simultaneous determination of acyclovir and valacyclovir...
TRANSCRIPT
Chapter-1
Page 17
SIMULTANEOUS DETERMINATION OF ACYCLOVIR AND
VALACYCLOVIR IN HUMAN PLASMA
CONTENTS
1.1 INTRODUCTION
1.2 EXPERIMENTAL
1.2.1 Study Objective
1.2.2 Reference Compounds
1.2.3 Chemicals, Reagents and Materials
1.2.4 Liquid Chromatographic Conditions
1.2.5 Mass Spectrometric Conditions and Data Processing
1.2.6 Standard Solutions (Calibration Standards and Quality Control Samples)
1.2.7 Extraction Procedure
1.3 RESULTS AND DISCUSSION
1.3.1 Method Development
1.3.2 Method Validation
1.3.2.1 Selectivity
1.3.2.2 Linearity
1.3.2.3 Recovery
1.3.2.4 Precision and Accuracy
1.3.2.5 Matrix Factor
1.3.2.6 Dilution Integrity
1.3.2.7 Stability Study
1.4 CONCLUSION
1.5 REFERENCES
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1.1 INTRODUCTION
Valacyclovir (VCV), the L-valyl ester of acyclovir, is an oral prodrug that is rapidly and
extensively metabolized by enzymatic hydrolysis probably in the liver and the intestine
to acyclovir (ACV) and L-valine1,2. VCV is used for the treatment of herpes virus
infections predominantly caused by the herpes simplex virus (HSV-1, HSV-2) and the
varicella zoster virus (VZV)3,4. ACV is the active antiviral component of VCV and has
also shown good activity against the Epstein-Barr virus and moderate efficacy against
cytomegalovirus1. The structural similarity of ACV and VCV with certain endogenous
substances requires a very selective analytical method, for the quantitative determination
of ACV and VCV in a range of extremely low concentrations. Radioimmunoassays
(RIA)5 and enzyme-linked immuno absorbent assays (ELISA)6 to determine the
concentration of ACV in serum and plasma are very sensitive, but these methods are
costly and time-consuming. HPLC with fluorescence or UV detection is a very common
method used for the determination of ACV7-10 and its structural analogue11. In literature,
most assays by LC7, 12-20 or RIA quantify only ACV7, 13, 16, 17, 21-26, but not VCV. Recently,
a few LC methods using isocratic and gradient elution have been published for the
analysis of VCV and ACV simultaneously27, 28. But, the methods reported by researchers
had set backs in terms of less sensitivity, long run time, processing large samples and
requiring large plasma volume7,9,29,. Moreover, there are no combination methods
available to estimate both ACV and VCV with better consistency31.
Acyclovir, 9-[(2-hydroxyethoxy)-methyl] methyl]-guanosine, is a synthetic purine
nucleoside derived from guanine, which exhibits a selective inhibition of herpes virus
replication with potent clinical antiviral activity against the herpes simplex and varicella-
zoster viruses28, 32. The pharmacokinetics of acyclovir is characterized by significant
individual variability33-35, leading to variability in the concentrations and therapeutic
efficacy achieved in patients. Understanding the factors that influence and contribute to
these differences, will allow for doses to be individually adjusted in order to achieve
optimal antiviral therapeutic efficacy.
Prompted by the importance of both ACV and VCV in combating viral aliments and
considering the availability of only LC methods on quantifying both, an attempt has been
made to develop a simple, sensitive and selective method for the simultaneous
quantification of ACV and VCV in human plasma using LC-MS/MS to overcome
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hurdles like long run time, less sensitivity, processing large number of samples and with
plasma volume.
1.2 EXPERIMENTAL
1.2.1 Study Objective
Purpose of the present study was to develop and validate an LC-MS/MS method for
simultaneous determination of acyclovir and valacyclovir in human plasma.
1.2.2 Reference Compounds : Acyclovir ; Valacyclovir
IUPAC Name : 9-[(2-hydroxyethoxy)-methyl] methyl]-guanosine; 2-[(2-
amino- 1, 6-dihydro-6-oxo-9H-purin-9-yl)methoxy]ethylestermonohydrochloride.
Molecular Formula : C8H11N5O ; C13H20N6O4. HCl
Molecular Weight : 225 ; 360.80
Purity : 99.82 % ; 89.9 %
Supplier : Arochem industries, (Thane, Maharashtra, India);
Dr.Reddy Laboratories, (Hyderabad, AP, India).
N
NNH
O
NH2
N
O
OH
N
NNH
N
O
NH2
OO
NH2
O
HC l
(I) (II)
Fig.1.1 Structures of Acyclovir (I) and Valacyclovir (II)
Reference Compound : Fluconazole; Molecular Formula : C13H12F2N6O
Molecular weight : 306.27; Purity : 99.32%
IUPAC Name : 1H-1, 2, 4-Triazole-1-ethanol, 1-(2, 4-difluorophenyl)-1-(1H-
1, 2, 4- triazol-1-ylmethyl)-.2, 4-Difluoro-1, 1-bis(1H-1, 2, 4-triazol-1 ylmethyl)
benzyl alcohol
Supplier : Dr.Reddys laboratories, (Hyderabad, AP, India).
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1.2.3 Chemicals, Reagents and Materials
1.2.3.1 Chemicals
All the solvents were of high purity and before analysis, all the glass ware
(flasks, tubes, pipettes) were carefully cleaned and rinsed with Milli Q water
(Type-1 grade). The reagents were obtained as stated:-
Methanol, from Merck (Darmstadt, Germany)
Formic acid from Merck (Darmstadt, Germany)
Hydrochloric acid from Merck (Darmstadt, Germany)
Water deionised and purified by a Milli-Q water purification system
from Millipore (Bedford, MA, USA)
Oasis HLB extraction cartridges 1cc (30mg) from Waters-India
(Bangalore, India)
Blank K2EDTA plasma bags (six different lots) from Karnavati blood
Bank (Ahmedabad, India)
1.2.3.2 Reagents
Mobile phase buffer: In a 500mL volumetric flask, 500µL of formic acid was
made up to mark with water and mixed thoroughly.
Mobile Phase: In a 1000mL volumetric flask 700mL of methanol and 300mL of
mobile phase buffer were properly mixed and filtered through 0.2µm filter
before use.
Extraction buffer: In a 100mL of volumetric flask, 850µL Hydrochloric acid
was made up to volume with water.
1.2.3.3 Materials
API 4000 triple Quadra pole Mass Spectrometer (Applied Biosystem-
SCIEX, Canada)
Shimadzu HPLC system with C18 Gemini column(150mm x 4.6mm,5µ)
Micropipettes from Eppendorf, Hamburg, Germany
Solvent filter, 0.2µm, Millipore, Banglore, India
Vortex mixer, Spinix, Tarson, India
Balance ME-5 from Sartorius, Germany
Positive pressure processor for SPE from Orochem technologies, USA
Class A calibrated glass ware from different supplier
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1.2.4 Liquid Chromatographic Conditions
A Shimadzu HPLC system with C18 Gemini column (150 mm x 4.6 mm, 5µ) that
contains packing of octadecylsilane chemically bonded to porous silica was used for
chromatographic separation. The mobile phase was prepared by addition of 0.1% formic
acid solution in methanol (30:70 v/v, pH 3.5).The flow rate of 0.8 ml/min and 70%
splitting for mobile phase were adjusted to carry out separation. The column temperature
was set at 45˚C, the auto-sampler was conditioned at 10°C and injection volume was
2µL with a run time around 3min.
1.2.5 Mass Spectrometric Conditions and Data Processing
The mass spectrometry was operated in positive ion detection mode. Nitrogen was used
as nebulizing turbo spry. Temperature of vaporizer was set at 400°C and the ESI needle
voltage was 5500V. The declustering potential was set at 60 volts for ACV, VCV and for
Internal Standard (IS). Collision energy for ACV, VCV and IS was 14, 18 and 28V
respectively. The mass spectrometer was operated at unit mass resolution with a dwell
time of 200 milli seconds per transition. Quantification was performed using multiple
reactions monitoring (MRM) of the transition m/z 226.30 (parent ion) → m/z 152.10
(product ion); m/z 325.40 (parent ion) → m/z 152.10 (product ion) and m/z 307.06
(parent ion) → m/z 220.20 (product ion) for ACV, VCV and IS respectively.
1.2.6 Standard Solutions (Calibration Standards and Quality Control Samples)
1.2.6.1 Stock Solution Preparation for ACV and VCV
Stock solutions (2mg/mL of ACV and 1mg/mL of VCV) were prepared for each
reference compound by dissolving 20mg of ACV in 10mL of methanol and 10mg of
VCV in 10mL of methanol separately. Spiking solutions of ACV and VCV were
prepared from stock solutions by serial dilution method in methanol: water (1:1, v/v).
Eight levels of calibration curve standards were prepared by adding the spiking solution
in plasma to achieve the concentration levels of 47.6, 95.3, 216.6, 492.2, 1230.6, 2563.8,
5127.6, 10255.1ng/mL for ACV and 5.0, 10.0, 22.7, 51.6, 129.0, 268.8, 537.6,
1075.2ng/mL for VCV respectively. Four levels of quality control samples were
prepared by adding the spiking solutions in plasma and the concentration levels were
49.1, 140.3, 779.4, 8856.7ng/mL for ACV and 5.1, 14.7, 81.7, 928.6ng/mL for VCV
respectively.
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1.2.7 Extraction Procedure
All the calibration standards (0.5mL) or QC samples (0.5mL) were taken in
polypropylene tubes. 50μL of internal standard (1µg/mL of fluconozole) was added and
vortexed for 10 seconds. 0.5mL of 0.1N hydrochloric acid was added to the plasma
samples, vortexed for 10 seconds and centrifuged for 2min at 4,500 rpm at 4°C. The
samples were transferred to a 1cc/30 mg Oasis HLB SPE column, which had been
conditioned with 1.0mL methanol, followed by 1.0mL water. After application of the
samples, the SPE column was dried for 1.0min by applying positive pressure at
maximum flow rate. The column was eluted with 0.2mL water followed by 0.3mL of
methanol (40:60 v/v) and vortexed for about 10 seconds. The SPE elutes were
transferred into 1mL LC vials for injection of 2μL into the LC system.
1.3 RESULTS AND DISCUSSION
1.3.1 Method Development
Method development was initiated with the aim to suitably optimize the chromatographic
and mass spectrometric conditions. Also, the extraction procedure should be simple and
efficient as VCV and ACV have structural similarity with endogenous components
present in human plasma. The inherent selectivity of MS/MS detection was expected to
be beneficial in developing a selective and sensitive method. Optimum mass acquisition
parameters were obtained by direct infusion of 500ng/mL solution of both the analytes
and IS at a flow rate of 10µL/min. An earlier report had suggested the use of negative
ionization mode and SIM (selected ion monitoring) for quantitation of both the analytes
at low levels31. However, in the present study a much higher response was obtained
under MRM (multiple reaction monitoring) with a signal to noise ratio ≥90 in the
positive ionization mode. The Q1 MS full scan mass spectra of VCV and ACV was
dominated by protonated precursor ions [M+H]+ at m/z 325 and 226 respectively. The
mass spectrometer was set up in multiple reaction monitoring mode to monitor the
transitions from the precursor ions to product ions. The MRM state file parameters like
nebulizer gas, CAD (collision associated dissociation) gas; ion spray voltage and
temperature were suitably optimized to obtain a consistent and adequate response for
both the analytes. A dwell time of 200ms was adequate and no cross talk was observed
between their MRMs.
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Due to similar MRMs for both the analytes it was essential to have a chromatographic
separation of the drugs so as to minimize any interference during quantitation.
Chromatographic analysis of VCV, ACV and IS was initiated under isocratic conditions
to obtain adequate response, sharp peak shape and a short run time. It has been
documented in an earlier report37 that stability of VCV is pH dependent, with maximum
stability at pH≤4 in aqueous media and gastrointestinal fluids. Thus, separation was
attempted using various combinations of methanol/acetonitrile, acidic buffers and
additives like formic acid on different reversed-phase columns [Chromolith RP18
(100mm×4.6mm, 5µm), Kromasil C18 (50/100mm×4.6mm, 5µm), Hypersil C18
(50/100mm×4.6mm, 5µm), Waters Acquity UPLC BEH C18 (2.1mm×50mm,1.7µm),
ACE C18 (50×2.1mm, 5µm) and Gemini C18 (50/150mm×4.6mm, 5µm)]. Best results
in terms of reproducibility, complete separation and peak shape were obtained with
Gemini C18 (150mm×4.6mm, 5µm) column compared to others and hence it was
selected for further study. The analytes showed poor reproducibility for proposed linear
range except for Gemini C-18 column which offered superior peak shape, efficient
separation, desired linearity and reproducibility for VCV, ACV and IS from endogenous
plasma matrix. This may be attributed to the large surface area (396m2/g) of Gemini C-
18 compared to other columns (300-187m2/g). A mobile phase (pH 3.5) consisting of
0.1% formic acid in water and methanol (30:70, v/v) was found most suitable for eluting
VCV, ACV and IS at 1.47, 2.11 and 2.51min respectively. A flow-rate of 0.8mL/min
with 70% flow splitting produced good peak shapes and permitted a run time of 3.0min
per analysis. Previous studies27,31 have reported longer run times (≥4min) for their
simultaneous separation.
Maria K et al.31 have reported a protein precipitation method to separate these polar
drugs from human plasma. Thus, the extraction was carried out initially via protein
precipitation with common solvents like acetonitrile, methanol and acetone, but the
sensitivity (especially for VCV) and reproducibility were poor in all the solvents. There
was frequent clogging of the column which required on-line flushing. Liquid-liquid
extraction technique was also tested to isolate the drugs from plasma using diethyl ether,
dichloromethane, methyl tert butyl ether and isopropyl alcohol (alone and in
combination) as extracting solvents.
However, the recovery was inconsistent with some ion suppression (greater than 15%
CV) for both the analytes. Finally, optimization of solid phase extraction process was
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initiated on Waters Oasis HLB,Waters Oasis MCX and Phenomenex Strata cartridges.
Addition of strong acid like HCl during sample preparation helped in maintaining the
analyte in the ionized form with better retention on Waters Oasis HLB as compared to
other cartridges. Further, use of strong acid during processing enhanced the stability of
VCV and subsequently gave consistent recovery, especially at the LLOQ level with
minimum matrix interference. Initially, different washing solvents like water, dilute
formic acid, acetic acid, hydrochloric acid (alone and in combination) were tried to
achieve desired specificity and reproducibility. But due to polar nature of analytes (VCV
and ACV) none of the washing solvents worked satisfactorily.
Moreover, the recoveries were low when compared with neat samples. Thus, washing
step was avoided; instead, direct elution was done sequentially with water and methanol
respectively. This helped in getting desired recovery, sensitivity, reproducibility with
minimum matrix effect and fast turns around for analysis.
By virtue of its similarity in chromatographic behavior ketoconazole and fluconazole
hydrochloride were tested to minimize analytical variation due to solvent evaporation,
integrity of the column and ionization efficiency. The results found, were superior with
fluconazole hydrochloride as compared to ketoconazole in terms of consistency and
reproducibility and hence it was selected as the internal standard.
1.3.2 Method Validation
1.3.2.1 Selectivity
Possible interferences at the retention times of ACV, VCV and IS from endogenous
compounds were checked during the validation by testing six different batches of
K2EDTA human plasma, one lipemic blank plasma and one lot of haemolysed blank in
order to check the absence of signals for the retention times of each compound.
Selectivity was carried out by analyzing the six blank plasma samples spiked with ACV
and VCV (LLOQ level) and IS.
1.3.2.2 Linearity
Linearity of the method was evaluated using bulk spiked plasma samples in the
concentration range as mentioned above using the method of least squares. Six such
linearity curves were analyzed. Each calibration curve consisted of a blank sample, a
zero sample (blank + IS) and eight concentrations. The standard curves were linear over
the concentration ranges of 47.6 -10255.1 and 5.0 - 1075.2ng/mL for ACV and VCV
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respectively. The mean correlation coefficient was 0.9966 for ACV and 0.9980 for VCV.
Samples were quantified using the ratio of peak area of analyte to that of IS. A weighting
factor linear regression (1/concentration) was performed with the nominal concentrations
of calibration levels. Peak area ratios were plotted against plasma concentrations. The
limits of quantitation were 47.6 and 5.0ng/mL for ACV and VCV respectively which
were better than the previously reported LC methods using gradient solution27.
1.3.2.3 Recovery
The recovery of drug and IS was evaluated at three concentration levels namely low,
medium and high quality control. Recovery was calculated by comparing its response in
replicate samples with that of neat standard solution responses. Analyte recovery from a
sample matrix (extraction efficiency) is a comparison of analytical response from an
amount of analyte added to that determined from sample matrix. The extraction
efficiency of ACV from human plasma at the concentrations of LQC, MQC and HQC
was found to be 50.1, 50.3 and 51.7% respectively. The extraction efficiency of VCV
from human plasma at the concentrations of LQC, MQC and HQC was found to be 46.0,
47.2 and 45.3%. The mean recovery for the internal standard was 79.0% (Table 1.1)
which was superior to the LC method published earlier using LLE27.
Table 1.1 Percentage Recovery of ACV, VCV and IS
Nominal concentrations (ng/mL) % Recovery
ACV VCV ACV VCV Internal Standard
140.3 14.7 50.12 45.96
78.98779.4 81.7 50.25 47.16
8856.7 928.6 51.69 45.28
1.3.2.4 Precision and Accuracy
Intra-day accuracy and precision were evaluated from replicate analyses (n = 6) of
quality-control samples containing ACV and VCV at different concentrations on the
same day. Inter-day accuracy and precision were also assessed from the analysis of the
same QC samples on separate occasions in replicate (n = 6). QC samples were analyzed
against calibration standards.
All calibration curves were found to be linear over the range of 47.6-10255.1 and
5.0 -1075.2ng/mL. The precision for six plasma samples spiked with ACV and
VCV at LLOQ concentration was 1.3 and 2.63% with a mean accuracy of 98.6
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and 101.8% respectively (Table 1.2 and 1.3). The inter-batch assay accuracy for
acyclovir and valacyclovir ranged between 96.0 - 106.3 and 97.4 - 105.5%
respectively, whereas intra-batch accuracy ranged between 98.8 - 105.9 and 91.9
- 102.7%. The inter-batch precision for acyclovir and valacyclovir ranged
between 4.0 - 4.8 and 4.5 - 8.9% and intra batch precision ranged between 3.3 -
3.9 and 2.8 - 5.3%. The results are presented in Table 1.4 and 1.5. Representative
chromatograms of ACV and VCV are depicted in Fig.1.2 - 1.5. All the results
were found within the acceptable limit of precision not more than 15.0% and
accuracy 85.0 - 115.0%, except LLOQ for which precision was not more than
20% and accuracy was between 80.0 - 120.0%.
Table 1.2 Results of Six Calibration Curves for Determination of ACV in Human
Plasma
Conc.added
(ng/mL)
Conc. determined
(mean ± S.D) (ng/mL)
Precision
(%)
Accuracy
(%)
47.6 47.0 ± 0.6 1.3 98.6
95.3 96.1 ± 3.5 3.7 100.9
216.6 219.4 ± 11.0 5.0 101.3
492.2 524.0 ± 19.5 3.7 106.5
1230.6 1254.7 ± 28.3 2.3 102.0
2563.8 2573.1 ± 78.9 3.1 100.4
5127.6 4948.0 ± 210. 4 4.3 96.5
10255.1 9631.2 ± 148.7 1.5 93.9
Table 1.3 Results of Six Calibration Curves for Determination of VCV in Human
Plasma
Conc.added
(ng/mL)
Conc. determined
(mean ± S.D) (ng/mL)
Precision
(%)
Accuracy
(%)
5.0 5.1 ± 0.1 2.6 101.8
10.0 9.7 ± 0.6 6.5 96.7
22.7 22.3 ± 0.7 3.1 98.0
51.6 53.3 ± 1.7 3.2 103.4
129.0 128.0 ± 3.4 2.7 99.2
268.8 267.1 ± 4.2 1.6 99.4
537.6 538.8 ± 13.7 2.5 100.2
1075.2 1089.9 ± 28.8 2.6 101.4
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Table 1.4 Results of Six Quality Control Batches for Determining ACV Concentrations in HumanPlasma Samples
Conc. added
(ng/mL)
Intra-batch (n=6) Inter-batch (n=36)
Conc. determined
(mean ± S.D)
(ng/mL)
Precision
(%)
Accuracy
(%)
Conc. determined
(mean ± S.D)
(ng/mL)
Precision
(%)
Accuracy
(%)
49.1 49.9 ± 1.7 3.3 101.6 49.9 ± 2.4 4.8 101.7
140.3 147.7 ± 5.0 3.4 105.3 149.1 ± 6.2 4.2 106.3
779.4 825.6 ± 31.3 3.8 105.9 824.2 ± 37.6 4.6 105.8
8856.7 8749.7± 340.8 3.9 98.8 8505.2 ± 342.4 4.0 96.0
Table 1.5 Results of Six Quality Control Batches for Determining VCV Concentrations in HumanPlasma Samples
Conc.
added
(ng/mL)
Intra-batch (n=6) Inter-batch (n=36)
Conc. determined
(mean ± S.D)
(ng/mL)
Precision
(%)
Accuracy
(%)
Conc. determined
(mean ±S.D )
(ng/mL)
Precision
(%)
Accuracy
(%)
5.1 4.7 ± 0.2 5.0 91.9 5.4 ± 0.5 9.0 105.5
14.7 14.2 ± 0.8 5.3 96.7 14.8 ± 1.1 7.1 100.7
81.7 84.0 ± 3.6 4.3 102.7 79.6 ±4.4 5.6 97.4
928.6 938.6 ± 26.6 2.8 101.1 921.5 ±41.7 4.5 99.2
1.3.2.5 Matrix Factor
The matrix of plasma constituents over the ionization of analyte was determined by
comparing the concentrations of the post-extracted plasma standard QC samples (n = 6)
with the concentration of analyte from neat samples at equivalent concentrations38-40.The
matrix effect intended method was assessed using chromatographically screened human
plasma. For VCV, the precision (%CV) at HQC and LQC levels was within 5.8 -2.5%.
For ACV, the precision (%CV) at HQC and LQC levels were between 11.5-11.8%
respectively (Table1.6 and 1.7).
1.3.2.6 Dilution Integrity
During the course of study, probability of encountering samples with concentrations
above the upper limit of quantitation (ULOQ) could not be ruled out and therefore
dilution with drug free plasma is necessary to bring them within the calibration range. To
establish the effect of dilution on the integrity of samples, six aliquots of 20,000 and
2,000ng/mL of ACV and VCV respectively were prepared. The samples were subjected
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to fivefold dilution (n = 6) and ten -fold dilution (n = 6) with drug free human plasma to
bring them within the calibration range. The samples were processed, analyzed and the
concentrations obtained were compared with theoretical values.
The precision for dilution integrity standards at 1:5 and 1:10 for ACV were 4.24 and
5.5% and for VCV were 4.98 and 8.26 % respectively. The mean accuracy for dilution
integrity of 1:5 and 1:10 for ACV were 101.00 and 101.23% while for VCV they were
102.63 and 100.21% respectively, which are within the acceptance limits of 85.00 -
115.00% (Table1.8 and 1.9).
Table1.6 Matrix Effect of ACV
Description
HQC LQC
Nominal Concentration (ng/mL)
8856.709 140.290
Calculated Concentrations (ng/mL)
Aliquot 1 8457.707 154.874
Aliquot 2 9382.712 154.277
Aliquot 3 8551.045 148. 682
Aliquot 4 8060.566 160.700
Aliquot 5 8709.871 154.349
Aliquot 6 8047.147 152.812
Mean 8534.841 154.282
SD 493.662 3.874
% CV 5.78 2.51
Table1.7 Matrix Effect of VCV
Description
HQC LQC
Nominal concentration (ng/mL)
928.550 14.708
Calculated Concentration (ng/mL)
Aliquot 1 966.636 16.548
Aliquot 2 1111.782 16.747
Aliquot 3 784.317 13.418
Aliquot 4 942.246 16.860
Aliquot 5 936.199 15.173Aliquot 6 882.452 19.224
Mean 937.272 16.328
SD 107.463 1.933% CV 11.47 11.84
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Table1.8 Dilution Integrity for ACV
Description
DI Spiked Concentration (23307.128ng/mL)
DI 1/5 sample (ng/mL) DI 1/10 sample (ng/mL)
Sample
conc.
With dilution
factor
Sample conc. With dilution factor
Aliquot 1 4644.496 23222.478 2244.179 22441.789
Aliquot 2 4986.045 24930.226 2518.017 25180.171
Aliquot 3 4448.933 22244.666 2374.490 23744.899
Aliquot 4 4570.042 22850.209 2179.428 21794.227
Aliquot 5 4886.601 24433.006 2474.476 24744.758
Aliquot 6 4713.307 23566.533 2365.341 23653.409
Mean 4708.2373 23541.1865 2359.3217 23593.217
SD 199.707 998.539 129.869 1298.697
% CV 4.24 4.24 5.50 5.50
% Mean Accuracy 101.000 101.230
Table1.9 Dilution Integrity for VCV
Description
DI Spiked Concentration (2443.554ng/mL)
DI 1/5 sample(ng/mL) DI 1/10 sample (ng/mL)
Sample conc. With dilution factor Sample conc. With dilution factor
Aliquot 1 501.515 2507.574 242.0497 2420.970
Aliquot 2 508.371 2541.853 252.905 2529.052
Aliquot 3 453.104 2265.521 256.159 2561.586
Aliquot 4 525.116 2625.580 205.566 2055.663
Aliquot 5 508.763 2543.813 260.737 2607.373
Aliquot 6 512.551 2543.813 260.737 2607.373
Mean 501.569 2507.849 244.872 2448.7233
SD 24.987 124.938 20.218 202.180
% CV 4.98 4.98 8.26 8.26
% Mean Accuracy 102.630 100.210
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Fig.1.2. Representative Chromatograms of Acyclovir Extracted Blank Sample
Fig.1.3. Representative Chromatograms of Acyclovir LLOQ Sample
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Fig.1.4. Representative Chromatograms of Valacyclovir Extracted Blank Sample
Fig.1.5. Representative Chromatograms of Valacyclovir LLOQ Sample
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1.3.2.7 Stability Study
Evaluation of the stability of samples was based on the comparison of various samples
against freshly prepared sample of the same concentration. Percentage difference
between the back calculated concentrations obtained for the sample under investigation
and freshly prepared sample was evaluated. Four aliquots, each of LQC and HQC
concentrations were used for stability study.
The bench top stability (at 20-30°C) was determined for 1h 41min by comparing the
accuracy of the mean concentrations for the low and high QCs for ACV and VCV were
found to be 102.1 and 99.7%, 96.3 and 93.6 respectively. The freeze-thaw stability was
determined at -50°C for the low and high QCs of ACV and VCV, which underwent three
freeze thaw cycles. In each freeze thaw cycle, the frozen plasma samples were thawed at
room temperature for 2-3h and refrozen for 12-24h. The accuracy of the mean
concentrations for the low and high QCs of ACV and VCV were found to be 101.4 and
99.5%, 103.4 and 100.3% respectively. Auto sampler stability of the plasma samples
were over 14h 20min was established. All the stability results were tabulated in Table
1.10, were found within the acceptable.
Table 1.10 Stability Results for ACV and VCV
Stability of ACV Stability of VCV
Sampleconcentrations(ng/mL) (n=6)
(nominal)
Conc. found(mean, n=6)
(ng/mL)
Precision(%)
Accuracy(%)
Sampleconcentrations(ng/mL) (n=6)
(nominal)
Conc. found(mean, n=6)
(ng/mL)
Precision(%)
Accuracy(%)
Bench top stability (01 hour 41 minutes)
141.9 (140.2) 144.8 2.9 102.1 14.5 (14.6) 14.4 2.7 96.3
8548.6(8851.8) 8518.3 2.5 99.7 953.6 (924.7) 892.5 2.7 93.6
Freeze thaw stability (after 3 cycles below -500c)
141.9 (140.2) 142.4 7.0 101.4 14.6 (14.6) 15.1 10.0 103.4
9185.6(8851.8) 9142.5 6.1 99.5 975.7 (924.7) 978.2 3.9 100.3
Auto sampler stability ( 14 hours 20 minutes at 50c)
153.1 (140.3) 146.0 6.4 95.4 15.6 (14.7) 16.1 4.1 103.3
8340.5(8856.7) 8016.1 2.9 96.1 961.7 (928.6) 962.7 2.1 100.1
Chapter-1
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1.4 CONCLUSION
The LC-MS-MS assay described here is simple, selective, precise and accurate for
quantification of acyclovir and valacyclovir in human plasma; fully validated according
to FDA guidelines. The developed assay method was validated and found to be precise
(inter batch 4.0-4.8% , 4.5-8.9% for ACV and VCV respectively; intra batch 3.3-3.9%,
2.8-5.3% for ACV and VCV respectively) and accurate (inter batch 96.0-106.3%, 97.4-
105% for ACV and VCV respectively; intra batch 98.8-105.9% ; 91.9-102.7% for CV
and VCV respectively) over a wide concentration range with no interference by
endogenous compounds. This method is highly sensitive (LLOQ 47.0ng/mL for ACV,
1089.9 for VCV) and produced consistent recovery even with less injection volume
(2µL). This method could be used for the routine therapeutic monitoring of the drug and
pharmacokinetic studies.
Chapter-1
Page 34
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