a. guenzi, g. fischer, a. gloge and b. lausecker

Microdosing with LC-MS Analysis:

Variations on the Theme

A. Guenzi, G. Fischer, A. Gloge and B. Lausecker

Regulatory environment

• The CHMP position paper (23 June 2004)

“…less than 1/100th of the dose calculated to yield a pharmacological

effect of the test substance based on primary pharmacodynamic data

obtained in vitro and in vivo (typically doses in, or below, the low

microgram range) and at a maximum dose of ≤ 100 microgram.”

• FDA Guidance for Industry, Investigators, and

Reviewers Exploratory IND studies (January 2006)

“…less than 1/100th of the dose of a test substance calculated (based on

animal data) to yield a pharmacologic of the test substance with a

maximum dose of ≤ 100 micrograms (for imaging agents, the latter

criterion applies).”

Roche has a long-standing interest in

microdosing

• Member of CREAM (Consortium for Resourcing and Evaluating

AMS Microdosing)

• Took part in the first published microdosing experiment1 in 2005

that used commercially available AMS by providing 2 marketed

drugs (diazepam and midazolam)

• Microdosing not used for decision making on developability or

hypothesis testing, but rather to obtain the maximum information

in special PK studies (mass balance, SAD) during normal

development

• Only microdosing with cold material and LC-MS analysis used so

far

1. G. Lappin et al., Clin. Pharmacol. Therap. 2006, 80, 203-215.

QUANTIFICATION OF AN I.V. MICRODOSE

OF A VERY LIPOPHILIC DRUG

WITH LONG TERMINAL HALF-LIFE

Case study 1

The drug and its p.o. PK profile

N

N

O

Cl

Cl

RR1

MW 463 amu

Chiral (at R)

Why microdosing?

Question: what is the absolute bioavailability of the

compound?

• The compound is highly insoluble in solvents suitable for i.v.

administration

5 mL of concentrate solution (containing 2-hydroxypropyl-beta-cyclodextrin

and PEG 400 in water) were diluted to 30 mL with 0.9% sodium chloride

and infused over 30 minutes

• The maximum achievable dose was 100 µg

Consequences for the bioanalytical lab

• The original method used to analyze the samples after p.o.

administration had LLOQ 500 pg/mL

The new LLOQ must be 5 pg/mL or lower

• The original method used protein precipitation followed by

column-switching (injection of 0.3 µL plasma equivalents) for

sample preparation

A more sophisticated sample preparation procedure is

needed, and larger volume of plasma equivalent is

injected

• An additional validation is needed for just a few samples

Analysts are born to suffer…

Modification of the assay

• Increase volume of extracted plasma

From 10 µL to 250 µL

• Introduce an off-line cleanup step

After addition of the internal standard in 50 µL water-ACN 1:1,

apply all the sample to an SPE plate, wash with water and elute

with ACN containing formic acid.

• Increase the volume of plasma equivalents injected

From 0.3 to 167 µL (500x increase)

• Introduce on-line dilution before on-line SPE (column switching)

100-µL of 100% organic SPE eluate injected with on-line

dilution with water on the trapping column.

Combined flow-rate to load the sample on the trapping

column:

3 mL/min


• The original method used to analyze the samples after p.o.

administration had LLOQ 500 pg/mL

The new LLOQ must be 5 pg/mL or lower

• The original method used protein precipitation followed by

column-switching (injection of 0.3 µL plasma equivalents) for

sample preparation

A more sophisticated sample preparation procedure is

needed, and larger volume of plasma equivalent is

injected

• An additional validation is needed for just a few samples

Analysts are born to suffer…

MS20-20110110_A005 - TIC - SM: 5 RT: 1.76 - 3.76 NL: 2.35E2F: + c ESI SRM ms2 463.000 [ 347.899-347.901]

1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 2.76

1.94

1.86

2.40

2.063.57 3.68

2.20 2.52

3.382.323.483.11

2.92 3.28

3.05

Double blank plasma


1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 2.79

1.88 2.002.09

2.392.21 3.31

3.563.452.532.293.12

2.94

LLOQ (2 pg/mL)


1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 2.80

2.953.532.081.85 3.17 3.752.55 3.062.191.96 3.322.45 3.41 3.652.32 2.65

Study sample (180 pg/mL)

Epimer (at R)

Results of the microdosing study

Fast initial

decline

Comparable

elimination

phase

Adequate

LLOQ


OF A VERY LIPOPHILIC DRUG

WITH LONG TERMINAL HALF-LIFE.

CO-ADMINISTRATION OF 14C-DRUG P.O.

AND 13C-DRUG I.V.

IN THE HUMAN MASS BALANCE STUDY

Case study 2

The drug and its p.o. PK profile

N

N

N

R

R1

FF

F

Why microdosing?


compound?

• The compound is highly insoluble in solvents suitable for i.v.

administration

• The maximum achievable dose was 100 µg

Study design:

p.o. administration of a 25-mg dose of 14C-labeled

compound (diluted with 12C-material)

i.v. administration of 100 µg 13C-labeled (6 times)

compound 4 hours later

Analytes and ISTDs

N

N

N

R

R1

FF

F

N

N

N

R

R1C

13

C13

FF

F

C13

C13 C

13

C13

C13

R1

N

C13 N

N

FFF

R (contains 3 2H atoms)

N

N

N

C14

R

R1

FF

F

Given p.o. +

ISTD for the p.o. assay

453413 m/z Given i.v.

N

N

N

R

R1

FF

ISTD for the i.v. assay

m/z 444 171

448 408 m/z 450 410 m/z

454 414 m/z

10 parts 1 part


• The original method is sufficiently sensitive (it was developed for

the SAD study with a LLOQ of 25 pg/mL)

• An additional full validation is needed for the i.v. dosing (13C-

labeled material is the new analyte and a new ISTD is used) with a

LLOQ of 10 pg/mL

MS20-20110124_A006 - TIC - SM: 5 RT: 0.57 - 2.57 NL: 7.51E3F: + p ESI SRM ms2 448.000 [ 408.150-408.250]

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 1.57

2.27

2.171.031.830.91 1.15 1.39 2.001.670.760.67 2.43


0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 1.56

1.79 2.151.981.03 2.29 2.470.92 1.411.140.74 1.29

C13

R1

N

C13 N

N

FFF

R (contains 3 2H atoms)

Single-blank human

plasma MS20-20110124_A022 - TIC - SM: 5 RT: 0.57 - 2.57 NL: 3.77E4F: + p ESI SRM ms2 448.000 [ 408.150-408.250]

0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e In

ten

sity

RT: 1.57

1.35 1.680.93 1.85 2.271.48 1.95 2.140.64 1.04 1.17 2.04 2.370.75


0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

In

ten

sity

RT: 1.56

1.720.93 1.831.41 2.06 2.181.971.321.23 2.361.020.64 1.12 2.460.76

LLOQ (25 pg/mL)

N

N

N

R

R1

FF

F

Assay for

p.o. samples


0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

2.94

1.500.93

2.001.79 3.622.601.11 1.360.550.26 3.370.76 2.37


0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e In

ten

sity

RT: 1.51

0.93 1.73 1.84 1.94 2.031.38 2.411.15 2.141.27 2.240.62 0.81

N

N

N

R

R1

FF


0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 1.57

1.35 1.680.93 1.85 2.271.48 1.95 2.140.64 1.04 1.17 2.04 2.370.75


0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e In

tens

ity

RT: 1.56

1.720.93 1.831.41 2.06 2.181.971.321.23 2.361.020.64 1.12 2.460.76

N

N

N

R

R1C

13

C13

FF

F

C13

C13 C

13

C13

Single-blank human

plasma

LLOQ (10 pg/mL)

Assay for

i.v. samples

Results of the mass balance study


OF A DRUG (AND TWO METABOLITES)

IN THE HUMAN MASS BALANCE STUDY

Case study 3

The drug and its metabolites

R3

R4

R2 R6

R5R1

Compound R1 R2 R3 R4 R5 R6 MW (amu)

Drug H H H H H CH3 386.4

Drug ISTD 2H 2H 2H 2H H CH3 390.4

M1 H H H H =O CH3 400.4

M1 ISTD 2H 2H 2H 2H =O CH3 404.4

M2 H H H H H COO

H

416.4

M2 ISTD 2H 2H 2H 2H H COO

H

420.4

13C6-Drug H H H H H CH3 392.4

13C6-Drug

ISTD

(= M1 ISTD)

2H 2H 2H 2H =O CH3 404.4

Analytes and ISTDs

D

D

D

D

C1 4

C1 3

C1 3

C1 3

C1 3C

1 3

C1 3

O

O HO

O HO

D

D

D

D

D

D

D

D

O

D

D

D

D

O

+

M1 Drug

ISTD for drug ISTD for M1 ISTD for M2

M2 p.o.

administratio

n

Drug ISTD for

drug

concomitant

i.v.

administratio

n

Why microdosing?


compound?

Study design:

p.o. administration of a 20-mg dose of 14C-labeled

compound (diluted with 12C-material)

i.v. infusion over 15 min of 100 µg 13C-labeled (6 times)

compound 45 min later


• The original method for the parent (12C) and metabolites is

sufficiently sensitive (it was developed for the SAD study with a

LLOQ of 200 pg/mL)

• An additional full validation is needed for the i.v. dosing (13C6-

labeled material with a different ISTD); needed LLOQ 10 pg/mL

• Changes to the original method to achieve LLOQ of 10 pg/mL:

– 300 µL plasma instead of 100 µL

– SPE used instead of protein precipitation

– Injection of 200-µL equivalent of plasma instead of 25 µL

– Use of a more sensitive instrument (Thermo TSQ Vantage

instead of Sciex API4000)

Results of the mass balance study

Validation approach

• Ultra-sensitive LC-MS methods for application to microdosing are

validated according to FDA guidance and Crystal City 3.

• The usual parameters (precision, accuracy, selectivity etc.) are all

addressed

• The only shortcut taken is that long-term stability is not

performed, since it is assumed that LTS for the 12C drug could be

extrapolated to the 13C drug. All other stabilities are done.

Rationale:

– Same chemical structure

– Stable isotopes

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a. guenzi, g. fischer, a. gloge and b. lausecker

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