Drug Metabolism Made Easy

usingLiquid Chromatography-Mass SpectrometryLiquid Chromatography-Mass Spectrometry

Andrew KicmanClick to edit Date/Venue

Use of Human Microsomes and Stable Isotope Substrates; An Alternative Approach for the

Identification of Novel Metabolites of Drugs by Mass Spectrometry in of Drugs by Mass Spectrometry in

Biological Matrices

Ketamine-a model drug

• Dissociative anaesthetic (t ½ = 4 hrs)

• ‘Party’ drug -music can sound heavy, weird and

strangely compelling, lights seem very intense…

• Readily available

• Implicated in Drug Facilitated Sexual Assault

Seized Ketamine

Extensively metabolised: ketamine, norketamine, dehydronorketamine,

hydroxynorketamine and phase II conjugates not all

available

• Implicated in Drug Facilitated Sexual Assault

Metabolism of ketamine

O

ClNHCH

3

O

ClNH

2

OH

O

ClNH

2

OH

O

ClNH

2

OH

OH

ketamine norketamine O

ClNH

2

OH

hydroxynorketamine

O

ClNH

2

OH

Primary

Metabolite

Secondary

metabolites

How can we use microsomes to aid

Identification of metabolites in biological matrices?

By producing stable isotopes ofBy producing stable isotopes of

the metabolites of the drug that

are not commercially available as

chromatographic and mass

spectrometric markers

Hydrogen

atomic mass

~1

Deuterium

atomic mass

~2

Image Adapted from NIST PHYSICS LABORATORY Web Page

Norketamine – Hydroxylation of cyclohexanone

NH3

O

OH

Cl

[M+H]+ = 240

OH

NH3

O

Cl

[M+H]+ = 224

hydroxylation

LC-MS/MS

NH3

O

OH

Cl

[M+H]+ = 244

D

D D

D

NH3

O

Cl

[M+H]+ = 228

D

D D

D

hydroxylation

LC-MS/MS

Norketamine – Hydroxylation of chlorobenzene ring

NH3

O

Cl

[M+H]+ = 240

NH3

O

Cl

[M+H]+ = 224

hydroxylation

OH

LC-MS/MS

NH3

O

Cl

[M+H]+ = 243

D

D D

NH3

O

Cl

[M+H]+ = 228

D

D D

D

hydroxylation

OH

LC-MS/MS

1 Hydroxynorketamine

m/z 240

Tetradeuterated (d4)-

hydroxynorketamine

m/z 244

(alcoholic metabolites)

Trideuterated (d3)-

hydroxynorketamine

NH3

O

OH

Cl

240NH3

O

OH

Cl

D

D D

D

O

Cl

Extracted ion chromatograms of selected metabolites

obtained from microsomal incubates using norketamine or

tetradeuterated norketamine as substrate

3

hydroxynorketamine

m/z 243

(phenolic metabolites)

244

D DNH3

Cl

HO D3243 Time (min)

Volunteer study

• 50 mg ketamine (orally)

• 6 volunteers (3 men, 3 women)

• Urine for up to 10 days post-dose

• ADD 200 µL deuterated microsomalmixture to urine

• ‘Look’ for isotopic doublets

Time (min)

O

Cl

NHCH3

OH

100

1000

195.05713

240.07867223.05209

205.04150141.01016

179.02585154.98944

195.05711

Microsome

mixture Accurate Mass

120 260m/z

0

100

205.04146 240.07867223.05214

141.01012179.02582

154.98944

Urine

UPLC-MS/MS MethodsSophie Turfus

Sophie was a PhD student who won the

‘Young Scientist’ prize (< 40 years) for the

best paper at The International Association

of Forensic Toxicologists International

Meeting in 2009.

Drug Metabolism & Disposition 2009;37:1769-1778;

The detection and quantification of lorazepam and its 3-O-glucuronide conjugate

in fingerprint deposits by LC-MS/MS

Ed Goucher, Sue Jickells, Andrew Kicman

Hypothesis

Given that both blood and sweat are polar matrices, glucuronideconjugates of drugs are partitioned from blood into sweat

Benzodiazepines: widely used for the treatment of anxiety but also used recreationally and in crimes involving drug facilitated sexual used recreationally and in crimes involving drug facilitated sexual assault (DFSA) - glucuronide conjugates are their major metabolites.

Model drug chosen was lorazepam.

Sample Collection

• Volunteers were administered with a standard therapeutic dose

(2 mg) of lorazepam

• A single forefinger deposit (in duplicate) was collected from

each volunteer at the following time points:

t = 0, 2, 4, 6, 8, 12 h and spot collected at 24 and 36 ht = 0, 2, 4, 6, 8, 12 h and spot collected at 24 and 36 h

• For 1 volunteer, all 10 prints were collected and combined at

each time point.

Extracted with solvent and then then analysed by LC-

MS/MS

Fingerprint Analysis

Loss of drug glucuronide moiety by MS/MS

HN

O

OH

OH

OH

O

OH

O[M + H – 176]+

NCl

Cl

O OH

Neutral loss of m/z 176

Control fingerprint - blank x 10 fingerprints – 4 h

> 1.5 pg LOD

Fingerprints – Control versus Treatment

CREATININE

LORAZEPAM

82 pg/print

LORAZEPAM-d4

LORAZEPAMGLUCURONIDE

Both lorazepam and its glucuronide were detected in the ten combined deposits

from one volunteer. Lorazepam-glucuronide was detected up to 12 h post

administration.

Lorazepam

Lorazepam

glucuronide

200

Co

nc

en

tra

tio

n (

pg

/10

pri

nts

)

glucuronide

0

100

Co

nc

en

tra

tio

n (

pg

/10

pri

nts

)

Time since administration (h)

2 4 6 8 12 240

Fingerprint Analysis

To our knowledge, this is the first report of detecting a glucuronide

conjugate of a drug in fingerprint deposits.

CONCLUSION

•LC-MS/MS permits the analysis of polar molecules,

including phase 2 metabolites of drugs (in vivo & in vitro)

•Alternative biological matrices

•Sufficiently sensitive for bioanalysis

(similar to immunoassay (<1 nmol/L; < 100 pg/mL) but with (similar to immunoassay (<1 nmol/L; < 100 pg/mL) but with

SPECIFICITY

•Quantification

•Mass spectrometry is for all, and not just for specialists!

Thanks for listeningThanks for listening

Andrew KicmanClick to edit Date/Venue