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Chapter 5 Valacyclovir - Introduction
5.1 Introduction
Valacyclovir is an antiviral drug. The chemical formula of Valacyclovir
hydrochloride is L-valine, 2-[(2-amino-1,6-dihydro-6oxo-9H-purin-9-yl) methoxy]
ethyl ester, mono hydrochloride with the molecular formula C13H20N6O4.HCl and a
molecular weight of 360.80 (Figure 5.1).
Figure 5.1 Chemical structures of Valacyclovir (A), Valacyclovir-D8 (B)
Valacyclovir hydrochloride is rapidly absorbed from the gastrointestinal tract and
nearly completely converted to acyclovir and L-valine by first-pass intestinal and
hepatic metabolism by enzymatic hydrolysis. Acyclovir is converted to a small extent
to inactive metabolites by aldehyde oxidase and by alcohol and aldehyde
dehydrogenase. Neither Valacyclovir nor acyclovir is metabolized by cytochrome P450
enzymes. Plasma concentrations of unconverted Valacyclovir are low and transient,
generally becoming non-quantifiable by 3 hours after administration. Peak plasma
concentrations of Valacyclovir are generally less than 0.5 µg/mL at all doses. The
absolute bioavailability of acyclovir after oral administration is 54.5% ± 9.1%. The
binding of Valacyclovir to human plasma proteins ranges from 13.5% to 17.9%.
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Chapter 5 Valacyclovir - Introduction
The binding of acyclovir to human plasma proteins ranges from 9% to 33% 51
Several techniques such as HPLC and LC-MS/MS methods have been
reported in the literature for the quantitative estimation of Valacyclovir and Acyclovir
in biological fluids 52-58
and pharmaceutical dosage forms59-61
,A number of methods
were developed in animals such as rabbit55
rat56
and horse57
plasma for quantification
of Valacyclovir and Acyclovir by LC-MS/MS. Only a few methods were reported in
human plasma for quantification of Valacyclovir and Acyclovir 52-54
by LC-MS/MS.
Among all Yadav M, Upadhyav V et al 52
achieved best results. They developed the
method with linearity between the concentration range of 5-1075 ng/mL for
Valacyclovir, 47.6-10225 ng/ mL for Acyclovir using the mobile phase ratio of 0.1%
formic acid: methanol (30:70) on Gemini C18 column . They compared the drug with
fluconazole as an internal standard. They have not achieved sensitive less than
5 ng/mL.52
In bioanalytical method development, usage of deuterated internal standard is
very helpful to find the exact matrix effect at analyte and internal standard retention
times. Till now, as of our knowledge, there is no method reported for comparision of
Valacyclovir with its deuterated internal standard.
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Chapter 5 Valacyclovir - Introduction
5.2 Experimental Investigations
5.2.1 Materials and reagents
Valacyclovir and Valacyclovir-D8 TFA salt, Acyclovir were obtained from
Synfine Research Canada. Formic acid, Acetic acid, Ammonium hydroxide, NH4OH,
Ammonium formate were obtained from Merck Mumbai. Methanol, Acetonitrile
(HPLC grade) were Obtained from J.T.Baker, Mumbai. Ammonium formate,
Ammonia solution (NH4OH, 25%, Reagent grade), Formic acid, Glacial acetic acid
(CH3COOH, reagent grade) were Obtained from sd.Fine chemicals Mumbai.
Dichloromethane, were purchased from Merck Speciality Chemicals Ltd, Mumbai,
India. Human plasma was procured from Navazeevan Blood bank, Hyderabad.
Millipore water was used from Milli-Q system.
5.2.2 Instrumentation and equipment
Refer Chapter - 3.2.2
5.2.3 Preparation of Reagents and Solvents
Table 5.1 Preparation of Reagents and Solvents
Reagents and Solvents preparation
0.1% Formic acid Dilute 1 mL of formic acid to 1000 mL with water.
30% Methanol in 0.1% formic acid Mix 300 mL of methanol with 700 mL of 0.1% formic acid.
1N Acetic acid Dilute 60 mL of glacial acetic acid to 1000 mL with water (prepare
daily).
30% Formic acid Dilute 30 mL of formic acid to 100 mL with water.
2.5% NH4OH in methanol Mix 10 mL of ammonia solution with 90 mL of methanol (prepare
daily).
10mM Ammonium formate, PH 5.0
Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust PH to
5.0 ± 0.05 with formic acid.
10mM Ammonium formate, PH 3.1
Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust PH to
3.1 ± 0.05 with formic acid.
50% Methanol Mix 500 mL of methanol with 500 mL of water.
Reconstitution solution Mix 800 mL of 10mM ammonium formate, P
H 3.1 with 200 mL of
methanol.
20% Methanol
(Auto sampler wash) Mix 200 mL of methanol with 800 mL of water.
Acidified plasma Add approximately 5 mL of 30% formic acid to 100 mL of plasma.
Mobile phase
Mix 10mM Ammonium formate PH 5.0 : Methanol in the ratio of 80:20
and
Filter through 0.45 m filter
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Chapter 5 Valacyclovir - Introduction
5.2.4 Preparation of Stock solutions
Table 5.2 Preparation of Stock solutions
Name of the solution Concentration Volume (mL) Diluent
Valacyclovir stock solution 100.o µg/mL 100 mL Methanol
Valacyclovir- D8 stock solution 100.0 µg/mL 100 mL Methanol
Acyclovir stock solution 1000.0 µg/mL 10 mL Methanol
5.2.5 Preparation of standards and quality control (QC) Samples
Standard stock solution of Valacyclovir (100 g/mL) was prepared in
50% methanol. From this stock, analytical standards were prepared at concentration
levels of 0.5, 1.0, 5.0, 35.0, 70.0, 140.0, 280.0, 420.0, 560.0 and 700.0 ng/mL by
appropriate dilution with human plasma.
From the standard stock solution, Quality control samples were prepared separately
at LLOQ (0.5 ng/mL) low (1.5ng/mL) medium, (210.0 ng/mL) and high
(490.0 ng/mL) concentrations. All the samples were stored in -80°C freezer until
analysis.
5.3 Method Development
The goal of this research is to develop and validate a simple, selective,
sensitive, rapid, rugged and reproducible assay method for the quantitative
determination of Valacyclovir from plasma samples. In the way to develop a simple
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Chapter 5 Valacyclovir - Introduction
and easy applicable method for Valacyclovir assay in human plasma for
pharmacokinetic study, HPLC with MS/MS detection was selected as the method of
choice.
Mass parameter Optimization,Chromatographic Optimization and Extraction
optimization to be optimized carefully to achieve the best results.
The MS optimization was performed by direct infusion of solutions of both
Valacyclovir and Valacyclovir-D8 into the ESI source of the mass spectrometer. Other
parameters, such as the nebulizer and the heater gases and Declustering potential(DP),
Entrance potential(EP),Collision energy(CE) was optimized to obtain a better spray
shape, resulting in better ionization and droplet drying to form the protonated ionic
Valacyclovir and Valacyclovir- D8 molecules.
A CAD product ion spectrum for Valacyclovir and Valacyclovir- D8 yielded
high-abundance fragment ions of m/z (amu) 152.0 and m/z (amu) 152.0 respectively
Shown in Figure 5.2 and Figure 5.3.
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Chapter 5 Valacyclovir - Introduction
Figure 5.2 Parent and Product ion mass spectra of Valacyclovir
128
Chapter 5 Valacyclovir - Introduction
Figure 5.3 Parent and Product ion mass spectra of Valacyclovir-D8
Chromatographic conditions, especially, selection of column, composition and
nature of the mobile phase were optimized through several trials to achieve best
resolution and increase the signal of Valacyclovir and Valacyclovir- D8. Separation
was tried using various combinations of mobile phase with variety of columns like
YMC Pack pro C18, RP-Amide, Ascentis Express RP-amide, X-Bridge, Discovery
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Chapter 5 Valacyclovir - Introduction
Cyano, Kromasil 100- 5CN. After the MRM channels were tuned, the mobile phase
was changed from more aqueous phase to organic phase to obtain a fast and selective
LC method. A good separation and elution were achieved using 10 mM ammonium
formate (pH 5.0): methanol (80:20 v/v) as the mobile phase, at a flow-rate of
0.25 mL/ min and injection volume of 5 µL. Chromatographic analysis of the analyte
and IS was initiated under isocratic conditions with an aim to develop a simple
separation process with a short run time.
Extraction was performed by different extraction techniques like SPE, LLE,
Precipitation methods. Finally a simple SPE technique was selected in the extraction
of Valacyclovir and Valacyclovir - D8 from the plasma samples.
Chromatographic conditions
Chromatographic separation was carried out on a reversed phase Zorbax, SB
C18, 4.6 x 75mm, 3.5 m column using a mixture of 10mM ammonium formate buffer
(PH 5) and methanol (80:20 v/v) as mobile phase with a flow-rate of 0.25 mL/min.
The column temperature was set at 45°C. Retention time of Valacyclovir and
Valacyclovir-D8 was found to be approximately 4.4 ± 0.2 min for both drug and IS.
Sample preparation
A Solid phase extraction procedure was used for extraction of drug and IS
from the plasma samples. For this purpose, 50µL of Valacyclovir- D8 (200ng/mL),
200 µL plasma ( respective concentration of plasma sample) was added into ria vials
then vortexed for 30 seconds followed by 200 µL of acetic acid solution was added
and vortexed briefly. SPE cartridges (Water Oasis, MCX LP, 3 cc, 60 mg) were
130
Chapter 5 Valacyclovir - Introduction
conditioned with 2 mL of dichloromethane, 1.5 mL of 2.5% NH4OH in methanol.
After that, samples from ria vials were loaded the onto SPE cartridge. Wash the
cartridges with 1.5 mL of water followed by 1.5 mL of methanol. Allowed to dry the
cartridge then eluted cartridges with 1.5 mL of 2.5% NH4OH in methanol into
prelabled ria vials. These samples were evaporated to dryness under the nitrogen
stream at 40°C. Finally, the residue was reconstituted with 400 µL of reconstitution
solution (10mM ammonium formate (PH 3.1): methanol 80:20) and vortexed briefly.
Then the samples were transferred into auto sampler vials injected into the
LC-MS/MS system.
Calibration curve parameters and regression model
The analytical curves of Valacyclovir were constructed in the concentrations
ranging from 0.5- 700.0 ng/mL in human plasma. Calibration curves were obtained by
weighted linear regression (weighing factor: 1/x2). The ratio of Valacyclovir peak area
to Valacyclovir-D8 peak area was plotted against the ratio of Valacyclovir
concentration in ng/mL. The fitness of calibration curve was confirmed by
back-calculating the concentrations of calibration standards.
Method Development Conclusion
The developed method is suitable for estimation of plasma concentrations for
Valacyclovir as a single analytical run, in clinical samples from Pharmacokinetic
studies. This was followed by method validation.
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Chapter 5 Valacyclovir - Introduction
5.4 Method Validation
The objective of the work is to validate specific HPLC- MS method for the
determination of Valacyclovir in human plasma for clinical / Pharmacokinetic study.
Chromatography
Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ,
LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 5.6 to 5.14.
Figure 5.4 MRM Chromatogram of Blank Human Plasma Sample
132
Chapter 5 Valacyclovir - Introduction
Figure 5.5 Chromatogram of Blank + IS
133
Chapter 5 Valacyclovir - Introduction
Figure 5.6 Chromatogram of LOQ Sample (Valacyclovir & IS)
134
Chapter 5 Valacyclovir - Introduction
Figure 5.7 Chromatogram of ULOQ Sample (Valacyclovir & IS)
135
Chapter 5 Valacyclovir - Introduction
Figure 5.8 Chromatogram of LLOQ Sample (Valacyclovir & IS)
136
Chapter 5 Valacyclovir - Introduction
Figure 5.9 Chromatogram of LQC Sample (Valacyclovir & IS)
137
Chapter 5 Valacyclovir - Introduction
Figure 5.10 Chromatogram of MQC Sample (Valacyclovir & IS)
138
Chapter 5 Valacyclovir - Introduction
Figure 5.11 Chromatogram of HQC Sample (Valacyclovir & IS)
Figure 5.12 Calibration Curve of Valacyclovir
139
Chapter 5 Valacyclovir - Introduction
Blank Matrix Screening
During validation, blank plasma samples from 10 different lots were processed
according to the extraction procedure and evaluate the interference at the retention
times of analyte and internal standard. The 6 free interference lots were selected from
the 10 lots. Results are presented in Table 5.3.
Table 5.3 Screening of Different batches of blank matrix
(Human K2EDTA Plasma) for interference free Valacyclovir blank plasma
Matrix identification
Blank plasma Area
Analyte
(Valacyclovir)
RT
Internal standard
RT
AP/3451/09/10 0 0
AP/3452/09/10 0 0
AP/3453/09/10 27 0
AP/3454/09/10 0 0
AP/3455/09/10 35 0
AP/3456/09/10 0 0
AP/3457/09/10 28 0
AP/3458/09/10 0 0
AP/3459/09/10 0 0
AP/3460/09/10 0 0
Blank +IS with AP/3451/09/10 0 557914
LOQ with AP/3451/09/10 4932 551115
Blank Matrix Specificity and Limit of Quantification
During specificity run, the LLOQ standard was prepared in one of the
screened blank plasma including the spiking of working range of internal standard.
Blank plasma samples from 10 different lots, 6 LLOQ standards were processed
according to the extraction procedure. The responses for the blank plasma from 10
different lots were compared to the LLOQ standard of the analyte and internal
140
Chapter 5 Valacyclovir - Introduction
standard. No significant response (≤ 20% for the analyte response and ≤ 5% of the
internal standard response) was observed at the retention times of the analyte or the
internal standard in blank plasma as compared to the LLOQ standard. Results are
presented in Table 5.5
The specificity experiment shall be considered for calculation of LOQ
experiment. Results are presented in Table 5.4
Table 5.4 Specificity of Different batches of blank matrix
(Human K2EDTA Plasma) for Valacyclovir
Matrix
Identification
LLOQ
Area
Internal
standard
(IS) area
Interference with
Analyte(% of
LLOQ Response)
Interference
with IS(% of IS
Response)
AP/3451/09/10 4930 551118 0 0
AP/3452/09/10 4876 568732 0 0
AP/3454/09/10 4798 563455 0 0
AP/3456/09/10 4689 558974 0 0
AP/3458/09/10 4768 557687 0 0
AP/3459/09/10 4812 568794 0 0
Acceptance criteria:
1. Analyte response should be ≤ 20% of LOQ Response in at least 75% of the
blank.
2. Internal standard response should be ≤ 5% of mean internal standard response
in at least 75% of the blank.
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Chapter 5 Valacyclovir - Introduction
Table 5.5 Limit of Quantitation for analyte (Valacyclovir)
Matrix identification Blank plasma area at
Analyte RT
LLOQ
response
LLOQ S/N
RATIO
AP/3451/09/10
0 4932 17.4
0 4836 15.9
0 4860 15.2
0 4734 17.8
0 4677 17.4
0 4505 13.6
N 6 6 6
Mean 0 4757 16.2
LLOQ was spiked in -AP/3451/09/10
Acceptance criteria:
1. Mean S/N ratio of LLOQ should be ≥ 5.
2. S/N ratio is analyst software generated data.
Intra Batch Accuracy and precision
Intra batch accuracy and precision evaluation were assessed by analyzing
1 calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from
precision and accuracy batch-1.
The Intra batch percentage of nominal concentrations for Valacyclovir was
ranged between 94.43% and 97.86%.
The Intra batch percentage of coefficient of variation is 0.74% to 4.09% for
Valacyclovir.
Results are presented in Table 5.6
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Chapter 5 Valacyclovir - Introduction
Table 5.6 Intra batch (Within-Batch) Accuracy and Precision for determination
of Valacyclovir levels in human plasma
Analytical
Run ID
LLOQ
0.50 ng/mL
Low QC
1.50 ng/mL
Mid QC
210.00 ng/mL
High QC
490.00 ng/mL
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
P&A Batch 1
0.49 97.00 1.43 95.33 206.00 98.10 478.00 97.55 0.47 94.20 1.43 95.33 204.00 97.14 482.00 98.37 0.47 94.80 1.55 103.33 208.00 99.05 476.00 97.14 0.48 96.00 1.42 94.67 206.00 98.10 472.00 96.33 0.49 97.60 1.44 96.00 204.00 97.14 486.00 99.18 0.44 87.00 1.44 96.00 205.00 97.62 478.00 97.55
N 6
6
6
6
Mean 0.47 1.45 205.50 478.67
SD (±) 0.02 0.05 1.52 4.84
CV (%) 4.09 3.36 0.74 1.01
%Accuracy 94.43
96.78
97.86
97.69
Acceptance criteria:
1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.
2. Mean % Nominal (100 ±15% and for LLOQ 100±20%).
Inter Batch Accuracy and Precision
Inter batch accuracy and precision evaluation were assessed by analyzing 5
sets of calibration curves for Valacyclovir and 5 sets of QC samples, 6 replicates each
of the LLOQ, LQC, MQC and HQC.
The inter batch percentage of nominal concentrations for Valacyclovir was
ranged between 95.07% and 102.15%.
The Inter batch percentage of coefficient of variation is 3.29% to 7.80% for
Valacyclovir.
Results are presented in Table 5.7
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Chapter 5 Valacyclovir - Introduction
Table 5.7 Inter batch (Between-Batch) Accuracy and Precision for determination
of Valacyclovir levels in human plasma
Analytical
Run ID
LLOQ
0.50 ng/mL
Low QC
1.50 ng/mL
Mid QC
210.00 ng/mL
High QC
490.00 ng/mL
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
P&A
Batch 1
0.49 97.00 1.43 95.33 206.00 98.10 478.00 97.55
0.47 94.20 1.43 95.33 204.00 97.14 482.00 98.37
0.47 94.80 1.55 103.33 208.00 99.05 476.00 97.14
0.48 96.00 1.42 94.67 206.00 98.10 472.00 96.33
0.49 97.60 1.44 96.00 204.00 97.14 486.00 99.18
0.44 87.00 1.44 96.00 205.00 97.62 478.00 97.55
P&A
Batch 2
0.47 94.40 1.40 93.33 199.00 94.76 458.00 93.47
0.49 97.00 1.41 94.00 185.00 88.10 426.00 86.94
0.47 93.60 1.45 96.67 192.00 91.43 462.00 94.29
0.46 92.60 1.33 88.67 202.00 96.19 452.00 92.24
0.47 94.20 1.37 91.33 199.00 94.76 424.00 86.53
0.47 93.80 1.49 99.33 203.00 96.67 428.00 87.35
P&A
Batch 3
0.54 107.20 1.66 110.67 216.00 102.86 497.00 101.43
0.56 111.60 1.53 102.00 201.00 95.71 458.00 93.47
0.56 111.60 1.49 99.33 203.00 96.67 456.00 93.06
0.55 109.40 1.49 99.33 202.00 96.19 476.00 97.14
0.53 105.60 1.48 98.67 210.00 100.00 470.00 95.92
0.58 116.60 1.46 97.33 203.00 96.67 470.00 95.92
P&A
Batch 4
0.50 100.20 1.47 98.00 189.00 90.00 429.00 87.55
0.49 98.60 1.38 92.00 199.00 94.76 418.00 85.31
0.53 105.40 1.46 97.33 202.00 96.19 457.00 93.27
0.53 106.40 1.55 103.33 212.00 100.95 469.00 95.71
0.53 106.00 1.51 100.67 212.00 100.95 482.00 98.37
0.52 103.40 1.51 100.67 199.00 94.76 466.00 95.10
P&A
Batch 5
0.54 107.60 1.55 103.33 211.00 100.48 504.00 102.86
0.50 99.00 1.38 92.00 202.00 96.19 486.00 99.18
0.59 118.40 1.40 93.33 196.00 93.33 464.00 94.69
0.50 100.00 1.43 95.33 201.00 95.71 478.00 97.55
0.56 112.40 1.42 94.67 202.00 96.19 481.00 98.16
0.56 112.80 1.40 93.33 197.00 93.81 493.00 100.61
N 30
30
30
30
Mean 0.51 1.46 202.33 465.87 SD(±) 0.04 0.07 6.65 22.27 % CV 7.80 4.68 3.29 4.78
%Nominal 102.15
97.18
96.35
95.07
Acceptance criteria: Same as presented in Table 5.6
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Chapter 5 Valacyclovir - Introduction
Calibration Curve
Calibration curves are found to be consistently accurate and precise for
Valacyclovir over 0.50 -700.00 ng/mL for calibration range. The correlation
coefficient is greater than 0.9988 for Valacyclovir. Back calculations were made from
the calibration curves to determine Valacyclovir concentrations of each calibration
standard.
Results are presented in Tables 5.8 & 5.9
Table 5.8 Summary of calibration curve parameters for Valacyclovir in human
plasma
Analytical Run ID A B C
Coefficient of
regression
(r2)
P&A Batch-1 -0.000001537 0.02206 0.0004753 0.9980
P&A Batch-2 -0.000004956 0.02394 -0.0003083 0.9994
P&A Batch-3 -0.000002361 0.02194 -0.0009788 0.9994
P&A Batch-4 -0.000002326 0.02453 -0.0002957 0.9987
P&A Batch-5 -0.000001149 0.02193 0.00008181 0.9989
N 5 5 5
Mean -000002.4658 0.02288 -0.00021 0.9988
SD (±) 000001.48562 0.001255 0.000539 0.00058
CV (%) -60.24 5.48 -262.82 0.05
Regression model y = ax2 +bx+c
where:
y = peak area ratio (PAR) of valacyclovir to internal standard.
x = concentration (ng/mL) of valacyclovir in plasma.
Acceptance criteria:
1. Coefficient of regression (r) ≥ 0.9980.
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Chapter 5 Valacyclovir - Introduction
Table 5.9 Back-calculated standard concentrations from each calibration curve
for Valacyclovir in human plasma.
Analytical Run
ID
Nominal Concentration (ng/mL)
CS1 CS2 CS3 CS4 CS5
0.50
ng/mL
1.00
ng/mL
5.00
ng/mL
35.00
ng/mL
70.00
ng/mL
P&A Batch-1 0.52 0.91 4.81 37.30 70.70
P&A Batch-2 0.51 0.99 4.77 36.50 70.90
P&A Batch-3 0.51 0.96 4.90 35.30 72.40
P&A Batch-4 0.51 0.98 4.75 35.40 72.20
P&A Batch-5 0.50 1.02 4.74 37.10 69.80
N 5 5 5 5 5
Mean 0.51 0.97 4.79 36.32 71.20
SD(±) 0.01 0.04 0.07 0.93 1.09
CV% 1.87 4.05 1.36 2.57 1.53
%Nominal 101.72 97.26 95.88 103.77 101.71
Analytical run
ID
Nominal Concentration (ng/mL)
CS6 CS7 CS8 CS9 CS10
140.00
ng/mL
280.00
ng/mL
420.00
ng/mL
560.00
ng/mL
700.00
ng/mL
P&A Batch-1 141.00 282.00 419.00 556.00 700.00
P&A Batch-2 139.00 284.00 416.00 556.00 702.00
P&A Batch-3 138.00 285.00 426.00 555.00 693.00
P&A Batch-4 147.00 280.00 395.00 561.00 715.00
P&A Batch-5 141.00 269.00 414.00 571.00 704.00
N 5 5 5 5 5
Mean 141.20 280.00 414.00 559.80 702.80
SD(±) 3.49 6.44 11.55 6.69 7.98
CV% 2.47 2.30 2.79 1.19 1.14
%Nominal 100.86 100.00 98.57 99.96 100.40
Acceptance criteria
1. Mean % Nominal (100±15%) except lowest calibration standard.
2. Mean % Nominal (100±20%) for lowest calibration standard (CS1).
3. % CV ≤ 15% except lowest calibration standard (CS1) for which it is ≤ 20%.
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Chapter 5 Valacyclovir - Introduction
Recovery
The percentage recovery of Valacyclovir was determined by comparing the
mean peak area of Valacyclovir in extracted LQC, MQC, HQC samples with freshly
prepared unextracted LQC, MQC, HQC samples respectively.
The mean % recovery for LQC, MQC, HQC samples of Valacyclovir were
88.48%, 105.88% and 103.15% respectively.
The mean recovery of Valacyclovir across QC levels is 99.17%.
The mean recovery of % CV recovery of Valacyclovir across QC levels is
10.9%.
For the internal standard, mean peak area of 18 extracted samples was
compared to the mean peak area of 18 unextracted IS solution. The mean %
recovery is 110.84%.
The % CV recovery of IS Valacyclovir- D8 for extracted is 7.9%.
Results are presented in Table 5.10
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Chapter 5 Valacyclovir - Introduction
Table 5.10 Recovery of Analyte Valacyclovir and Valacyclovir-D8 from human
plasma
Standard
Extracted peak
response
Unextracted peak
response
Drug IS Drug IS
Low QC: 1.50 ng/mL
21804 614170 27316 729049
19882 608307 23875 655807
22415 708869 22084 599935
24321 769709 24652 657996
18076 575189 22374 628841
20168 651502 22846 630215
N 6 6 6 6
Mean 88.48
SD (±) 9.193
%CV 10.4
Medium QC: 210.00 ng/mL
3082852 666064 3005197 619888
2873999 667969 2830170 580764
2700670 619936 2357070 480503
3164543 729545 3024239 607338
2813385 623809 2504035 506765
2500981 575139 2460142 505789
N 6 6 6 6
Mean 105.88
SD(±) 9.071
%CV 8.6
High QC: 490.00 ng/mL
6632999 643479 6561725 603186
6093578 637962 5900211 535684
6016152 632263 5769571 514291
6211171 626532 6337750 572815
6070756 620561 5607332 504914
5914901 604012 5636899 509386
N 6 6 6 6
Mean 103.15
SD(±) 4.225
%CV 4.1
Drug IS
Mean recovery of across QC levels 99.17 110.84
Mean SD(±) of across QC levels 10.77 8.742
The Mean % CV across QC levels 10.9 7.9
Acceptance criteria:
1. The coefficient of variation for mean recovery across LQC, MQC and HQC
shall not exceed 25%.
2. The coefficient of variation for mean recovery of IS shall not exceed 25%.
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Chapter 5 Valacyclovir - Introduction
Matrix Effect
Samples were prepared at LQC & HQC level in triplicate in each of 6 different
lots of human plasma. A calibration curve and 6 replicates of LQC & HQC samples in
triplicate for each matrix were freshly prepared and analyzed in single run.
No significant matrix effect found in different sources of human plasma tested
for Valacyclovir, Valacyclovir- D8.
Results are presented in Tables 5.11 and 5.12.
Table 5.11 Assessment of Matrix Effect on determination of Valacyclovir
at LQC levels in human plasma
Identification of
matrix
Drug response
in Matrix at
LQC Level
Internal
standard
response
Matrix factor
AP/3451/09/10 4866 404298 0.011 AP/3451/09/10 4917 407166 0.012
AP/3451/09/10 5614 446360 0.0121 AP/3452/09/10 6080 457956 0.0126 AP/3452/09/10 6903 541129 0.0133
AP/3452/09/10 5836 428604 0.0128 AP/3453/09/10 6100 434136 0.0136 AP/3453/09/10 6634 450758 0.0141
AP/3453/09/10 6793 483352 0.0147 AP/3454/09/10 6848 454347 0.0141
AP/3454/09/10 6771 449004 0.0151 AP/3454/09/10 6781 442115 0.0151 AP/3455/09/10 6061 425255 0.0153 AP/3455/09/10 6255 415656 0.0143
AP/3455/09/10 6465 423037 0.015 AP/3456/09/10 5907 406537 0.0153
AP/3456/09/10 6285 425181 0.0145 AP/3456/09/10 4866 404298 0.0148
N 18 18 18
Grand Mean 0.013872 SD(±) 0.0013 % CV 9.36
Acceptance criteria:
1. Mean % Nominal 100±15% of nominal value.
2. % CV ≤ 15%.
149
Chapter 5 Valacyclovir - Introduction
Table 5.12 Assessment of Matrix Effect on determination of Valacyclovir
at HQC levels in human plasma
Identification of matrix Drug response
in Matrix at
HQC Level
Internal
standard
response
Matrix factor
AP/3451/09/10 4785575 368086 13.0012
AP/3451/09/10 4841203 364895 13.2674
AP/3451/09/10 4935020 373153 13.2252
AP/3452/09/10 5219207 393230 13.2727
AP/3452/09/10 5629933 415291 13.5566
AP/3452/09/10 5456931 404696 13.484
AP/3453/09/10 5464913 403621 13.5397
AP/3453/09/10 5544012 401873 13.7954
AP/3453/09/10 5191929 383555 13.5363
AP/3454/09/10 5464629 396315 13.7886
AP/3454/09/10 5427333 397085 13.6679
AP/3454/09/10 5489749 400656 13.7019
AP/3455/09/10 5231784 384228 13.6164
AP/3455/09/10 4965675 369029 13.456
AP/3455/09/10 5052930 371975 13.5841
AP/3456/09/10 5468764 392570 13.9307
AP/3456/09/10 5360425 391526 13.6911
AP/3456/09/10 5422345 390640 13.8807
N 18 18 18
Grand Mean 13.55533 SD(±) 0.2438 % CV 1.80
Acceptance criteria:
1. Mean % Nominal 100 ±15% of nominal value.
2. % CV ≤ 15%.
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Chapter 5 Valacyclovir - Introduction
Dilution Integrity
Dilution integrity experiment was carried out at six replicate of two times
diluted (1 in 2 dilution) and four times diluted of approx 1.5 × ULOQ (1 in 4 dilution)
samples were prepared and concentrations were calculated including the dilution
factor against the freshly prepared calibration curve.
The % accuracy of Valacyclovir nominal concentrations ranged between
90.50% and 92.43% for 1 in 2 dilutions and 1 in 4 dilutions respectively.
The % CV is 4.29% to 4.59%.
Results are presented in Table 5.13.
Table 5.13 Assessment of Dilution integrity for Valacyclovir
at DQC Conc (ng/mL)
DQC
Dilution factor: ½
Nominal conc: 1125.00 ng/mL
DQC
Dilution factor: ¼
Nominal conc: 1125.00 ng/mL
Conc. Found % Nominal Conc. Found %Nominal
1090.00 96.89 1090.00 96.89
986.00 87.64 1050.00 93.33
1040.00 92.44 1040.00 92.44
976.00 86.76 1050.00 93.33
1030.00 91.56 949.00 84.36
987.00 87.73 1060.00 94.22
N 6
6
Mean
%Nominal 90.50 92.43
SD (±) 43.73 47.71
CV (%) 4.29 4.59
Acceptance criteria:
1. % CV ≤ 15%.
2. Mean % Nominal (100 ±15%).
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Chapter 5 Valacyclovir - Introduction
Whole Batch Reinjection Reproducibility
To evaluate the whole batch reinjection reproducibility experiment, samples of
P & A batch-2 were kept at auto sampler temperature for approx. 26 hrs after the
initial analysis and were re-injected again after approx. 26 hrs. Concentrations were
calculated to determine precision and accuracy after reinjection.
The Accuracy of Valacyclovir QC samples in reinjection was between
100.23% and 106.84%.
The Precision (% CV) of Valacyclovir QC samples in reinjection was between
3.27 % and 5.47%.
Valacyclovir was found to be stable at autosampler temperature post
extraction (in reconstitution solution) for approx. 26 hrs and reproducible after
reinjection.
Results are presented in Table 5.14
Table 5.14 Assessment of Whole Batch Re-injection Reproducibility during
estimation of Valacyclovir in human plasma
Analytical
Run ID
Low QC 1.50 ng/mL High QC 490.0 ng/mL
Comp sample Reinjection
sample Comp sample
Reinjection
sample
1.47 1.53 429 516 1.38 1.45 418 486 1.46 1.55 457 479 1.55 1.43 469 464 1.51 1.45 482 488 1.51 1.49 466 474
N 6 6 6 6
Mean 1.48 1.48 453.50 484.50 SD(±) 0.06 0.05 24.83 17.71 %CV 3.96 3.27 5.47 3.65
%NOM 100.23 106.84
Acceptance criteria:
1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.
2. Mean % Nominal (100 ±15% and for LLOQ 100 ±20%).
3. 67% 0f the re-injected QCs at each level shall be within ± 20% of their previous
concentration.
152
Chapter 5 Valacyclovir - Introduction
Ruggedness-Different Analyst
To evaluate ruggedness experiment with different analysts, one P&A batch
(P&A-3) was processed by different analyst. The run consisted of a calibration curve
standards and 6 replicates of each LLOQ, LQC, MQC, HQC samples.
The Accuracy of Valacyclovir QC samples within the range of 98.43% to
116.90%.
The Precision of Valacyclovir QC samples within the range of 0.74% to
4.81%.
These results indicated that the method is rugged and reproducible by different
analyst.
Results are presented in Table 5.15
Table 5.15 Ruggedness of the method for estimation of Valacyclovir Plasma
levels in human plasma with different Analyst.
Analytical
Run ID
LLOQ
0.50 ng/mL
Low QC
1.50 ng/mL
Mid QC
210.00 ng/mL
High QC
490.00 ng/mL
Analyst
ID 1
Analyst
ID 2
Analyst
ID 1
Analyst
ID 2
Analyst
ID 1
Analyst
ID 2
Analyst
ID 1
Analyst
ID 2
P&A Batch
3
0.49 0.54 1.43 1.66 206.00 216.00 478.00 497.00
0.47 0.56 1.43 1.53 204.00 201.00 482.00 458.00
0.47 0.56 1.55 1.49 208.00 203.00 476.00 456.00
0.48 0.55 1.42 1.49 206.00 202.00 472.00 476.00
0.49 0.53 1.44 1.48 204.00 210.00 486.00 470.00
0.44 0.58 1.44 1.46 205.00 203.00 478.00 470.00
N 6 6 6 6 6 6 6 6
Mean 0.47 0.55 1.45 1.52 205.50 205.83 478.67 471.17
SD (±) 0.02 0.02 0.05 0.07 1.52 5.91 4.84 14.81
CV (%) 3.93 3.16 3.36 4.81 0.74 2.87 1.01 3.14
%Accuracy 116.90
104.59
100.16
98.43
Acceptance criteria:
1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.
2. Mean % Nominal (100±15% & for LLOQ 100 ±20%).
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Chapter 5 Valacyclovir - Introduction
Ruggedness-Different Column
To evaluate ruggedness experiment with different column, samples of P&A
batch-5 were reinjected on different columns with same and specifications,
Concentrations were calculated to determine precision and accuracy.
The Accuracy of Valacyclovir QC samples within the range of 98.05% to
114.44%.
The Precision of Valacyclovir QC samples within the range of 0.74% to
6.65%.
These results indicated that the method is rugged and reproducible by different
analyst.
Results are presented in Table 5.16
Table 5.16 Ruggedness of the method for estimation of Valacyclovir Plasma
levels in human plasma with different Analytical column
Analytical
Run ID
LLOQ
0.50 ng/mL
Low QC
1.50 ng/mL
Mid QC
210.00 ng/mL
High QC
490.00 ng/mL
Column
ID
LC/312
Column
ID
LC/345
Column
ID
LC/312
Column
ID
LC/345
Column
ID
LC/312
Column
ID
LC/345
Column
ID
LC/312
Column
ID
LC/345
P&A Batch
5
0.49 0.54 1.43 1.55 206.00 211.00 478.00 504.00
0.47 0.50 1.43 1.38 204.00 202.00 482.00 486.00
0.47 0.59 1.55 1.40 208.00 196.00 476.00 464.00
0.48 0.50 1.42 1.43 206.00 201.00 472.00 478.00
0.49 0.56 1.44 1.42 204.00 202.00 486.00 481.00
0.44 0.56 1.44 1.40 205.00 197.00 478.00 493.00
N 6 6 6 6 6 6 6 6
Mean 0.47 0.54 1.45 1.43 205.50 201.50 478.67 484.33
SD(±) 0.02 0.04 0.05 0.06 1.52 5.32 4.84 13.63
CV (%) 3.93 6.65 3.36 4.29 0.74 2.64 1.01 2.81
%Accuracy 114.44
98.51
98.05
101.18
Acceptance criteria:
1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.
2. Mean % Nominal (100±15% & for LLOQ 100 ±20%).
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Chapter 5 Valacyclovir - Introduction
Bench Top Stability (at room temp for 41.0 hrs)
Spiked LQC and HQC samples were retrieved from deep freezer and were kept at
room temperature for 41.0 hrs and were processed and analyzed along with freshly prepared
calibration standards, comparison LQC and HQC samples. Concentrations were calculated to
determine mean % change during stability period.
The mean Accuracy for LQC & HQC samples of Valacyclovir from
comparison samples were 96.55% and 97.48% respectively.
The plasma samples of Valacyclovir were found to be stable for
approximately 41.0 hrs min at room temperature.
Results are present in Table 5.17
Table 5.17 Assessment of stability of Analyte (Valacyclovir) in Biological matrix
at Room temperature
Low QC 1.50 ng/mL High QC 490.0 ng/mL
Comparison
samples
(0.00 hr)
Stability samples
(41.0 hrs)
Comparison
samples
(0.00 hr)
Stability samples
(41.0 hrs)
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
1.49 99.33 1.46 97.33 521.00 106.33 485.00 98.98 1.36 90.67 1.34 89.33 501.00 102.24 479.00 97.76 1.44 96.00 1.58 105.33 490.00 100.00 451.00 92.04 1.43 95.33 1.45 96.67 511.00 104.29 476.00 97.14 1.47 98.00 1.43 95.33 512.00 104.49 483.00 98.57 1.38 92.00 1.43 95.33 504.00 102.86 492.00 100.41
N 6
6
6
6
Mean 1.43 1.45 506.50 477.67 SD(±) 0.05 0.08 10.67 14.17
CV (%) 3.53 5.34 2.11 2.97 %Accuracy 95.22 96.55 103.37 97.48
Acceptance criteria:
1. % Ratio (stability/comparison) should be within 85-115 %.
2. %CV ≤ 15%.
3. Mean % Nominal (100 ±15%).
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Chapter 5 Valacyclovir - Introduction
Freeze and Thaw Stability (after 3rd
cycle at -30°C)
Samples were prepared at LQC and HQC levels, aliquoted and frozen at -
30±5°C six samples from each concentration were subjected to three freeze and thaw
cycles (stability samples). These samples were processed and analyzed along with
freshly prepared calibration standards, LQC and HQC samples (comparison samples).
Concentrations were calculated to determine mean % change after 3 cycles.
The mean Accuracy for LQC & HQC samples of Valacyclovir from
comparison samples were 95.22% and 98.37% respectively.
The plasma samples of Valacyclovir were found to be stable after 3 cycles at -
30 ±5°C.
Results are present in Table 5.18
Table 5.18 Assessment of Freeze-Thaw stability of Analyte (Valacyclovir)
at -30±5°C
Low QC 1.50 ng/mL High QC 490.0 ng/mL
Comparison
samples
Stability sample
at 4th
cycle
Comparison
samples
Stability sample at
4th
cycle
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
1.49 99.33 1.45 96.67 521 106.33 486 99.18 1.36 90.67 1.36 90.67 501 102.24 470 95.92 1.44 96.00 1.4 93.33 490 100.00 467 95.31 1.43 95.33 1.49 99.33 511 104.29 475 96.94 1.47 98.00 1.48 98.67 512 104.49 499 101.84 1.38 92.00 1.39 92.67 504 102.86 495 101.02
N 6
6
6
6
Mean 1.43 1.43 506.50 482.00 SD(±) 0.05 0.05 10.67 13.36
CV (%) 3.53 3.69 2.11 2.77 %Accuracy
95.22 95.22 103.37 98.37
Acceptance criteria
Same as presented in Table 5.17
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Chapter 5 Valacyclovir - Introduction
Autosampler stability at 2-8°C in autosampler
LQC and HQC samples were prepared and processed. These processed
samples were analyzed and kept in auto sampler for 79 hrs at 2-8°C and analyzed
along with freshly prepared calibration standard samples. Concentrations were
calculated to determine mean % change during stability period.
The mean Accuracy change for LQC & HQC samples of Valacyclovir from
comparison samples were 96.89% and 99.09% respectively.
Valacyclovir samples were stable for 79 hrs at 2-8°C in autosampler.
Results are present in Table 5.19
Table 5.19 Assessment of Autosampler stability of Analyte (Valacyclovir)
at 2-8°C
Low QC 1.50 ng/mL High QC 490.00 ng/mL
Comparison
samples (0.0 hr)
Stability samples
(79 hr)
Comparison
samples (0.0 hr)
Stability samples
(79 hr)
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
1.49 99.33 1.47 98.00 521 106.33 517 105.51 1.36 90.67 1.43 95.33 501 102.24 480 97.96 1.44 96.00 1.47 98.00 490 100.00 463 94.49 1.43 95.33 1.48 98.67 511 104.29 472 96.33 1.47 98.00 1.46 97.33 512 104.49 487 99.39 1.38 92.00 1.41 94.00 504 102.86 494 100.82
N 6
6
6
6
Mean 1.43 1.45 506.50 485.50
SD(±) 0.05 0.03 10.67 18.90
CV (%) 3.53 1.88 2.11 3.89
%Accuracy 95.22 96.89 103.37 99.08
Acceptance criteria: Same as presented in Table 5.17
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Chapter 5 Valacyclovir - Introduction
Long-term stability (at -30°C temp for 55 days)
Spiked LQC and HQC samples were retrieved from deep freezer after 34 days
and were processed and analyzed along with freshly prepared calibration standards,
comparison LQC and HQC samples. Concentrations were calculated to determine
mean % change during stability period.
The mean Accuracy for LQC and HQC samples of Valacyclovir from
comparison samples were 93.56% and 101.53% respectively.
The plasma samples of Valacyclovir were found to be stable for
approximately 34 days at -30°C temp.
Results are present in Table 5.20
Table 5.20 Assessment of Long term plasma stability of Analyte
(Valacyclovir) at -30°C.
Low QC 1.50 ng/ml High QC 490.0 ng/ml Comparison
samples (0.0 hr)
Stability samples
(34 days)
Comparison samples
(0.0 hr)
Stability samples
(34 days)
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
Conc.
found
%
nominal
1.49 99.33 1.5 100.00 521 106.33 506 103.27 1.36 90.67 1.38 92.00 501 102.24 496 101.22 1.44 96.00 1.41 94.00 490 100.00 481 98.16 1.43 95.33 1.38 92.00 511 104.29 498 101.63 1.47 98.00 1.42 94.67 512 104.49 504 102.86 1.38 92.00 1.33 88.67 504 102.86 500 102.04
N 6
6
6
6
Mean 1.43 1.40 506.50 497.50 SD(±) 0.05 0.06 10.67 8.89 CV (%) 3.53 4.05 2.11 1.79 %Accuracy 95.22 93.56 103.37 101.53
Acceptance criteria: Same as presented in Table 5.17
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Chapter 5 Valacyclovir - Introduction
Short Term Stock Solution Stability of Valacyclovir, and Valacyclovir- D8 at
Room Temperature
Stock solution stability was determined by comparing the peak areas of freshly
prepared stock solutions (comparison samples) with stability stock solutions. Main
Stock solutions of Valacyclovir and Valacyclovir-D8 were freshly prepared and
aliquots of stocks were kept at room temperature for 9.0 hr (stability samples).
Aqueous equivalent highest calibration standard of Valacyclovir and solution of
Valacyclovir- D8 were prepared from the stability samples and analyzed. Areas of
stability samples and freshly prepared samples were compared to determine mean %
change during stability period.
The % CV for of Valacyclovir stock solution from comparison samples was
0.86% and % Ratio (stability/comparison) was 101.25
The % CV for of Valacyclovir- D8 stock solution from comparison samples
was 1.06% and % Ratio (stability/comparison) was 100.08
The % CV for Valacyclovir- D8 working solution (Internal standard spiking
solution) from comparison samples was 1.86% and % Ratio (stability/
comparison) was 101.57
Valacyclovir, Valacyclovir- D8 stock solutions and Valacyclovir- D8 spiking
solutions were found to be stable at room temperature for 9.0 hr.
Results are present in Tables 5.21 and 5.22
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Chapter 5 Valacyclovir - Introduction
Table 5.21 Assessment of Short term stock solution stability of Analyte
(Valacyclovir) and Internal standard (Valacyclovir- D8) at Room temperature
Analyte Internal standard Comparison
Standard stock
solution response
(0.0 hr)
Stability stock
solution response
(9.0 hr)
Comparison stock
solution response
(0.0 hr)
Stability
Standard stock
Response
(9.0 hr)
1125523 1164383 247074 242627 1148859 1161817 238708 245090 1140661 1158323 244735 249679 1157040 1136488 243203 243594 1112872 1152873 240936 243848 1160514 1157245 240507 243193
N 6 6 6 6
Mean 1140911.50 1155188.17 242527.17 244671.83
SD (±) 18604.47 9975.97 3072.58 2586.71
CV (%) 1.63 0.86 1.27 1.06
%
Ratio
101.25
100.88
Acceptance criteria:
1. % change should be ± 15 %
Table 5.22 Assessment of Short term solution stability of internal standard
spiking solution (Valacyclovir- D8) at refrigerated conditions
Comparison solution (Internal
standard Spiking solution)
Response (0.0 hr)
Stability solution (Internal
standard spiking solution)
Response (9.0 hr)
240177 242981 238872 248870 232906 236707 241784 241009 231518 239699 238928 237213
N 6 6
Mean 237364.17 241079.83
SD (±) 4152.57 4478.55
CV (%) 1.75 1.86
% Ratio 101.57
Acceptance criteria:
1. % change should be ± 5%
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Chapter 5 Valacyclovir - Introduction
Method validation Conclusion
As all the values obtained were within the Acceptance criteria. The method
stands validated and is suitable for estimation of plasma concentrations of
Valacyclovir in a single analytical run. The rugged, efficient Solid phase extraction
method provides exceptional sample clean up and constant recoveries using 200µl of
plasma. The high extraction efficiency, low limit of quantification, and wide linear
dynamic range make this a suitable method for use in clinical samples from
Pharmacokinetic studies following oral administration of Valacyclovir fixed dose
(1000/1000 mg) tablets in healthy human subjects.
5.5 Application
In order to evaluate the practical applicability of the developed method, we used it for
drug analysis throughout a project designed for bioequivalence analysis of Valacyclovir
generic product (Test tablet) with the innovator product. For this purpose, twenty healthy,
non-alcoholic, non-smoking, male volunteers were enrolled in this study. These volunteers
were, contracted in APL Research Pvt.Lt.D, Hyderabad, India. The clinical protocol was
approved by the IEC (Institutional Ethics Committee) as per ICMR (Indian council for
medical research) guidelines. The volunteers gave written informed consent after they had
received detailed instructions about the aims, restrictions and possible adverse effect, which
could be experienced as a result of taking the drug. Volunteers were healthy and had no
history of kidneys and metabolic diseases. Also they had a routine physical examination and
the routine laboratory tests found them to be normal. Subjects did not receive any medication
during the 2 weeks period prior to the start and also were not undergoing any pharmacological
treatment during the study period. The study was an open, randomized, two-period, two-
161
Chapter 5 Valacyclovir - Introduction
group crossover design over a 7days washout period between doses. The tablets were
administered to the volunteers in the next morning after an overnight fast, with 200 ml of
water. No other food was permitted for consumption during the sampling period. Blood
samples (2 ml) were collected via the catheter into K2EDTA anticoagulant containing tubes at
0, 0.33, 0.5, 0.67, 0.83, 1.0, 1.25, 1.5, 1.75, 2.0, 2.33, 2.67, 3.0, 3.33, 3.67, 4.0, 4.5, 5.0 and
6.0 h post-dosing. The blood samples were centrifuged at 4000 rpm for 10 min and the
plasma was separated stored at -80 °C until assayed for Valacyclovir content 40,41
The Mean Plasma concentration data for 20 volunteers is represented in Table
5.23 with respective concentration-time curve is shown in Figure 5.13.
Table 5.23. Valacyclovir Mean concentration (ng/mL) data for the subject samples obtained from
the LC-MS/MS
Time in hours Mean Plasma Concentration data
Test Reference
0 0 0
0.33 73.38014 81.51986
0.5 104.2249 98.44586
0.67 111.6632 105.0226
0.83 119.4778 105.2104
1 115.1178 114.2283
1.25 131.4125 143.7394
1.5 129.6616 157.2031
1.75 115.9391 139.2379
2 98.02067 125.6938
2.33 77.84638 93.85057
2.67 40.61176 55.72605
3 22.69424 29.17157
3.33 14.01033 16.90043
3.67 10.23967 11.39776
4 7.706762 8.646
4.5 5.779857 5.691524
5 4.134095 4.206143
6 2.475619
2.457048
162
Chapter 5 Valacyclovir - Introduction
Figure 5.13 Mean plasma Concentration Vs Time curve for Valacyclovir
5.6 Pharmacokinetic Studies
Pharmacokinetic parameters from the human plasma concentration samples
were calculated by a non compartmental statistics model using WinNon-Lin5.0
software (Pharsight, USA). Blood samples were taken for a period of 3 to 5 times of
the terminal elimination half-life (t1/2) and it was considered as area under the
concentration time curve (AUC) ratio higher than 80% as per FDA guidelines8-10
.
Plasma Valacyclovir concentration-time profiles were visually inspected Cmax and
Tmax values were determined. The AUC0–t was obtained by trapezoidal method.
AUC0-∞ was calculated up to the last measureable concentration and extrapolations
were obtained using the last measureable concentration and the terminal elimination
rate constant (Kel). The terminal elimination rate constant (Kel), was estimated from
the slope of the terminal exponential phase of the plasma of Valacyclovir
163
Chapter 5 Valacyclovir - Introduction
concentration-time curve by means of the linear regression method. The terminal
elimination half-life, t1/2, was then calculated as 0.693/Kel. Regarding AUC0–t, AUC0-∞
and Cmax bioequivalence was assessed by means of analysis of variance (ANOVA)
and calculating the standard 90% confidence intervals (90% CIs) of the ratios
test/reference (logarithmically transformed data). The bioequivalence was considered
when the ratio of averages of log-transformed data was within 80 to 125% for AUC0–t,
AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 5.24 and Table 5.25.
Table 5.24 Valacyclovir Pharmacokinetic data
Valacyclovir Pharmacokinetic data
Pharmacokinetic
Parameter
Test Reference
Mean±SD Mean±SD
Cmax
(ng/mL ) 157.20 ± 56.09 131.41 ± 51.74
AUC 0-t
(ng h/mL) 322.32 ± 10.85 286.78 ± 12.78
AUC 0-inf
(ng h/mL) 325.78± 8.44 290.89± 10.21
Tmax(hr) 1.5 1.25
t1/2 0.9760 1.125
Table 5.25 Valacyclovir Pharmacokinetic data (Test/Reference)
Pharmacokinetic
Parameter Cmax AUC 0-t
AUC 0-∞
Test/Reference 88.97 89.29
83.59
164
Chapter 5 Valacyclovir - Introduction
Pharmacokinetic Studies Conclusion
The present study provides firm evidence to support that the in house
Valacyclovir 1000 mg was bioequivalent with Valtrex Tablets (GSK, Australia) 1000
mg tablet under fasting conditions.
In vivo data was predicted by using Solid Phase Extraction procedure and
concentrations were found through Liquid Chromatography Tandem Mass
Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,
AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidence
intervals whose acceptance range is in between 80% -125%.
The results obtained for Valacyclovir was within the acceptance range.
Therefore, it can be concluded that the two Valacyclovir formulations (reference and
test) analyzed were bioequivalent in terms of rate and extent of absorption.