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

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 LUCHEChimica Analitica III - 2 Luglio 2012

“Quantification of cidofovir in human serum by LC–MS/MS for children”André Breddemann, Linda Hsien, Edith Tot, Stephanie Läer

Journal of Chromatography B, 861 (2008) 1–9

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Analitical issueCitofovir(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


● Plasmatic protein precipitation850 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)

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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


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)

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II)““Quantification of cidofovir in human serum by LC–MS/MS for children”André Breddemann, Linda Hsien, Edith Tot, Stephanie Läer


● 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

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● Vortex







● Vortex


● HPLCMS/MS

9-(2-phosphonylmethoxyethyl)guanine

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● Vortex







● Vortex


● HPLCMS/MS

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● Vortex







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:

● CDV278.1->234.9

● IS288.1->133.1

● Negative electronspray ionization

9





● Vortex







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:

● CDV278.1->234.9

● IS288.1->133.1

● Negative electronspray ionization

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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.

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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%

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Reagents stability HPLC-MS-MS Breddeman et al.

● 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 ISCalibration 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

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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%

>>>


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Precision HPLC-MS-MS Breddeman et al.

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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





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● 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.

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4 concentration levels (lower limit of quatification, low, mid, high 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.

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Trueness and accuracy HPLC-MS-MS Breddeman et al.





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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


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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


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FittingHILIC-UV Lecomte et al.

quadratic model weighted 1/x

quadratic model

weighted 1/x^2quadratic model

weighted 1/xlinear model

linear model

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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.


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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.

HPLC-MS-MS Breddeman et al.

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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.


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● 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

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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.

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(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

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LOD e LOQ

28







...e il LOD?

LOD e LOQ

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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%


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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?


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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.

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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

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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.

HILIC-UV Lecomte et al. Routine performance

150 ng/mL

500 ng/mL

850 ng/mL

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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.

HILIC-UV Lecomte et al. Routine performance

150 ng/mL

500 ng/mL

850 ng/mL

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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

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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

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Fitness for purposeHILIC-UV Lecomte et al. HPLC-MS-MS Breddeman et al.

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

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● 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).

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● 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).

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● 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.

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

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