Method Validation: Comparison among two analitical methods for the determination of cidofovir in human plasma

Comparison among validations of two
analitical methods for the determination
of cidofovir in human plasma
“Comparison of the quantitative performances
and measurement uncertainty estimates
obtained during method validation versus
routine applications of a novel hydrophilic
interaction chromatography method for the
determination of cidofovir in human plasma”
F. Lecomte, C. Hubert, S. Demarche, C. De Bleye, A. Dispas, M. Jost , F. Frankenne,Journal of
Pharmaceutical and Biomedical Analysis
Journal of Pharmaceutical and Biomedical Analysis 57 (2012) 153– 165
GRETA DALLE LUCHE
Chimica Analitica III - 2 Luglio 2012
“Quantification of cidofovir in human serum by
LC–MS/MS for children”
Andr´e Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aer
Journal of Chromatography B, 861 (2008) 1–9

2
Analitical issue
Citofovir(CVD, HPMPC, VISTIDE®) is a
nucleotide analogue of cytosine with antiviral
activity against a broad spectrum of DNA
viruses. It has been approved for the treatment
of Cytomegalovirus retinis in AIDS patients. Its
therapeutic use is still under investigation, as it
could be applied to a much greater number of
patologyes. Information about the
pharmacokinetic properties of CVD are
urgently required to determine the individual
drug exposure for different metabolism
patients(children or patients affected by renal
insufficiency) or different topic
treatments(papilloma virus infection).
.
2.15
7.00
4.57
An accurate quantification of CDV in human plasma in needed to run pre-clinical trials and to avoid
toxic dosing. The analytical method should quantify CDV in human plasma over, at least a
concentration v range of 100-1000 ng/mL..

3
Analitical procedure
I)“Comparison of the quantitative
performances and measurement
uncertainty estimates obtained during
method validation versus routine
applications of a novel hydrophilic
interaction chromatography method for
the determination of cidofovir in human
plasma”
F. Lecomte, C. Hubert, S. Demarche, C. De Bleye, A. Dispas, M.
Jost , F. Frankenne,Journal of Pharmaceutical and Biomedical
Analysis
Journal of Pharmaceutical and Biomedical Analysis 57 (2012) 153–
165
● Plasmatic protein precipitation
850 μL plasma + 850 μL HPO3(5% w/v)
● Centrifugation
● SPE Plexa PCXcatridges packed
with mixed mode polimeric cation
exchange sorbent
conditionment:
1mL AcN, 500 μL ammonium formate (Ph
3;20mM)
washing:
250 μL AcN-water(85:15v/v)
elution:
1.0 mL AcN-ammonium carbonate
(Ph 10.0;20 mM)
● HP HILIC-UV(275 nm)

4
I)“Comparison of the quantitative
performances and measurement
uncertainty estimates obtained during
method validation versus routine
applications of a novel hydrophilic
interaction chromatography method for
the determination of cidofovir in human
plasma”
F. Lecomte, C. Hubert, S. Demarche, C. De Bleye, A. Dispas, M.
Jost , F. Frankenne,Journal of Pharmaceutical and Biomedical
Analysis
Journal of Pharmaceutical and Biomedical Analysis 57 (2012) 153–
165
Alltima HP HILIC
● Volume of injection 5 μL
● Column 150mm x 2.1 mm i.d., 3 μm
● Pre-column 7.5mm x 2.1 mm i.d., 5 μm
● isocratic separation
AcN-NH4HCO3(72:28)
(pH7; 20mM)
● 25°C
● Flow rate 0.21 mL/min
● UV(275 nm)

5
II)““Quantification of cidofovir in
human serum by LC–MS/MS for
children”
André Breddemann, Linda Hsien, Edith Tot, Stephanie L¨aer
● IS:300 μL plasma + 30 μL PMEG (100
μg/mL in H2O)->1000 μL
● Vortex
● SPE catridges strong anion
exchange(SAX) Varian Bond Elut
conditionment:
2 mL MeOH, 2 mL H2O
washing:
2 x1 mL H2O
elution:
2 X1.0 mL MeOH containg 3%HCl
● Dryness 40°C (N2)
● ->300 μL MeOH-H2O(50:50, v/v)
modified with 3%ammonia
● Vortex
● Centrifugated 20 min 3220 x g
(4000 rpm)
● HPLCMS/MS

6
children”
● Vortex
conditionment:
2 mL MeOH, 2 mL H2O
washing:
2 x1 mL H2O
elution:
● ->300 μL MeOH-H2O(50:50, v/v)
● Vortex
(4000 rpm)
● HPLCMS/MS
9-(2-phosphonylmethoxyethyl)guanine

7
children”
● Vortex
conditionment:
2 mL MeOH, 2 mL H2O
washing:
2 x1 mL H2O
elution:
● ->300 μL MeOH-H2O(50:50, v/v)
● Vortex
(4000 rpm)
● HPLCMS/MS

8
children”
● Vortex
conditionment:
2 mL MeOH, 2 mL H2O
washing:
2 x1 mL H2O
elution:
● ->300 μL MeOH-H2O(50:50, v/v)
HPLCMS/MS
Shimazu controller SCL10Avp with 2 separate
Shimazu pumps LC10Avp
● Triple quadrupole mass
spectrometer API 2000(Applied
Biosystems/SCIEX, Concord)
● Column(tetraalkoxysilane
column): Purospher Star RP-18 (125
mm x 2 mm, 5um, Merk KgaA,
Darmstadt)
● Pre-column: 4 mm x 4 mm, ame
features
● T= 30 °C
● Isocratic
elution:50:49:1(v/v)methanol,water,a
mmonia Ph=10.5
● 0.3 mL/min
MRM, m/z:
● CDV
278.1->234.9
● IS
288.1->133.1
● Negative electronspray ionization

9
children”
● Vortex
conditionment:
2 mL MeOH, 2 mL H2O
washing:
2 x1 mL H2O
elution:
● ->300 μL MeOH-H2O(50:50, v/v)
HPLCMS/MS
Shimazu controller SCL10Avp with 2 separate
Shimazu pumps LC10Avp
● Triple quadrupole mass
spectrometer API 2000(Applied
Biosystems/SCIEX, Concord)
● Column(tetraalkoxysilane
column): Purospher Star RP-18 (125
mm x 2 mm, 5um, Merk KgaA,
Darmstadt)
● Pre-column: 4 mm x 4 mm, ame
features
● T= 30 °C
● Ph=10.5
● 0.3 mL/min
MRM, m/z:
● CDV
278.1->234.9
● IS
288.1->133.1
● Negative electronspray ionization

10
Selectivity
● 6 indipendent source of plasma were
analysed;
No endogenous source of interference was
observed at the ritention time of CVD.
Chromatograms of (A) a blank plasma chromatogram, (B) a spiked plasma at 100
ng/mL and (C) a spiked plasma at 1000 ng/mL of cidofovir. Peak identification:
(1) cidofovir, (2) endogenous compound.
HILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.
● Interferring drug or endogenous compound
was considered if there was a signal close
to 0.3 min of the retention time of the
analyte or the IS
7 serum extracts from 7 different pediatric
cancer patients not receiving CDV therapy
were analysed;
● Possible “cross-talk” between the MS/MS
channels
This was achieved by separately injecting:
CDV at the highest concentrtion of the
calibration line(10'000 ng/mL) and
monitoring the response in IS channel; by
injecting a serum spiked only with internal
standard and monitoring the response with
the CDV channel.
Interferences were not observed.

11
Matrix effect
HPLC-MS-MS Breddeman et al.
To analize possible matrix effect on the
quantitative essays, sample extracts with the
analyte of interest added post extraction were
compared with pure solutions prepared in
mobile phase, containing equivalents amounts
of the analyte of interest.
Plasma spiked after extraction - Pure solution x100%
Pure solution
Ion-suppressive matrix effect on the ionisation
of the IS is consistent with the matrix effect of
the analyte. Therefore, the technical necessity
for the reliable quantification may not be
adversely affectedby this matrix effect.
.
(b)Typical ion chromatogram of blank human serum extract. (b) Typical ion
chromatogram of LLOQ-human serum extract (spiked with 78.125 ng/ml CDV)
for CDV.
78 ng/mL -72.94%
1'250 ng/mL -72.14%
10'000 ng/mL -74.96%
IS 10'000 ng/mL -69.68%

12
Reagents stability
● Relevant data regarding the stability of
CDV and IS at different concentration
were not avaiable. Fo this reaon, stability
tests were conducted in the following
different matrices:
Conditions CDV IS
Calibration solution (Ringer's
solution)
After 1 month storing at 4°C -1.91% -0.14%
Extracted QC samples (4 conc
levels; 5 replicates)
After 24h stoing at room T +(11.51-0.92)%
Human plasma (4 conc levels; 5
replicates)
After 1 month storing at -20°C 94.36-98.72% of
nominal
concentration
After 3 freeze-thaw cycles 106.15-110.15% of
nominal
concentration

13
Precision
● Precision of this analitical method was
determined by computing relative standard
deviationr(RSD%) for repeatability and
time-different intermediate precision at
each concentration level.
5 concentration levels of spiked plasma
ranging from 50 to 1020 ng/mL
4 days of analysis, 5 repetitions each day
● Precision espressed as relative standard
deviation(CV%)
Accuracy and precision were assested by
determing quality control samples at the:
4 concentration levels (lower limit of
quatification, low, mid, high
concentration); five samples each
concentration
on 3 subsequent validation days
Intra- and inter-day precisions(CV%) were
less than 7.8%
>>>

14
Precision

15
Trueness and accuracy
● Trueness espressed in terms of relative
bias(%)
5 concentration levels of spiked plasma
ranging from 50 to 1020 ng/mL
4 days of analysis, 5 repetitions each day
● Accuracy espressed as percentages(%)of
nominal concentrations
concentration

16
● Accuracy takes into account the
total(systematic and random)error. This
interval defines a region where each future
results generated by the bioanalytical
procedure has 95% chance to fall.
The accuracy profile is obtained by linking
on one hand the lower bounds and on the
other hands the upper bounds of β-
expectation tolerance limits calculated at
each concentration levels for the validation
standards.
β-expectation tolerance limits:
X ± ks σˆ
“β-Expectation and β-Content Tolerance Limits for Balanced
One-Way ANOVA Random Model Robert” W. Mee,
Technometrics, Vol. 26, No. 3 (Aug., 1984), pp. 251-254
HILIC-UV Lecomte et al.

17
concentration); 5 samples each
concentration
Intra- and inter-day accuracies(CV%) were
within ± 12.1%
>>>
● Accuracy takes into account the
total(systematic and random)error. This
interval defines a region where each future
results generated by the bioanalytical
procedure has 95% chance to fall.

18
concentration

19
Calibration
● For the method calibration, calibration and
validation standards were prepared spiking
blank human plasma.
Calibration stadards were prepared at 7
concentration levels, ranging from 50 to
1000 ng/mL.
4 series were performed by injecting all
the calibration stadards in triplicate.
● The CDVstandard solution were
preparedby dissolving the accurately
weighed reference standard in Ringer's
solution to obtain a final concentration of
100 μg/mL. The standard solution was then
serially diliuted with Ringer's solution to
obtain 8 working solutions, ranging from
781.25 ng/mL to100 μg/mL.
The calibration standards were then
prepared by spiking 270 μL blank human
plasma with 30 μL of the working
solutions. The 8 calibration solution
obtained range from 78.125 ng/mL to 10
μg/mL

20
Fitting
● Response function coresponds to the assessment of the
relationship between the chromatographic response and
the concentration of the analyte. The optimal regression
model should be the one that firstly allows to
accurately quantify CDV over the widest concetration
range and secondly provides the smallest bias over this
concentration range.
From each response function tested, the
concentration of the spiked plasma
validation standards were back calculated
in order to determine the upper and lower
expectation limits at β=95%.The
acceptance limit were set at +30%.
The only fuction that complied with the
defined criteria is the weighted quadratic
model using the weight 1/X^2

21
Fitting
quadratic
model
weighted 1/x
quadratic model
weighted 1/x^2
quadratic model
weighted 1/x
linear model
linear model

22
Fitting
● In order to demostrate the linearity of the
results, a regression line was fitted between
the back-calculated concentration of
validation standards versus the introduced
concentration applaying a linear regression
model.

23
Fitting
● The calibration curve was obtained by fitting the ratio of the integrated mass peak
area of CDV to the integrated mass peak of the IS (y-axis) against the range of
added analyte concentration(x-axis) using 1/X linear regression plot.

24
LOD e LOQ
● The lower limit of quantification (LLOQ)
is defined as the smallest quantity that can
be quantitatiely determined uner the
experimental conditions with the well
defined accuracy.
Using the accuracy profile, the LLOQ was
estimated as 92.7 ng/mL. The LLOQ was
obtained calculating the smallest
concentration for which the β-expectation
tolerance limit cross the acceptance limits.

25
● The lower limit of quantification (LLOQ)
is defined as the smallest quantity that can
be quantitatiely determined uner the
experimental conditions with the well
defined accuracy.
Using the accuracy profile, the LLOQ was
estimated as 92.7 ng/mL. The LLOQ was
obtained calculating the smallest
concentration for which the β-expectation
tolerance limit cross the acceptance limits.
The limit of detection(LOD) was estimated
using the mean intercept of the calibration
model and the residual variance of the
regression and was evaluated to be 28.1
ng/mL.
LOD = Sb + 3 σb
LOD e LOQ

26
According to FDA, Food and Drug
Administration:
“the lowest standard on the calibration curve
should be accepted as the limit of quantification
if the following conditions are met:
● The analyte response at the LLOQ should
be at least 5 times the response compared
to blank response.
● Analyte peak (response) should be
identifiable, discrete, and reproducible
with a precision of 20% and accuracy of
80-120%”
U.S. Department of Health and Human Services, Food and Drug
Administration,
Center for Drug Evaluation and Research (CDER), 2001.
http://www.fda.gov/cder/guidance/4252fnl.htm.
.
(b)Typical ion chromatogram of blank human serum extract. (b) Typical ion
chromatogram of LLOQ-human serum extract (spiked with 78.125 ng/ml CDV)
for CDV.
LOD e LOQ

27
Administration:
to blank response.
80-120%”
Administration,
LOD e LOQ

28
Administration:
to blank response.
80-120%”
Administration,
...e il LOD?
LOD e LOQ

29
Recovery
● The recoveries of CDV were determined
using blank plasma sample spiked at three
different concentrations ranging from 100
to 1020 ng/mL
Recoveries were found to be constant
around 85% over the entire range studied.
● The absolute recovery was determined by a
comparison of the peak areas derived from
serum samples(QC sample at 3
concentrations with 5 samples for each
concentration) spiked before extraction
with the peak areas from serum samples
spiked after the extraction procedure.
Plasma spiked before extraction x100%
Plasma spiked after extraction
The recovery of IS(PMEG) was
determined similarly
78 ng/mL 62.69%
1'250 ng/mL 51.38%
10'000 ng/mL 52.45%
IS 10'000 ng/mL 60.94%

30
Recovery
● The recoveries of CDV were determined
using blank plasma sample spiked at three
different concentrations ranging from 100
to 1020 ng/mL
Recoveries were found to be constant
around 85% over the entire range studied.
● The absolute recovery was determined by a
comparison of the peak areas derived from
serum samples(QC sample at 3
concentrations with 5 samples for each
concentration) spiked before extraction
with the peak areas from serum samples
spiked after the extraction procedure.
Plasma spiked before extraction x100%
Plasma spiked after extraction
The recovery of IS(PMEG) was
determined similarly
78 ng/mL 62.69%
1'250 ng/mL 51.38%
10'000 ng/mL 52.45%
IS 10'000 ng/mL 60.94%
...e le dev standard?
riproducibilità?

31
Risk assessment
The risk of having future measurements falling
outside the specified acceptance limits was
evaluated using the β-expectation tolerance intervals
obtained with the previously selected regression
model. This risk is computed for each concentration
level investigated, as the sum of the proportion of
results effectively lying outside the upper
acceptance limit on one hand and under the lower
acceptance limit on the other hand. The maximum
risk tolerated was set to 5%, meaning that it is
accepted that at most each future result provided by
the developed method will have five chances out of
100 to fall outside the acceptance limits of ±30%.
The risk was clearly smaller than 5% over the valid
concentration range. However, this risk was about
36% for the smallest concentration level (50 ng/mL)
thus confirmingthe inaccuracy of the results
generated by the bioanalytical method at this
concentration level.
Illustrates the risk profile for CDV results obtained at each validationstandard
concentration level.

32
Routine performance
The validated method was applied routinely to the
quantitative determination of CDV in human plasma
samples from a pre-clinical trial.
The estimate concentration level, of a patient after a
defined time T5, is equal to 184.9 ng/mL and
demonstrating the good selectivity of the
currentmethod at low concentration levels (two
times the LLOQ).
In each trial apart from the calibration standards and
the real unknownsamples, each analytical run
involved quality control (QC) samples prepared in
blank plasma spiked with CDV in order to reach
three concentration levels: 150, 500 and 850 ng/mL.
Fig. 8 illustrates chromatograms obtained from incurred blank sample at T0 (pre-
dose) and a real unknown sample from the same patient at T5

33
For the first trial:
● 10 routine runs (one run per day)were
performed, leading to the analysis of
– 40 QC samples (spikedplasma
samples) at each of the three
concentration levels
– as well as 192 real samples.
For the second trial:
● 15 routine runs were realized.
– The numbers of QC samples for this
trial add up to 172
– the number of real samples to 252.
Each level of concentration was analysed in
quadruplicates.
Routine performance
150 ng/mL
500 ng/mL
850 ng/mL

34
As can be seen, for the two highest QC levels, all
(100%) QC samples fell within the acceptance
limits, thereby confirming the validation step
prediction.
For the smallest QC level (150 ng/mL) only two
samples out of 106 at this level fell outside the
acceptance limits of ±30% resulting in a proportion
of 98.1% which is above the minimum requirement
of95% defined during the method validation.
The routine performance of this
bioanalyticalmethod demonstrates that this method
is clearly appropriate forits final use since at least
95% of the QC samples are included in theroutine
acceptance limits.
Routine performance
150 ng/mL
500 ng/mL
850 ng/mL

35
Routine performance
Ion chromatogram of a quality control extract (spiked with 1250 ng/ml CDV) for
CDV and the internal standard PMEG. (b) Ion chromatogram of apae diatric
patient sample extract (blood withdrawal 1 h after infusion of 2.5 mg/kg CDV,
the measured CDV concentration amounted to 5270 ng/ml) for CDV andthe
internal standard PMEG.
To apply and test the new LC–MS/MS method
under realistic application conditions, we
analysed blood samples of a paediatric cancer
patient who intravenously received 2.5 mg per
kg bodyweight CDV. Blood samples were
collectedat 0 h (pre-treatment), 1 h, 2 h, 3 h, 4 h
and 6 h after infusion.
...no quantitative data

36
Conclusions
SELECTIVITY
MATRIX EFFECT
REAGENTS STABILITY
PRECISION
ACCURACY
TRUTHNESS
LOQ E LOD
RECOVERY
RISK ASSESSMENT
CONTROL CHART
✔
-
-
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
-
✔
X
-
-

37
Conclusions
SELECTIVITY
MATRIX EFFECT
REAGENTS STABILITY
PRECISION
ACCURACY
TRUTHNESS
LOQ E LOD
RECOVERY
RISK ASSESSMENT
CONTROL CHART
✔
X
-
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
-
✔
X
-
-
3.4-9.7(RSD%) 3.0-4.0(RSD%)
100.0 ng/mL e 28.09 78.125 ng7mL.. no LOD
Range 78-10.000 ng/mLRange 93-1020 ng/mL

38
Fitness for purpose
Both the proposed methods seem to satisfy the fundamental prerequisite for the investigation
of the pharmacokinetic properties of CDV:
● With a linear signal response in the concentration range from 78.125 ng/ml to 10'000
and from 100.0 to 1'000 ng/ml, the developed methods covers the expected
concentrations in patients at least up to four half-lives.
● The required plasma volume for the analysis is restricted for both the method: 300 μL

39
Fitness for purpose
●
Lecomte et al. writes:”As can be seen the LC–MS/MS method of Breddemann et al.
is only able to provide accurate results 95 times out of 100 over the concentration range
2000–4870 ng/mL of CDV. Indeed, for concentration levels of CDV smaller than 2000
ng/mL, the risk to obtain results out of the±30% acceptance limits is greater than 5 %.
This means that there are more than five chances out of 100 that future results will fall
outside the acceptance limits recommended by the Food and Drug Administration
(FDA).

40
Fitness for purpose
●
Lecomte et al. Denounce :”As can be seen the LC–MS/MS method of Breddemann
et al. is only able to provide accurate results 95 times out of 100 over the concentration
range 2000–4870 ng/mL of CDV. Indeed, for concentration levels of CDV smaller than
2000 ng/mL, the risk to obtain results out of the±30% acceptance limits is greater than 5
%. This means that there are more than five chances out of 100 that future results will
fall outside the acceptance limits recommended by the Food and Drug Administration
(FDA).

41
Fitness for purpose
● However, the adequate treatment often requires polymedication(e.g. cancer patients)
which is related to potential analytical interference. Therefore, analytical methods for the
determination ofCDV in human serum necessitate a selective detection.
●
Moreover matrix effect has not been investigated in the HILIC-UV.
●
In the HPLC-MS-MS method, the conducted tests to investigate these effects clearly
indicatea pronounced ion-suppressive matrix effect. But this effect wasconsistent for both
the analyte and the internal standard. Therefore, the technical necessity for the reliable
quantification may not be adversely affected by this matrix effect.

Method Validation: Comparison among two analitical methods for the determination of cidofovir in human plasma

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