development and validation of a sensitive and rugged spe

www.wjpps.com Vol 4, Issue 11, 2015.

1267

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 ×


1277


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)


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


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


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


1285


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.


1286


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


1287


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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