensitivity for PK studies at therapeutic doses
- LC-ESI-MS-MS
- Measures  both F-ddA and F-ddI
- Simple sample preparation and rapid analysis
- High sensitivity and specificity
- Backup assay for high volume of clinical samples

## Example 3: LC/MS/MS CYP GLP Assays

- 12 Semi-automated assays for 10 human CYP450 enzymes described
- Microsomes pooled from 54 human livers
- Microsomes, NADPH, substrate in 96 well plate; stable isotope internal standards added with quenching solvent
- Recombinant CYP450 enzymes (Sf9 cells) from PanVera run in parallel; reference values published
- High speed LC/MS/MS conditions established for each analyte and internal standard (2 min/assay)
- Interassay precision of reaction velocity <10%
- Validated Assays for Human Cytochrome P450 Activities**, **RL Walsky and  RS Obach, *Drug Metab Disp* 32:647-660, 2004

## CYP 450 Validated AssaysSubstrates and Metabolites

- From RL Walsky &  RS Obach

## Hydroxybupropion - ESI-MS

- 8

- 0

- 9

- 0

- 1

- 0

- 0

- 1

- 1

- 0

- 1

- 2

- 0

- 1

- 3

- 0

- 1

- 4

- 0

- 1

- 5

- 0

- 1

- 6

- 0

- 1

- 7

- 0

- 1

- 8

- 0

- 1

- 9

- 0

- 2

- 0

- 0

- 2

- 1

- 0

- 2

- 2

- 0

- 2

- 3

- 0

- 2

- 4

- 0

- 2

- 5

- 0

- 2

- 6

- 0

- 2

- 7

- 0

- m

- /

- z

- 0

- 1

- 0

- 0

- %

- MH+ 256

- m/z

- Int

- [D6]-MH+ 262

- From RL Walsky &  RS Obach

- + [D6]-hydroxybupropion

## Hydroxybupropion - CID of MH+ 256

- 8

- 0

- 9

- 0

- 1

- 0

- 0

- 1

- 1

- 0

- 1

- 2

- 0

- 1

- 3

- 0

- 1

- 4

- 0

- 1

- 5

- 0

- 1

- 6

- 0

- 1

- 7

- 0

- 1

- 8

- 0

- 1

- 9

- 0

- 2

- 0

- 0

- 2

- 1

- 0

- 2

- 2

- 0

- 2

- 3

- 0

- 2

- 4

- 0

- 2

- 5

- 0

- 2

- 6

- 0

- m

- /

- z

- 0

- 1

- 0

- 0

- %

- 139

- MH+ 256

- m/z

- Int

- MH+ 262

- From RL Walsky &  RS Obach

- [D6]-Hydroxybupropion - CID of MH+ 262

## Example: CYP2B6 AssayBupropion substrate 

- 0

- 0

- %

**[D6]Hydroxybupropion**

**Hydroxybupropion**

***1.97 r.t.***
***628 area***

***1.96 r.t.***
***96538 area***

*retention time (min)*

- From RL Walsky &  RS Obach

- 0.20		   1.00                                             2.00	                     2.80

## Example: CYP2B6 Results

- From RL Walsky &  RS Obach

## Partial Summary of CYP Activities RL Walsky and  RS Obach, Drug Metab Disp 32:647-660, 2004

| Enzyme | Assay | Inhibitor | IC50 (μM)
Human      Recomb |  |
| --- | --- | --- | --- | --- |
| CYP1A2 | Phenacetin O-deethylase | Furafylline | 1.76±0.28 | 1.54±0.16 |
| CYP2A6 | Coumarin 7-hydroxylase | Tranylcyp-romine | 0.449±.073 | 0.895±.262 |
| CYP2B6 | Buproprion hydroxylase | PPP | 7.74±0.47 | 2.02±0.19 |
| CYP2C8 | Amodiaquine N-deethylase | Quercetin | 3.06±0.31 | 3.33±0.20 |
| CYP2C9 | Diclofenac 4’-dydroxylase | Sulfaphen-azole | 0.272±.031 | 0.169±.004 |

## Example 4: Cyclosporin A (CsA)

- HPLC - UV (210 nm) method first used for clinical analyses
- LOQ - 20-45 μg/L (therapeutic range 80-300 μg/L)
- LC/MS/MS method for fingerprick samples
- 25 μL; LOQ 10 μg/L
- Keevil BG, Ther Drug Monitor 24: 757-67 (2002

*Potent immunosuppresive drug for transplantation; irreversible kidney damage if dose too high *

## Cyclosporin Immuno Assays

- Florescence polarization immunoassay (FPIA)
- Homogeneous immunoassay
- Fluorescein tagged drug competes with patient drug for monoclonal Ab
- Polarized light excites Ab-tagged drug complex most efficiently
- LOQ 25 μg/L; analysis of 20 samples in 19 min
- Enzyme monitored Immunoassay Technique (EMIT) and Cloned Enzyme Donor Immunoassay (CEDIA)
- Competitive: enzyme labeled antigen competes with sample antigen; enzyme labeled antigen-Ab complex changes rate
- Multiple cyclosporin metabolites exhibit cross-reactivity in immunoassays

## WWW Sites (1) HPLC & Drug Metabolism

- Tutorial for HPLC
- http://kerouac.pharm.uky.edu/asrg/hplc/HPLCMYTRY.HTML
- Prediction Software
- http://www.acdlabs.com/products/phys_chem_lab/ (pK, metabolite structures)
- Human Drug Metabolizing Enzymes:
- Human Biologics International (www.humanbiologics.com)
- InVitro Tech (www.invitrotech.com)

## WWW Sites (2): Mass Spectrometry Information Education

- http://ull.chemistry.uakron.edu/classroom.html
- Excellent introductory tutorials in analytical methods including chromatography and mass spectrometry
- http://www.i-mass.com/
- Site with very useful links for mass spectrometry including tutorial movies
- http://www.sisweb.com/mstools.htm
- Calculation tools free ware (isotope calculations, elemental compositions)