Variations in internal standard
response
Some thoughts and real-life cases
Olivier Le Blaye
Inspector
21 November 2013
EBF 6th Open Meeting
Disclaimer
 The
views expressed in this presentation are
mine and may not represent those of either
ANSM, EMA, the PKWP or the GCP IWG
Agence nationale de sécurité du médicament et des produits de santé
1
Preliminary observation: choice of IS
A
minority of the studies I inspect use a
stable-isotope labelled IS (SIL IS)
 Very few have a structural analogue
 Most IS used are not structurally or chemically
related to the analyte
 Some have the same indication: so what ?
Agence nationale de sécurité du médicament et des produits de santé
2
WTF ?
Analyte: ethinyl estradiol
Derivatisation with dansyl chloride
IS: diltiazem
No derivatisation
Agence nationale de sécurité du médicament et des produits de santé
3
Variations in internal standard response
 Different
situations
 Isolated variation (limited number of isolated
samples)
 Systematic differences, trends…
Agence nationale de sécurité du médicament et des produits de santé
4
Variations in internal standard response
 Isolated
variation
 Easy to detect and to handle
 Often seen in SOPs: repeat if IS response deviates
by more than x % from mean
(of CC / QC ? of whole run ?)
 Influence on overall study results limited, unless
Cmax sample
Agence nationale de sécurité du médicament et des produits de santé
5
IS variation: individual samples
50000
Double spike ?
Sample processing problem ?
40000
30000
CC
QC
Subject
20000
10000
Sample processing problem ?
Air bubble ?
0
0
10
20
30
40
50
60
70
Agence nationale de sécurité du médicament et des produits de santé
6
IS variation: individual samples

Usually well described in SOPs, monitored by labs, few
inspection findings

If variation due to IS addition error: stable isotope
labelled IS will not compensate

If variation due to other reasons: SIL IS may
compensate. But:
 Low response: analyte S/N ? LLOQ ?
 High response: linearity ?
Agence nationale de sécurité du médicament et des produits de santé
7
Variations in internal standard response
 Systematic
differences, trends…
 Multiple possible reasons
 May or may not affect the results
 More difficult to capture in SOP, may require lab
investigations, scientific approach
Agence nationale de sécurité du médicament et des produits de santé
8
Variations in internal standard response
Millions
Systematic differences
3,00
2,50
2,00
1,50
1,00
0,50
0,00
0
Standard
QC
S14
S15
S16
Agence nationale de sécurité du médicament et des produits de santé
160
9
Systematic differences
SOP with ± 50 % rule may not help !
± 50 % of mean IS response of CC / QC samples
50000
40000
30000
20000
10000
0
1
Standard
QC
S10
S11
Agence nationale de sécurité du médicament et des produits de santé
118
10
Systematic differences
SOP with ± 50 % rule may not help !
± 50 % of mean IS response of all samples
50000
40000
30000
20000
10000
0
1
Standard
QC
S10
S11
Agence nationale de sécurité du médicament et des produits de santé
118
11
Variations in internal standard response
Systematic differences
 Applying
blindly an SOP with ± x %
acceptance criteria may result in the blind
acceptance of possibly inaccurate results !
Agence nationale de sécurité du médicament et des produits de santé
12
Variations in internal standard response
Trends – case 1
100000
80000
60000
CC
QC
Subject
40000
Cmax
ISR: fails -70 %
20000
0
0
10
20
30
40
50
60
70
Agence nationale de sécurité du médicament et des produits de santé
13
Variations in internal standard response
Trends – case 1
 Lab
did not monitor IS response
 SOP
with ± x % rule may not help anyway
 IS response obviously results in inaccurate results
 Tmax at 10’ with bolus IV administration; other
datasets: 1-2’
 ISR: 70 % difference
 What % difference in IS would mean inaccurate
results ?
Agence nationale de sécurité du médicament et des produits de santé
14
Variations in internal standard response
Trends – case 2
400000
350000
300000
CC
QC
Subject
250000
200000
150000
0
10
20
30
40
50
60
Agence nationale de sécurité du médicament et des produits de santé
15
Variations in internal standard response
Trends – case 2
 Gradual
increase in subject IS, decrease in
QC IS
 Are QCs representative of subject samples ?
Accuracy of subject samples ?
 Subject samples with highest IS result were re-
analysed
 No IS response trend in repeat run
 Repeat result confirmed initial value, data
accepted
Agence nationale de sécurité du médicament et des produits de santé
16
Variations in internal standard response
Trends – case 3
30000
25000
20000
CC
QC
Subject
15000
10000
5000
0
0
10
20
30
40
50
60
70
Agence nationale de sécurité du médicament et des produits de santé
17
Variations in internal standard response
Trends – case 3
 IS
response systematically lower every 11
and 12 samples
 Post-inspection root-cause analysis
 IS added with Gilson 401 dilutor,




series of 12 fractions
Dilutor checked periodically,
but only 10 fractions
Due to piston seal problem:
fractions 11 and 12 too low
Incorrect IS addition resulted in inaccurate
results
Affected all runs, possibly other studies
Agence nationale de sécurité du médicament et des produits de santé
18
Variations in internal standard response
Trends – case 4, ISR run
90000
80000
70000
CC
QC
Subject
60000
50000
40000
30000
0
10
20
30
40
50
60
70
Agence nationale de sécurité du médicament et des produits de santé
19
Variations in internal standard response
Trends – case 4, ISR run
90000
80000
70000
60000
CC
QC
Subject
3 ISR results fail, result higher
than initial
50000
4 QCs fail, result too high.
IS too low ?
40000
30000
0
10
20
30
40
50
60
70
Agence nationale de sécurité du médicament et des produits de santé
20
Variations in internal standard response
Trends – case 4, ISR run

Low IS response could be the reason for QC and ISR
failure in this run

Variations in IS response appeared to be the reason for
failure of several run / extraction batches in this study –
but not always: some runs with low or high IS response
in QC sets, result OK

IS not monitored during study, issue not identified, not
investigated
Agence nationale de sécurité du médicament et des produits de santé
21
Variations in internal standard response
Trends – case 5
220000
Extraction batch 1
200000
180000
CC
QC
Subject
160000
140000
120000
Extraction batch 2
100000
0
10
20
30
40
50
60
70
Agence nationale de sécurité du médicament et des produits de santé
22
Variations in internal standard response
Trends – case 5

Samples processed in 2 batches per run, injected in the
processing order
 CC, 2 sets QC, period 1
 2 sets QC, period 2 from the same subject

Difference in IS response between the two extraction
batches

EMA guideline: acceptance criteria for QC samples
 First for the whole run
 Then for each extraction batch
Agence nationale de sécurité du médicament et des produits de santé
23
Variations in internal standard response
Systematic differences, trends
 Multiple
possible situations
 Multiple possible reasons
 Some may affect accuracy, others not
 Difficult to plan / describe in SOP
 Criteria such as ± x % may not be relevant
 May not be sensitive enough: ± x % inaccuracy ?
 If limits too tight: may result in numerous unneeded
repeats
 ISR
results can help in discussion
(too late ?)
Agence nationale de sécurité du médicament et des produits de santé
24
Variations in internal standard response
Systematic differences, trends
 “We
are using an SIL IS anyway, gold
standard, will compensate for any variation,
no concern, no need to check IS response”
Agence nationale de sécurité du médicament et des produits de santé
25
Variations in internal standard response
Systematic differences, trends
 “We
are using an SIL IS anyway, gold
standard, will compensate for any variation,
no concern, no need to check IS response”
 Not an acceptable answer if the root cause for the
variation has not been identified
 SIL IS can compensate for some sources of
variations, but not all !
 If you don’t know why the IS varies, you don’t know
whether the SIL IS will compensate
Agence nationale de sécurité du médicament et des produits de santé
26
Variations in internal standard response
Systematic differences, trends
 IS
variations may trigger laboratory
investigations to identify root cause
 Depending on the result: may require the re-analysis
of study samples
 We, inspectors, should be ready to accept decisions
which are science-driven and not just based on
SOPs, if:
 Based on facts and solid scientific arguments
 Well documented
 Lack of SOP or inadequate criteria are not valid
reasons to accept obviously inaccurate results
Agence nationale de sécurité du médicament et des produits de santé
27
See Tan et al., Journal of Chromatography B, 877
(2009) 3201-3209 for 12 more cases !
Thank you for your attention !
Agence nationale de sécurité du médicament et des produits de santé
28
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Variations in internal standard response