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
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…
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
MS20-20110110_A021 - TIC - SM: 5 RT: 1.79 - 3.79 NL: 3.88E2F: + 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: 2.79
1.88 2.002.09
2.392.21 3.31
3.563.452.532.293.12
2.94
LLOQ (2 pg/mL)
MS20-20110110_A023 - TIC - SM: 5 RT: 1.80 - 3.80 NL: 1.40E4F: + 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: 2.80
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
QUANTIFICATION OF AN I.V. MICRODOSE
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?
Question: what is the absolute bioavailability of the
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
Consequences for the bioanalytical lab
• 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
MS20-20110124_A006 - TIC - SM: 5 RT: 0.56 - 2.56 NL: 5.02E5F: + p ESI SRM ms2 453.000 [ 413.150-413.250]
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: 1.56
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
MS20-20110124_A022 - TIC - SM: 5 RT: 0.56 - 2.56 NL: 5.42E5F: + p ESI SRM ms2 453.000 [ 413.150-413.250]
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
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
MS20-20110127_A004 - TIC - SM: 7 RT: 0.00 - 4.00 NL: 2.66E2F: + c ESI SRM ms2 454.100 [ 414.095-414.105]
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
MS20-20110127_A004 - TIC - SM: 7 RT: 0.51 - 2.51 NL: 4.98E4F: + c ESI SRM ms2 444.150 [ 170.995-171.005]
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
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
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
MS20-20110124_A022 - TIC - SM: 5 RT: 0.56 - 2.56 NL: 5.42E5F: + p ESI SRM ms2 453.000 [ 413.150-413.250]
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: 1.56
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
QUANTIFICATION OF AN I.V. MICRODOSE
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?
Question: what is the absolute bioavailability of the
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
Consequences for the bioanalytical lab
• 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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