development and validation of a sensitive and rugged spe
TRANSCRIPT
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Patel et al. World Journal of Pharmacy and Pharmaceutical Sciences
VALIDATION OF SENSITIVE AND RUGGED SPE-LC-MS/MS
METHOD FOR DETERMINATION OF ACYCLOVIR IN HUMAN
PLASMA: APPLICATION TO FOUR PIVOTAL
BIOEQUIVALENCE STUDIES
Nirav P. Patela,b*
, Mallika Sanyalb,c
, Naveen Sharmaa, Pranav S. Shrivastav
d,
Bhavin N. Patela*
, Dinesh S. Patel a
aBio-Analytical Laboratory, Cliantha Research India Ltd., Bodakdev, Ahmedabad-380054,
Gujarat, India.
bKadi Sarva Viswavidyalaya, Sector-15, Ghandhinagar-382715, Gujarat, India.
cDepartment of Chemistry, St. Xavier‟s College, Navrangpura, Ahmedabad-380009,
Gujarat, India.
dDepartment of Chemistry, School of Sciences, Gujarat University, Navrangpura,
Ahmedabad-380009, Gujarat, India.
ABSTRACT
A selective, sensitive and rugged liquid chromatography- tandem mass
spectrometry (LC-MS/MS) assay for the determination of acyclovir in
human plasma is developed using Acyclovir-d4 as an internal standard
(IS). The analyte and IS were extracted from 200µL of human plasma
via solid phase extraction on Water Oasis HLB cartridges.
Chromatographic separation is achieved on a BDS Hypersil C18 (150
mm×4.6 mm, 3µm) column under isocratic conditions. Detection of
analyte and internal standard is done by tandem mass spectrometry,
operating in positive ion and multiple reaction monitoring (MRM)
acquisition mode. The method is fully validated for its selectivity,
sensitivity, carryover check, linearity, precision and accuracy,
recovery, matrix effect, ion suppression/enhancement, stability and
dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method
were 0.2500ng/mL and 5.000ng/mL respectively with a linear dynamic range of 5.000-
2500ng/mL for acyclovir. The intra- and inter- batch precision (%CV) and relative recovery
across quality control levels is <3.4% and >73.4% respectively. The method is successfully
Article Received on
29 Aug 2015,
Revised on 20 Sep 2015,
Accepted on 12 Oct 2015
*Correspondence for
Author
Nirav P. Patel
Bio-Analytical
Laboratory, Cliantha
Research India Ltd.,
Bodakdev, Ahmedabad-
380054, Gujarat, India.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 5.210
Volume 4, Issue 11, 1267-1288 Research Article ISSN 2278 – 4357
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applied to four pivotal bioequivalence studies of 800 mg tablet and 200 mg capsule of
acyclovir in 48 and 30 healthy Indian male subjects under fasting and fed condition
respectively. The reproducibility of assay method in the measurement of study data is
demonstrated by incurred sample reanalysis.
KEYWORDS: Acyclovir; LC-MS/MS; solid phase extraction; human plasma;
bioequivalence; incurred sample reanalysis.
INTRODUCTION
Acyclovir (ACV) is a guanosine nucleoside analogue having antiviral activity.[1]
It is a
synthetic deoxyguanosine analog and it is the prototype antiviral agent that is activated by
viral thymidine kinase. It is primarily used for the treatment of herpes simplex virus
infections and varicella zoster virus infections. [2, 3]
The selective activity of ACV is due to its
affinity for the thymidine kinase enzyme encoded by Herpes simplex virus (HSV) and
varicella zoster virus (VZV). ACV involves the highly selective inhibition of herpes virus
DNA replication, via enhanced uptake in herpes virus-infected cells and phosphorylation by
viral thymidine kinase. The substrate specificity of acyclovir triphosphate for viral rather than
cellular, DNA polymerase contributes to the specificity of the drug .[4, 5]
Chemically know as
2-Amino-1, 9-dihydro-9-((2-hydroxyethoxy) methyl)-6Hpurin-6-one. The oral bioavailability
of ACV is 10% to 20%, and decreases with increasing dose. Food does not affect the
absorption of ACV. Protein binding of ACV is 9–33%. It is metabolized to 9-
[(carboxymethoxy) methyl] guanine (CMMG) and 8 hydroxy-acyclovir (8-OH-ACV) by
oxidation and hydroxylation. Half life of ACV is 2.5-3.3 hours, it is excreted unchanged by
the kidneys via active tubular secretion.[6, 7, 8, 9, 10, 11]
Molecular mass of ACV is 225.21
g·mol−1. In previous studies ACV was analysed by high performance liquid chromatography
with UV detection in human plasma,[12, 13, 14, 15, 16]
Also, determination of ACV in human
serum by high-performance liquid chromatography using liquid–liquid extraction was
done.[17]
Moreover, ACV was measured in maternal plasma, amniotic fluid, fetal and
placental tissues by high-performance liquid chromatography.[18]
A LC–MS/MS method
based on hydrophilic interaction liquid chromatography has been reported for the
determination of ACV in pregnant rat plasma and tissues.[19]
A Liquid
chromatography/positive-ion electro spray ionization mass spectrometry (LC–ESI-MS/MS)
method has been reported for simultaneous determination of valacyclovir and ACV in human
plasma,[20, 21, 22]
A liquid chromatography/negative-ion electro spray ionization mass
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spectrometry method reported for the quantification of valacyclovir and its metabolite in
human plasma.[23]
A liquid chromatography–tandem mass spectrometry method reported for
the determination of valacyclovir-HCl and ACV in tsetse flies.[24]
Present paper describes
selective, sensitive and rugged liquid chromatography- tandem mass spectrometry (LC-
MS/MS) assay for the determination of acyclovir in human plasma compared to reported
assay method with linear dynamic range of 5.000-2500ng/mL for ACV. This validated assay
method has been successfully applied on four bioequivalence studies for different dose and
form of ACV.
EXPERIMETAL
Chemicals and materials
Reference standard material of acyclovir (94.5%) was procured from pharmaceutical sponsor,
while acyclovir-d4 (IS, 98.05%) was purchased from clearsynth labs (P.) Ltd. HPLC grade
methanol, acetonitrile and n-Hexane were obtained from S.D.Fine Chemicals Ltd. (Mumbai,
India) and trifluoroacetic acid ammonia salt(98%) was obtained from fisher scientific
(Mumbai, India). Deionized water used for LC-MS/MS was prepared using Milli Q water
purification system from Millipore (Bangalore, India). Oasis HLB (1 cc, 30 mg) extraction
cartridges were procured from Water Corporation (Milford, MA, USA). Control buffered
(K2-EDTA) human plasma was procured from Clinic Department of Cliantha Research India
Limited (Ahmedabad, India) and was stored at -20°C. Centrifuge was of Eppendrof 5810
(Hamburg, Germany).
Mobile Phase solution for SPE: (Acetonitrile: Deionized water: Ammonium trifluoroacetate
Solution (1.0 M)) (80:20:0.05 v/v)
LC-MS/MS Instrumentation and conditions
The liquid chromatography system from Shimadzu (Kyoto, Japan) consisted of a LC-
10ADvp pump, an auto sampler (SIL-HTc) and an on-line degasser (DGU-14A).
Chromatographic column used was BDS Hypersil C18 (150 mm length × 4.6 mm inner
diameter, with 3.0 µm partical size) from Thermo Fisher Scientific Pvt. Ltd. (USA). The
mobile phase consisted of (Acetonitrile: Deionized water: Ammonium trifluoroacetate
Solution (1.0 M)) (80:20:0.05 v/v). Separation of analyte and IS was performed under
isocratic condition at a flow rate 0.7mL/min. The auto sampler temperature was maintained at
4°C and injection volume was kept at 3.0 µL. The total LC run time was 3.6 min. Ionization
and detection of analyte and IS was performed on a triple quadrupole mass spectrometer
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(API-4000) equipped with Turbo Ion spray® from MDS SCIEX (Toronto, Canada) operating
in the positive ion mode. Quantitation was done using MRM mode to monitor protonated
precursor product ion transition of m/z 226.1 152.1 for acyclovir and 230.1 152.1 for
IS (Figure 1a and 1b). All the parameters of LC and MS were controlled by Analyst
software version 1.4.2.
For ACV and IS the source dependant parameters maintained were Gas 1(Nebulizer gas): 55
psi, Gas 2(heater gas): 50 psi, ion spray voltage (ISV): 5500 V, turbo heater temperature
(TEM): 550 °C, entrance potential (EP): 10 V, collision activation dissociation (CAD): 6 psi,
curtain gas (CUR): 30 psi. The compound dependent parameters like declustering potential
(DP), collision energy (CE) and cell exit potential (CXP) were optimized at 45, 20 and 10 V
for acyclovir and 45, 20 and 10 V for IS respectively. Quadrupole 1 and quadrupole 3 were
maintained at unit resolution. A dwell time of 600 ms was set for ACV and IS.
Preparation of standard stock and plasma samples
The ACV standard stock solution of 1000µg/mL was prepared by dissolving requisite amount
in acetonitrile: deionized water (50:50, v/v). This was further diluted in deionized water to get
an intermediate solution of 50.00µg/mL. The working solution of ACV for spiking plasma
calibration standards and quality control samples were subsequently prepared using the
standard and intermediate stock solutions in deionized water. The IS stock solution of
100µg/mL was prepared by dissolving requisite amount of acyclovir-d4 in acetonitrile:
deionized water (50:50, v/v). IS working solution (300.0ng/mL) was prepared using the stock
solution in deionized water. All the above stock solutions were stored at -20°C±10°C until
use and intermediate working solutions were stored at 4°C until use. Drug free plasma, i.e.
control (blank) plasma was withdrawn from the deep freezer and allowed to get completely
thawed before use. The calibration standards (CS) and quality control (QC) samples (LLOQ
QC, lower limit of quantitation quality control; LQC, low quality control; MQC-1 & MQC-2
& MQC-3, medium quality control; HQC, high quality control; ULOQ QC, upper limit of
quantitation quality control) were prepared by spiking blank plasma with respective working
solutions (5% of total volume of plasma). CSs were made at 5.000, 10.00, 20.00, 50.00,
150.0, 300.0, 600.0, 1200, 2000, 2500ng/mL for ACV. QCs were prepared at 5.000ng/mL
(LLOQ), 15.00ng/mL (LQC), 125.0ng/mL (MQC-3), 275.0ng/mL (MQC-2), 1000ng/mL
(MQC-1), 1875ng/mL (HQC) and 2500ng/mL (ULOQ) concentrations. The spiked plasma
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samples at all the levels were stored at -20°C±10°C for validation and subject sample
analysis.
Protocol for sample preparation
Prior to analysis, spiked plasma samples were thawed and allowed to equilibrate at room
temperature. The samples were adequately vortexed before pipetting. Aliquots of 200 µL
plasma solutions containing 10 µL of working solutions of ACV and 190 µL of blank human
plasma were transferred into ria vials. Further, 200 µL working solution of IS (300.0ng/mL)
was added and vortexed to mix. All these procedure were Performed in wet ice bath. Prior to
loading plasma samples, SPE cartridges were pre-washed with 1.0mL of methanol, followed
by 1.0mL deionized water and centrifuged for 1 minute at 3000 rpm at 4°C. Plasma samples
were then applied to these conditioned cartridges and after centrifuged for 2 minutes at 3000
rpm at 4°C, washing was done with 0.5mL of n-Hexane followed by centrifugation for 1
minute at 3000 rpm at 4°C. Elution was carried out with 2×1mL of mobile phase solution
followed by centrifugation for 1 minute at 3000 rpm at 4°C after each step and 3.0 µL of
eluent was used for injection in LC-MS/MS, in partial loop mode.
Methodology for validation
A thorough and complete method validation of acyclovir in human plasma was done
following the USFDA guidelines. The method was validated for selectivity, interference
check, carryover check, linearity, precision and accuracy, reinjection reproducibility,
recovery, ion suppression /enhancement, matrix effect, stability and dilution integrity.
Test for selectivity was carried out in 12 different lots of blank human plasma including
haemolysed and lipemic plasma collected with K2-EDTA as an anticoagulant. From each of
these 12 different lots, two replicates each 190 µL were spiked with 10 µL of deionized
water. In the first set, the blank human plasma was directly injected after extraction (without
analyte and IS), while the other set was spiked with only IS before extraction (total 24
samples). Further, one system suitability sample (SSS) at CS-2 concentration and two
replicates of LLOQ concentration (CS-1) were prepared by spiking 190 µL blank human
plasma with 10 µL of respective working aqueous standards of ACV. The blank human
plasma used for spiking of SSS and LLOQ were chosen from one of these 12 lots of plasma.
The acceptance criterion requires that at least 90% of selectivity should be free from any
interference at the retention time of analyte and IS.
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The interference due to commonly used medications by human volunteers was done for
acetaminophen, aspirin, caffeine, cetrizine, chlorpheniramine maleate, ibuprofen and
pseudoephedrine. Their stock solutions (100µg/mL) were prepared by dissolving requisite
amount in methanol. Further, working solutions (20.0µg/mL) were prepared in deionized
water, spiked in plasma and analyzed under the same conditions at LQC and HQC levels in
triplicate. These sets were processed along with freshly prepared calibration curve standards
(CS) and two sets (8 samples) of qualifying QC samples (HQC, MQC-1, MQC-2 and LQC).
As per the acceptance criteria, the % accuracy should be within 85 to 115%.
Carry over experiment was performed to verify any carryover of analyte, which may reflect
in subsequent runs. The design of the study comprised of the following sequence of injections
i.e. double blank plasma sample two samples of LLOQ double blank plasma ULOQ
sample double blank plasma ULOQ sample double blank plasma, to check for any
interference due to carry over.
The linearity of the method was determined by analysis of six calibration curves containing
ten non-zero concentrations. The area ratio response for ACV/IS obtained from multiple
reaction monitoring was used for regression analysis. Each calibration curve was analyzed
individually by using least square weighted (1/x2) linear regression which was finalized
during pre-method validation. A correlation coefficient (r2) value >0.99 was desirable for all
the calibration curves. The lowest standard on the calibration curve was accepted as the
LLOQ, if the analyte response was at least ten times more than that of drug free (blank)
extracted plasma.
For the determining the intra-batch accuracy and precision, replicate analysis of plasma
samples of analytes was performed on the same day. The run consisted of a calibration curve
and six replicates of LLOQ QC, LQC, MQC-3, MQC-2, MQC-1, HQC, ULOQ QC samples.
The inter-batch accuracy and precision were assessed by analyzing three precision and
accuracy batches on three consecutive validation days. The deviation at each concentration
level from the nominal concentration was expected to be within ±15% except for the LLOQ
where it can be ±20% of the nominal concentration. Further, the reinjection reproducibility
was performed by re-injecting one complete validation batch.
The relative recovery, matrix effect and process efficiency were assessed as recommended by
Matuszewski et al.[25]
All three parameters were evaluated at HQC, MQC-1, MQC-2, MQC-3
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and LQC levels in six replicates. Relative recovery (RE) was calculated by comparing the
mean area response of pre-spiked samples (spiked before extraction) to that of extracts with
post-spiked samples (spiked after extraction) at each QC level. The recovery of IS was
similarly estimated. Absolute matrix effect (ME) was assessed by comparing the mean area
response of unextracted samples (spiked after extraction) with mean area of neat standard
solutions (in mobile phase). The overall „process efficiency‟ (%PE) was calculated as (ME ×
RE)/100. Further, the effect of plasma matrix (relative matrix effect) on analyte quantification
was also checked in eight different batches/lots of K2-EDTA plasma including haemolysed
and lipemic plasma. From each batch, four samples at LQC and HQC levels was prepared
(spiked after before extraction) and checked for the % accuracy and precision (%CV). The
deviation of standards and QCs should not be more than ±15 %. Matrix ion suppression
effects on the MRM LC-MS/MS sensitivity were evaluated by the post column analyte
infusion experiment. A standard solution containing 1000ng/mL of ACV and 300.0ng/mL of
IS in mobile phase was infused post via a „T‟ connector into the mobile phase at 3.0µL/min
employing Harvard infusion pump. Aliquots of 3.0µL of extracted blank plasma samples
(without ACV and IS) was then injected and MRM LC-MS/MS chromatograms were
acquired for acyclovir and IS. Any dip in the baseline upon injection of double blank plasma
would indicate ion suppression, while a peak at the retention time of ACV or IS indicates ion
enhancement.
All stability results were evaluated by measuring the area response (ACV / IS) of stability
samples against freshly prepared comparison standards at LQC and HQC levels. Stock
solutions of ACV and IS were checked for short term stability at room temperature and long
term stability at -20°C±10°C. The solutions were considered stable if the deviation from
nominal value was within ±10.0%. Bench top stability, processed sample stability at room
temperature and at refrigerated temperature (4°C), freeze thaw stability and long term
stability at -20°C were performed at LQC and HQC levels using six replicates at each level.
To meet the acceptance criteria the %CV and % accuracy should be within ±15%. Also, at
least 2/3 quality control samples should meet the criteria of ±15% of nominal concentration.
The dilution integrity experiment was performed with an aim to validate the dilution test to
be carried out on higher analyte concentrations (above ULOQ), which may be encountered
during real subject sample analysis. Dilution integrity experiment was carried out at 5 times
the ULOQ concentration i.e. 12500ng/mL and at HQC level for ACV. Six replicate samples
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each of 1/10 of 5×ULOQ (2500ng/mL) and 1/10 of HQC (1875ng/mL) concentration were
prepared and their concentrations were calculated by applying the dilution factor of 10
against the freshly prepared calibration curve for ACV.
Bioequivalence study design and incurred sample reanalysis
The design of study comprised of “An open label, randomized, two period, two treatment,
two sequence, crossover, balanced, single dose, evaluation of relative oral bioavailability of
test (800mg of acyclovir tablets of an Indian company) and reference formulation
(ZOVIRAX® 800mg acyclovir tablets of GlaxoSmithKline, USA) in 48 healthy Indian
subjects under fast condition and in 30 healthy Indian subjects under fed condition.” The
study was also conducted to evaluate the relative oral bioavailability for test formulation
(200mg acyclovir tablet of an Indian company) and reference formulation (ZOVIRAX®
200mg acyclovir tablets of GlaxoSmithKline, USA) in 48 healthy Indian subjects under fast
condition and in 30 healthy Indian subjects under fed condition. All the subjects were
informed of the aim and risk involved in the study and written consent were obtained. The
inclusion criteria for volunteer selection was based on the age (18 to 45 years old, both
inclusive), body mass index (between 18.5 and 24.9 kg/height2), general physical
examination, electrocardiogram and laboratory tests like hematology, blood chemistry, urine
examination and immunological tests. The exclusion criteria included allergic responses to
ACV, volunteers with history of alcoholism, smokers and having a disease which may
compromise the haemopoietic, gastrointestinal, renal, hepatic, cardiovascular, respiratory,
central nervous system, diabetes, psychosis or any other body system. The work was
approved and subject to review by Institutional Ethics Committee, an independent body
comprising of eight members which includes a lawyer, medical doctors, social workers,
pharmacologists and academicians. The procedures followed while dealing with human
subjects were based on International Conference on Harmonization, E6 Good Clinical
Practice (ICH, E6 GCP) guidelines and 21 CFR. The subjects for all the studies were fasted
10h before administration of the drug formulation. Further, under fed conditions the subjects
were given high fat and high calorie breakfast (consisting of 200mL milk with 16 gm sugar,
two slices of bread with butter and two cheese cutlets, total 939 calories ) 30 min prior to
giving the drug under investigation. Blood samples were collected in vacutainers containing
K2EDTA anticoagulant before (0.0h) and at 0.333, 0.667, 1.0, 1.25, 1.5, 1.75, 2.0, 2.333,
2.667, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0,10.0, 12.0, 18.0, 24.0, 36.0, 48.0 h and at 0.333, 0.667, 1.0,
1.333, 1.667, 2.0, 2.25, 2.5, 2.75, 3.0, 3.333, 3.667, 4.0, 4.5, 5.0, 6.0, 8.0, 10.0, 12.0, 18.0,
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24.0, 36.0 h of administration of drug for 800mg tablet dose under fasting and fed condition
respectively. Blood samples were collected in vacutainers containing K2EDTA anticoagulant
before (0.0h) and at 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 5.0, 6.0,
8.0, 10.0, 12.0, 18.0, 24.0 h and at 0.5, 1.0, 1.333, 1.667, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5,
3.75, 4.0, 4.5, 5.0, 6.0, 8.0, 10.0, 12.0, 18.0, 24.0 h of administration of drug for 200mg
capsule dose under fasting and fed condition respectively. Blood samples were centrifuged at
1811*g at 4°C for 15 min and plasma was separated, stored at -20°C until use. An incurred
sample reanalysis (ISR) was also conducted by computerized random selection of 587 subject
samples (10% of total study samples analyzed) near Cmax and the elimination phase for all
four studies. The results obtained were compared with the data obtained earlier for the same
sample using the same procedure. The percent change in the value should not be more than
±20%
Statistical analysis
The pharmacokinetic parameters of ACV were estimated by non-compartmental model using
WinNonlin software version 5.2.1 (Pharsight Corporation, Sunnyvale, CA, USA). The Cmax
values and the time to reach maximum plasma concentration (Tmax) were estimated directly
from the observed plasma concentration vs. time data. The area under the plasma
concentration-time curve from time for 800 mg tablet under fasting and fed condition 0 to
48h (AUC0-48) and 0 to 36 h (AUC0-36) respectively and for 200 mg capsule under fasting and
fed condition 0 to 24 (AUC0-24) were calculated using the liner trapezoidal rule. The AUC0-inf
was calculated as: AUC0-inf = AUC0-t + Ct/Kel, where Ct is the last plasma concentration
measured and Kel is the elimination rate constant; Kel was determined using linear regression
analysis of the logarithm linear part of the plasma concentration-time curve. The t1/2 of ACV
was calculated as: t1/2 = ln2/ Kel. To determine whether the test and reference formulations
were pharmacokinetically equivalent , Cmax ,AUC0-48, AUC0-24, AUC0-36 and AUC0-inf and
their ratios (test/reference) using long transformed data were assessed; their means and 90%
CIs were analyzed by using SAS® software version 9.1 or higher version (SAS Institute Inc.,
Cary, NC, USA). The drugs were considered pharmacokinetically equivalent if the difference
between the compared parameters was statistically non-significant (P ≥ 0.05) and the 90%
confidence intervals (CI) for these parameters fell within 0.8 to 1.25.
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RESULTS AND DICSCUSSION
Method development
The objective of the present work was to develop and fully validate a simple, rugged,
selective and sensitive method for ACV in human plasma by turbo ion spray LC-MS/MS for
routine sample analysis. Also, the sensitivity should be adequate enough to monitor at least
five half lives of ACV concentration with good accuracy and precision for subject samples.
To realize this aim the extraction procedure, mass spectrometry and chromatographic
conditions were suitably optimized based on the outcome of previous reports. During method
development, the electro spray ionization of ACV and acyclovir-d4 were conducted in
positive ionization mode as both the drug and internal standard are acidic in nature, using
0.1ppm tuning solution. The analyte and IS gave predominant singly charged protonated
precursor [M+H]+ ions at m/z of 226.1 and 230.1 for ACV and IS respectively in Q1 full scan
spectra. Further, fragmentation was initiated using sufficient nitrogen for CAD and by
applying 20 V collision energy to break the precursor ions. The most abundant and consistent
ion found in the product ion mass spectra of ACV was at m/z152.1, resulting from the
cleavage of heterocyclic ring to give a neutral fragment CH3OCH2CH2OH. Similarly, for
acyclovir-d4 the most stable and reproducible product ion was observed at m/z 152.1, due to
elimination of CH3OCD2CD2OH fragment. To attain an ideal Taylor cone and a better impact
on spectral response, nebulizer gas (GS1) pressure was optimized at 55 psi. Fine tuning of
nebulizer gas and CAD gas was done to get a consistent and stable response. Ion spray
voltage and temperature did not have any significant effect on analyte response and hence
were maintained at 5500 V and 550 °C respectively. A dwell time of 600 ms was found
adequate for ACV and IS. Also no cross talk was observed between the MRMs of analytes.
The chromatographic conditions were aimed to achieve an efficient separation and resolution
from endogenous peaks. Also, the response should be adequate with sharp peak shape and a
short run time for ACV and IS. This included mobile phase selection, flow rate, column type
and injection volume. Different washing solutions viz n-Hexane and deionized water at
different volumes 0.500mL and 1.0mL were tried. Different elution solutions with different
volume ratios (100, 90:10, 80:20, 50:50, 10:90 v/v) of deionized water-methanol and
deionized water-acetonitrile combinations were also tried as mobile phase, along with formic
acid, ammonium trifluoroacetate, ammonium acetate and ammonium formate buffers in
varying strength (2-20mM) on Hypurity C8 (100mm × 4.6mm, 3µm), BDS Hypersil C18
(50mm × 4.6mm, 3µm), BDS Hypersil C18 (100mm × 4.6mm, 3µm), Ascentis Si (100mm ×
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3.0mm, 3µm), ACE 3 C8-300(100mm × 4.0mm, 3µm). In addition, the effect of flow rate
was also studied from 0.3 to 1.0mL/min, which was also responsible for acceptable
chromatographic peak shapes. The use of BDS Hypersil C18 chromatography column helped
in the separation and elution of both analytes within 4.0 min. The mobile phase consisting
(Acetonitrile: Deionized water: Ammonium trifluoroacetate Solution (1.0 M)) (80:20:0.05
v/v) was most appropriate for faster elution, improved efficiency and peak shape. The
retention time for ACV and IS was 2.05 and 2.02 min respectively at a flow rate of
0.7mL/min. The maximum on-column loading (at ULOQ) of ACV per sample injection
volume was 7.5ng. The reproducibility of retention times for ACV, expressed as %CV was
≤5.0% for 100 injections on the same column. Ideally, to minimize analytical variation due to
evaporation, integrity of the column and ionization efficiency, a deuterated analogue is the
first-choice internal standard.
Extraction methods based on either liquid-liquid extraction (LLE) or solid phase extraction
(SPE) have been used to extract ACV under different extraction conditions. SPE on different
extraction cartridges like Oasis HLB, Oasis MAX, Oasis WAX have been successfully
carried out for ACV by using different washing and elution solutions. Similarly, LLE with
different solvents like methyl tert butyl ether, n-Hexane also combinations namely hexane-
dichloromethane, ethyl acetate-n-Hexane, diethyl ether-dichloromethane, n-Hexane- methyl
tert butyl ether has been demonstrated; however, in some methods the samples obtained were
not clear in either of the solvents with poor recovery and considerable ion suppression and
poor chromatography. Due to less protein binding of ACV protein precipitation method is not
so useful. Methods based on SPE with Oasis WAX have employed acidic conditions (formic
acid) for quantitative and less recovery. Thus, SPE was tried on Oasis hydrophilic-lipophilic
balance (HLB) under neutral conditions with washing n-Hexane and deionized water. Precise
and quantitative recoveries with minimum matrix interference were obtained in both the
cases, however due to comparatively higher recoveries in n-Hexane as washing media; the
latter conditions were finalized in the present work.
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Figure Captions
FIGURE 1: Product ion mass spectra of (a) Acyclovir (m/z 226.1 → 152.1, scan range
100-300 amu) and (b) Acyclovir-d4 (IS, m/z 230.1 → 152.1, scan range 100-250 amu) in
positive ionization mode.
FIGURE 2: MRM ion-chromatograms of Acyclovir (m/z 226.1 → 152.1) and Acyclovir-
d4 (IS, m/z 230.1 → 152.1) in (a) double blank plasma (without analyte and IS), (b)
blank plasma with IS, (c) Acyclovir at LLOQ and IS (d) real subject sample at Cmax
after administration of 800 mg Tablet dose of Acyclovir.
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FIGURE 3: Representative post column analyte infusion MRM LC-MS/MS overlaid
chromatograms for Acyclovir and Acyclovir-d4 (a) Exact ion current (XIC)
chromatogram of Acyclovir (m/z226.1→ 152.1) (b) XIC of Acyclovir-d4 (IS, m/z 230.1 →
152.1)
FIGURE 4: Mean plasma concentration-time profile of Acyclovir after oral
administration of test (800 mg Acyclovir orally disintegrating tablet of an Indian
Company) and a reference (ZOVIRAX®, 800 mg Acyclovir orally disintegrating tablet
of GlaxoSmithKline, USA) formulation to 48 and 30 healthy Indian subjects under (a)
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fast and (b) fed conditions. Figure (c) and (d) shows the profile for 200 mg Acyclovir
Capsule in 48 and 30 healthy volunteers under fasting and fed condition respectively.
TABLE 1: Intra-batch & inter-batch accuracy and precision for Acyclovir
QC ID
Conc.
added
(ng/mL)
Intra-batch Inter-batch
n
Mean Conc.
found
(ng/mL)a
Accuracy
(%)
CV
(%) n
Mean Conc.
found
(ng/mL) b
Accuracy
(%)
CV
(%)
LLOQ 5.00 6 4.45 89.1 3.1 18 4.57 91.4 3.4
LQC 15.0 6 13.69 91.3 2.8 18 13.99 93.3 2.4
MQC-3 125 6 117.2 93.8 1.1 18 117.5 94.0 1.1
MQC-2 275 6 259.2 94.3 0.9 18 258.7 94.1 1.0
MQC-1 1000 6 956.1 95.6 3.2 18 941.2 94.1 2.2
HQC 1875 6 1706 91.0 0.9 18 1714 91.4 1.2
ULOQ 2500 6 2314 92.6 1.3 18 2308 92.3 1.5 n: total number of observations
CV: coefficient of variation
a mean of six replicate observations at each concentration
bmean of eighteen replicate observations over three different analytical runs
TABLE 2: Absolute matrix effect, relative recovery and process efficiency for Acyclovir
and Acyclovir d4 (IS)
CV: coefficient of variation
amean area response of six replicate samples prepared in mobile phase (neat samples)
bmean area response of six replicate samples prepared by spiking in extracted blank plasma
cmean area response of six replicate samples prepared by spiking before extraction
100 A
Bd
Aa
(%CV)
Bb
(%CV)
Cc
(%CV)
Absolute matrix
effect (% ME)d
Relative recovery
(% RE)e
Process efficiency
(% PE)f
LQC
78808 (3.14)
48467 (4.28)
35583 (2.80)
61.5 (69.8)
g 73.4 (78.0)
g 45.2 (54.4)
g
MQC-3
564581 (2.35) 385044 (3.73) 285066 (3.02) 68.2 (70.9)g 74.0 (75.8)
g 50.5 (53.7)
g
MQC-2
1240768 (3.89) 801536 (4.16) 631303 (4.54) 64.6 (67.3)g 78.8 (77.2)
g 50.9 (51.9)
g
MQC-1
5256593 (4.66) 3012028 (5.24) 2229258 (2.34) 57.3 (65.4)g 74.0 (77.3)
g 42.4 (50.6)
g
HQC
8441958 (5.21) 5202752 (6.54) 4059204 (3.53) 61.6 (68.2)g 78.0 (79.0)
g 48.1 (53.9)
g
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100 B
Ce
100RE ME=100 A
Cf
g values for internal standard, Acyclovir d4
TABLE 3: Relative matrix effect in different lots of human plasma at LQC and HQC
levels for Acyclovir (n=4)
amean of four replicate observations at each concentration
CV: coefficient of variation
TABLE 4: Stability results for Acyclovir under different conditions (n=6)
Stability Storage
Condition Level
Mean stability
sample (ng/mL) % CV % change
Bench top stability (In Ice
water bath)
Room
temperature (24h)
LQC 13.92 1.5 -7.2
HQC 1707 1.5 -9.0
Processed sample stability
(extracted samples)
Auto sampler
(4C, 94h)
LQC 14.34 1.2 -4.4 HQC 1761 0.5 -6.1
Processed sample stability
(extracted samples)
Room
temperature (51h)
LQC 14.60 1.3 -2.7 HQC 1736 1.2 -7.4
Freeze and thaw stability After 6
th cycle at
- 20C
LQC 13.85 1.4 -7.7
HQC 1715 1.4 -8.5
Long term stability 107 days at
- 70C
LQC 15.37 4.3 2.5
HQC 1894 4.9 1.0
Long term stability 107 days at
- 20C
LQC 14.73 5.1 -1.8
HQC 1888 3.5 0.7
CV: coefficient of variation
n: number of replicates at each level
100samples comparisonMean
samples comparisonMean – samplesstability Mean %Change
Plasma lots
LQC
(15.00ng/mL)
HQC
(1875ng/mL)
Mean calculated
conc.a (%CV)
Mean calculated
conc.a (%CV)
Lot-1 15.49 (1.4) 1814 (1.6)
Lot-2 15.19 (0.9) 1896 (2.4)
Lot-3 16.72 (1.8) 1802 (2.4)
Lot-4 15.32 (3.7) 1840 (1.5)
Lot-5 (haemolysed) 15.44 (1.4) 1835 (2.5)
Lot-6 (lipemic) 15.52 (1.7) 1838 (2.7)
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T, test formulation; R, reference formulation; ACV, Acyclovir; SD, standard deviation, Cmax, maximum plasma concentration; Tmax, time point
of maximum plasma concentration; t1/2, half life of drug elimination during the terminal phase; AUC0-t: area under the plasma concentration-
time curve from zero hour to 24/36/48h; AUC0-inf: area under the plasma concentration-time curve from zero hour to infinity
Table 5 Summary of mean pharmacokinetic parameters for bioequivalence studies with Acyclovir in healthy Indian volunteers
Formulation and dose
strength
Study condition; No. of
subjects; measurement
time period
Cmax ± SD (ng/mL) Tmax ± SD (h)
AUC0-t ± SD (ng.h/mL)/
AUC0-inf ± SD (ng.h/mL)
t½ ± SD (h)/Kel ± SD (1/h)
T R T R T R T R
T- 800 mg ACV tablet,
USP, R-800 mg ACV
tablet,ZOVIRAX®
Fasting; 48 subjects;
0-48 h
854.0 +
217.0 914.9 + 219.9 1.90 + 0.808
1.85 + 0.
769
5330.8 + 1708.6/
5557.5 + 1748.5
5534.7 + 1625.1/ 5819.4 + 1793.2
9.57 + 8.81/ 0.096 + 0.038
10.04 + 7.15/
0.096 + 0.048
T- 800 mg ACV tablet,
USP, R-800 mg ACV
tablet,ZOVIRAX®
Fed; 30 Indian subjects;
0-36 h
1076.9±185.
9 1122.1± 224.3 3.31±1.20 3.14±1.27
6887.7±1257.9/
7046.9±1267.7
6961.9±1441.3/
7118.8±1412.3
5.85± 2.38/
0.134±0.043
5.66± 2.53/
0.138±0.043
T- 200mg ACV Capsule,
R-200mg ACV Capsule,
ZOVIRAX®
Fasting; 48 Indian
subjects; 0-24 h 454.6±155.6 507.8±211.5 1.70±0.519 1.69±0.749
2374.0±731.0/
2472.4±721.5
2615.1±950.8/
2716.0±957.9
5.20±2.27/
0.150±0.044
5.16±1.94/
0.150±0.048
T- 200mg ACV Capsule,
R-200mg ACV Capsule,
ZOVIRAX®
Fed; 30 Indian subjects;
0-24 h 354.8±93.24 378.5± 100.3 2.54±0.607 2.37±0.678
2110.7±597.7/
2180.0±502.5
2115.2±552.4/
2186.1±560.2
4.25± 0.851/
0.169±0.095
4.64± 1.24/
0.159±0.099
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TABLE 6(a): Comparison of treatment ratios and 90% CIs of natural log (Ln)-
transformed parameters for800 mg Acyclovir tablet test and reference formulations
under fast and fed condition
Parameter
Ratio
(test/reference),%
90% CI
(Lower – Upper) Power
Intra subject
variation,
% CV
Fast Fed Fast Fed Fast Fed Fast Fed
Ln Cmax (ng/mL) 93.8 96.7 86.0 – 102.4 91.3-102.4 0.99 1.00 26.6 12.9
Ln AUC0-t
(h.ng/mL) 96.7 99.3 87.1 – 107.3 95.6-103.1 0.97 1.00 29.8 8.4
Ln AUC0-inf
(h.ng/mL) 96.0 99.2 87.1 – 105.7 95.4-103.1 0.98 1.00 27.5 8.7
CI: confidence interval; CV: coefficient of variation
TABLE 6(b): Comparison of treatment ratios and 90% CIs of natural log (Ln)-
transformed parameters for 200 mg Acyclovir capsule test and reference formulations
under fast and fed condition
Parameter Ratio
(test/reference),%
90% CI
(Lower – Upper)
Power Intra subject
variation,
% CV
Fast Fed Fast Fed Fast Fed Fast Fed
Ln Cmax (ng/mL) 91.0 96.7 84.6-97.8 91.8-101.8 0.99 1.00 20.4 11.5
Ln AUC0-t
(h.ng/mL) 92.0 98.8 86.0-98.4 95.2-102.5 0.99 1.00 18.8 8.2
Ln AUC0-inf
(h.ng/mL) 92.5 98.9 86.8-98.5 95.5-102.4 0.99 1.00 17.8 7.8
CI: confidence interval; CV: coefficient of variation
Assay performance and validation
System suitability and carryover check
During method validation, the precision (%CV) of system suitability test was observed in the
range of 0.45 to 3.27 % for the retention time and 0.28 to 2.98 % for the area response of
ACV and IS. The signal to noise ratio for system performance was ≥ 25 for both the analytes
and IS. Carry-over evaluation was performed in each analytical run so as to ensure that it
does not affect the accuracy and precision of the proposed method. There was negligible
carry over observed during auto-sampler carry-over experiment. No enhancement in the
response was observed in extracted blank plasma (without IS and analytes) after subsequent
injection of higher calibration standard (ULOQ) at the retention time of ACV or IS.
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Selectivity and interference study
The aim of performing selectivity check with 12 different plasma samples was to determine
the extent to which endogenous plasma components might contribute to the interference at
the retention time of analyte and the IS and thus, ensure the authenticity of the results for
study sample analysis. All samples studied were found free from any endogenous
interference. Demonstrates the selectivity results with the chromatograms of double blank
plasma (without IS), blank plasma (with IS), peak response of ACV at LLOQ concentration.
No interference was observed for commonly used medications by healthy volunteers like
acetaminophen, aspirin, caffeine, cetrizine, chlorpheniramine maleate, ibuprofen and
pseudoephedrine as evident from the real subject sample chromatogram for acyclovir at 2.333
h after oral administration of 800 mg orally disintegrating tablet formulation (Figure 2d).
Linearity, sensitivity, accuracy and precision
All seven calibration curves were linear over the concentration range of 2500-5.000ng/mL
with correlation coefficient r ≥ 0.9996856. A straight-line fit was made through the data
points by least square regression analysis to give the mean linear equation y = (0.00398093) x
– (0.00147262), where y is the peak area ratio of the analyte/IS and x the concentration of the
analyte. The accuracy and precision (%CV) observed for the calibration curve standards
ranged from 97.5 to 102 % and 0.4 to 2.9% respectively. The lowest concentration (LLOQ)
in the standard curve that can be measured with acceptable accuracy and precision was found
to be 5.000ng/mL at a signal-to-noise ratio (S/N) of ≥ 25, with the limit of detection (LOD)
of 0.2500ng/mL.
The intra-batch and inter-batch precision and accuracy were established from validation runs
performed at LLOQ QC, LQC, MQC-3, MQC-2, MQC-1, HQC and ULOQ QC levels (Table
1). The intra-batch precision (%CV) ranged from 0.9 to 3.1 and the accuracy was within 89.1
to 95.6 %. For the inter-batch experiments, the precision varied from 1.0 to 3.4 and the
accuracy was within 91.4 to 94.1%.
Recovery, matrix effect and post-column analyte infusion study
The relative recovery, absolute matrix effect and process efficiency data for ACV and IS at
LQC, MQC-3, MQC-2, MQC-1 and HQC levels is presented in Table 2. The relative
recovery of the analyte is the „true recovery‟, which is unaffected by the matrix as it
calculated by comparing the area response (analyte/IS) of extracted (spiked before extraction)
and unextracted (spiked after extraction) samples. The process efficiency/absolute recovery
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obtained for acyclovir and IS was > 42.4 % at all QC levels. Further, the relative matrix effect
which compares the precision (%CV) values between different lots (sources) of plasma
(spiked after extraction) samples varied from 0.9 to 3.7 for ACV at the LQC level (Table 3).
Results of post-column analyte infusion experiment in Figure 3 indicate no ion suppression
or enhancement at the retention time of ACV and IS. The average matrix factor value
calculated as the response of post spiked sample/response of neat solution (in mobile phase)
at the LQC level was 1.02, which indicates a minor enhancement of about 2.0%.
Stability, dilution integrity and ruggedness study
Stability experiments were performed to evaluate the analyte stability in stocks solutions and
in plasma samples under different conditions, simulating the same conditions which occurred
during study sample analysis. The stock solution of ACV was stable at room temperature for
7 h and at -20°C for 23 days for ACV and 16 days for IS. The intermediate stock solutions of
ACV in deionized water was stable at room temperature for 25 h and at 4°C for 10 days with
% change of 0.8% and -3.5% respectively. ACV was found stable in controlled blank plasma
at room temperature up to 24 h and for six freeze and thaw cycles. The analyte in extracted
plasma samples were stable for 94 h under refrigerated conditions (4°C) and for 51 h under
room temperature. The spiked plasma samples of ACV stored at -20°C and -70°C for long
term stability were found stable for a minimum period of 107 days. The values for the percent
change for all the stability experiments are complied in Table 4.
The precision values for dilution integrity of 1/10 of 5×ULOQ (2500ng/mL) and 1/10 of
HQC (1875ng/mL) concentration were 2.0 and 1.3%, while percent bias results were within -
6.4 and -7.7 % respectively, which is within the acceptance limit of 15% for precision (%CV)
and 85 to 115% for accuracy.
Method ruggedness was evaluated using re-injection of analyzed samples on two different
columns of the same make and also with different analysts. The precision (%CV) and
accuracy values for two different columns ranged from 0.1 to 3.0 % and 97.5 to 104%
respectively at all six quality control levels. For the experiment with different analysts, the
results for precision and accuracy were within 0.4 to 3.6% and 89.5 to 97.3% respectively at
these levels.
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Application of the method in healthy subjects
The validated method was applied to a bioequivalence study of ACV in 48 healthy Indian
male subjects who received 800 mg test and reference formulations of ACV tablet under fast
and 30 healthy Indian male subjects who received 800 mg test and reference formulations of
ACV tablet under fed conditions along with 200 mg capsule in 48 and 30 Indian male
subjects under fast and fed conditions respectively. This was studied to investigate the effect
of dose strength and impact of food on the pharmacokinetics of acyclovir. Figure 4a, 4b
shows the plasma concentration vs. time profile of ACV 800 mg tablet in healthy subjects
under fast and fed conditions and Figure 4c, 4d shows the plasma concentration vs. time
profile of ACV 200 mg capsule in healthy subjects under fast and fed conditions. The method
was sensitive enough to monitor their plasma concentration up to 48h. In all approximately
8471 samples including the calibration, QC and volunteer samples were run and analysed
successfully. The precision and accuracy for calibration and QC samples were within the
acceptable limits. Table 5 compares the important pharmacokinetic parameters obtained for
the bioequivalence studies conducted with healthy volunteers for ACV. The effect of food
was negligible in the studies carried out with 800 mg tablet dose and 200 mg capsule dose
under fast and fed conditions. Comparison of dose strength (800 mg and 200 mg) revealed
dose dependent pharmacokinetics. The equivalence statistics of bioavailability for the
pharmacokinetic parameters of the two formulations are summarized in Table 6a and 6b. No
statistically significant differences were found between two formulations in any parameter.
The mean log-transformed ratios of the parameters and their 90% CIs were all within the
defined bioequivalence range. These observations confirm the bioequivalence of the test
sample with the reference product in terms of rate and extent of absorption. The % change in
the randomly selected samples for incurred samples (assay reproducibility) analysis was
within ±20 %. This authenticates the reproducibility and ruggedness of the proposed method.
Further, there was no adverse event during the course of the study.
CONCLUSION
The objective of this work to develop a selective, sensitive, rugged and a high throughput
method for the estimation of ACV in human plasma, especially to meet the requirement for
subject sample analysis. The solid phase extraction employed in the present work using
Waters Oasis HLB cartridge gave consistent and reproducible recoveries for ACV. The run
time per sample analysis of 3.6 min suggests high throughput of the proposed method. The
maximum on-column loading at ULOQ was 7.5ng for 3µL injection volume. This was
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considerably less compared to all other reported procedures, which helps in maintaining the
efficiency and lifetime of the column. Moreover, the limit of quantification is low enough to
monitor at least five half-lives of ACV concentration with good intra and inter-assay
reproducibility (%CV) for the quality controls. The sensitivity of the proposed method is
adequate to support a wide range of pharmacokinetic/bioequivalence studies.
ACKNOWLEDGEMENTS
The authors are indebted to Mr. Vijay Patel, Executive Director, Cliantha Research Ltd.,
Ahmedabad for providing necessary facilities to carry out this work. We gratefully
acknowledge Mr. Anshul Dogra, Head Of Director, Cliantha Research Ltd. for his continuous
support, motivation and assistance during the course of this project.
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