Chapter-1

Page 17

SIMULTANEOUS DETERMINATION OF ACYCLOVIR AND

VALACYCLOVIR IN HUMAN PLASMA

CONTENTS

1.1 INTRODUCTION

1.2 EXPERIMENTAL

1.2.1 Study Objective

1.2.2 Reference Compounds

1.2.3 Chemicals, Reagents and Materials

1.2.4 Liquid Chromatographic Conditions

1.2.5 Mass Spectrometric Conditions and Data Processing

1.2.6 Standard Solutions (Calibration Standards and Quality Control Samples)

1.2.7 Extraction Procedure

1.3 RESULTS AND DISCUSSION

1.3.1 Method Development

1.3.2 Method Validation

1.3.2.1 Selectivity

1.3.2.2 Linearity

1.3.2.3 Recovery

1.3.2.4 Precision and Accuracy

1.3.2.5 Matrix Factor

1.3.2.6 Dilution Integrity

1.3.2.7 Stability Study

1.4 CONCLUSION

1.5 REFERENCES

Chapter-1

Page 18

1.1 INTRODUCTION

Valacyclovir (VCV), the L-valyl ester of acyclovir, is an oral prodrug that is rapidly and

extensively metabolized by enzymatic hydrolysis probably in the liver and the intestine

to acyclovir (ACV) and L-valine1,2. VCV is used for the treatment of herpes virus

infections predominantly caused by the herpes simplex virus (HSV-1, HSV-2) and the

varicella zoster virus (VZV)3,4. ACV is the active antiviral component of VCV and has

also shown good activity against the Epstein-Barr virus and moderate efficacy against

cytomegalovirus1. The structural similarity of ACV and VCV with certain endogenous

substances requires a very selective analytical method, for the quantitative determination

of ACV and VCV in a range of extremely low concentrations. Radioimmunoassays

(RIA)5 and enzyme-linked immuno absorbent assays (ELISA)6 to determine the

concentration of ACV in serum and plasma are very sensitive, but these methods are

costly and time-consuming. HPLC with fluorescence or UV detection is a very common

method used for the determination of ACV7-10 and its structural analogue11. In literature,

most assays by LC7, 12-20 or RIA quantify only ACV7, 13, 16, 17, 21-26, but not VCV. Recently,

a few LC methods using isocratic and gradient elution have been published for the

analysis of VCV and ACV simultaneously27, 28. But, the methods reported by researchers

had set backs in terms of less sensitivity, long run time, processing large samples and

requiring large plasma volume7,9,29,. Moreover, there are no combination methods

available to estimate both ACV and VCV with better consistency31.

Acyclovir, 9-[(2-hydroxyethoxy)-methyl] methyl]-guanosine, is a synthetic purine

nucleoside derived from guanine, which exhibits a selective inhibition of herpes virus

replication with potent clinical antiviral activity against the herpes simplex and varicella-

zoster viruses28, 32. The pharmacokinetics of acyclovir is characterized by significant

individual variability33-35, leading to variability in the concentrations and therapeutic

efficacy achieved in patients. Understanding the factors that influence and contribute to

these differences, will allow for doses to be individually adjusted in order to achieve

optimal antiviral therapeutic efficacy.

Prompted by the importance of both ACV and VCV in combating viral aliments and

considering the availability of only LC methods on quantifying both, an attempt has been

made to develop a simple, sensitive and selective method for the simultaneous

quantification of ACV and VCV in human plasma using LC-MS/MS to overcome

Chapter-1

Page 19

hurdles like long run time, less sensitivity, processing large number of samples and with

plasma volume.

1.2 EXPERIMENTAL

1.2.1 Study Objective

Purpose of the present study was to develop and validate an LC-MS/MS method for

simultaneous determination of acyclovir and valacyclovir in human plasma.

1.2.2 Reference Compounds : Acyclovir ; Valacyclovir

IUPAC Name : 9-[(2-hydroxyethoxy)-methyl] methyl]-guanosine; 2-[(2-

amino- 1, 6-dihydro-6-oxo-9H-purin-9-yl)methoxy]ethylestermonohydrochloride.

Molecular Formula : C8H11N5O ; C13H20N6O4. HCl

Molecular Weight : 225 ; 360.80

Purity : 99.82 % ; 89.9 %

Supplier : Arochem industries, (Thane, Maharashtra, India);

Dr.Reddy Laboratories, (Hyderabad, AP, India).

N

NNH

O

NH2

N

O

OH

N

NNH

N

O

NH2

OO

NH2

O

HC l

(I) (II)

Fig.1.1 Structures of Acyclovir (I) and Valacyclovir (II)

Reference Compound : Fluconazole; Molecular Formula : C13H12F2N6O

Molecular weight : 306.27; Purity : 99.32%

IUPAC Name : 1H-1, 2, 4-Triazole-1-ethanol, 1-(2, 4-difluorophenyl)-1-(1H-

1, 2, 4- triazol-1-ylmethyl)-.2, 4-Difluoro-1, 1-bis(1H-1, 2, 4-triazol-1 ylmethyl)

benzyl alcohol

Supplier : Dr.Reddys laboratories, (Hyderabad, AP, India).

Chapter-1

Page 20

1.2.3 Chemicals, Reagents and Materials

1.2.3.1 Chemicals

All the solvents were of high purity and before analysis, all the glass ware

(flasks, tubes, pipettes) were carefully cleaned and rinsed with Milli Q water

(Type-1 grade). The reagents were obtained as stated:-

Methanol, from Merck (Darmstadt, Germany)

Formic acid from Merck (Darmstadt, Germany)

Hydrochloric acid from Merck (Darmstadt, Germany)

Water deionised and purified by a Milli-Q water purification system

from Millipore (Bedford, MA, USA)

Oasis HLB extraction cartridges 1cc (30mg) from Waters-India

(Bangalore, India)

Blank K2EDTA plasma bags (six different lots) from Karnavati blood

Bank (Ahmedabad, India)

1.2.3.2 Reagents

Mobile phase buffer: In a 500mL volumetric flask, 500µL of formic acid was

made up to mark with water and mixed thoroughly.

Mobile Phase: In a 1000mL volumetric flask 700mL of methanol and 300mL of

mobile phase buffer were properly mixed and filtered through 0.2µm filter

before use.

Extraction buffer: In a 100mL of volumetric flask, 850µL Hydrochloric acid

was made up to volume with water.

1.2.3.3 Materials

API 4000 triple Quadra pole Mass Spectrometer (Applied Biosystem-

SCIEX, Canada)

Shimadzu HPLC system with C18 Gemini column(150mm x 4.6mm,5µ)

Micropipettes from Eppendorf, Hamburg, Germany

Solvent filter, 0.2µm, Millipore, Banglore, India

Vortex mixer, Spinix, Tarson, India

Balance ME-5 from Sartorius, Germany

Positive pressure processor for SPE from Orochem technologies, USA

Class A calibrated glass ware from different supplier

Chapter-1

Page 21

1.2.4 Liquid Chromatographic Conditions

A Shimadzu HPLC system with C18 Gemini column (150 mm x 4.6 mm, 5µ) that

contains packing of octadecylsilane chemically bonded to porous silica was used for

chromatographic separation. The mobile phase was prepared by addition of 0.1% formic

acid solution in methanol (30:70 v/v, pH 3.5).The flow rate of 0.8 ml/min and 70%

splitting for mobile phase were adjusted to carry out separation. The column temperature

was set at 45˚C, the auto-sampler was conditioned at 10°C and injection volume was

2µL with a run time around 3min.

1.2.5 Mass Spectrometric Conditions and Data Processing

The mass spectrometry was operated in positive ion detection mode. Nitrogen was used

as nebulizing turbo spry. Temperature of vaporizer was set at 400°C and the ESI needle

voltage was 5500V. The declustering potential was set at 60 volts for ACV, VCV and for

Internal Standard (IS). Collision energy for ACV, VCV and IS was 14, 18 and 28V

respectively. The mass spectrometer was operated at unit mass resolution with a dwell

time of 200 milli seconds per transition. Quantification was performed using multiple

reactions monitoring (MRM) of the transition m/z 226.30 (parent ion) → m/z 152.10

(product ion); m/z 325.40 (parent ion) → m/z 152.10 (product ion) and m/z 307.06

(parent ion) → m/z 220.20 (product ion) for ACV, VCV and IS respectively.

1.2.6 Standard Solutions (Calibration Standards and Quality Control Samples)

1.2.6.1 Stock Solution Preparation for ACV and VCV

Stock solutions (2mg/mL of ACV and 1mg/mL of VCV) were prepared for each

reference compound by dissolving 20mg of ACV in 10mL of methanol and 10mg of

VCV in 10mL of methanol separately. Spiking solutions of ACV and VCV were

prepared from stock solutions by serial dilution method in methanol: water (1:1, v/v).

Eight levels of calibration curve standards were prepared by adding the spiking solution

in plasma to achieve the concentration levels of 47.6, 95.3, 216.6, 492.2, 1230.6, 2563.8,

5127.6, 10255.1ng/mL for ACV and 5.0, 10.0, 22.7, 51.6, 129.0, 268.8, 537.6,

1075.2ng/mL for VCV respectively. Four levels of quality control samples were

prepared by adding the spiking solutions in plasma and the concentration levels were

49.1, 140.3, 779.4, 8856.7ng/mL for ACV and 5.1, 14.7, 81.7, 928.6ng/mL for VCV

respectively.

Chapter-1

Page 22

1.2.7 Extraction Procedure

All the calibration standards (0.5mL) or QC samples (0.5mL) were taken in

polypropylene tubes. 50μL of internal standard (1µg/mL of fluconozole) was added and

vortexed for 10 seconds. 0.5mL of 0.1N hydrochloric acid was added to the plasma

samples, vortexed for 10 seconds and centrifuged for 2min at 4,500 rpm at 4°C. The

samples were transferred to a 1cc/30 mg Oasis HLB SPE column, which had been

conditioned with 1.0mL methanol, followed by 1.0mL water. After application of the

samples, the SPE column was dried for 1.0min by applying positive pressure at

maximum flow rate. The column was eluted with 0.2mL water followed by 0.3mL of

methanol (40:60 v/v) and vortexed for about 10 seconds. The SPE elutes were

transferred into 1mL LC vials for injection of 2μL into the LC system.

1.3 RESULTS AND DISCUSSION

1.3.1 Method Development

Method development was initiated with the aim to suitably optimize the chromatographic

and mass spectrometric conditions. Also, the extraction procedure should be simple and

efficient as VCV and ACV have structural similarity with endogenous components

present in human plasma. The inherent selectivity of MS/MS detection was expected to

be beneficial in developing a selective and sensitive method. Optimum mass acquisition

parameters were obtained by direct infusion of 500ng/mL solution of both the analytes

and IS at a flow rate of 10µL/min. An earlier report had suggested the use of negative

ionization mode and SIM (selected ion monitoring) for quantitation of both the analytes

at low levels31. However, in the present study a much higher response was obtained

under MRM (multiple reaction monitoring) with a signal to noise ratio ≥90 in the

positive ionization mode. The Q1 MS full scan mass spectra of VCV and ACV was

dominated by protonated precursor ions [M+H]+ at m/z 325 and 226 respectively. The

mass spectrometer was set up in multiple reaction monitoring mode to monitor the

transitions from the precursor ions to product ions. The MRM state file parameters like

nebulizer gas, CAD (collision associated dissociation) gas; ion spray voltage and

temperature were suitably optimized to obtain a consistent and adequate response for

both the analytes. A dwell time of 200ms was adequate and no cross talk was observed

between their MRMs.

Chapter-1

Page 23

Due to similar MRMs for both the analytes it was essential to have a chromatographic

separation of the drugs so as to minimize any interference during quantitation.

Chromatographic analysis of VCV, ACV and IS was initiated under isocratic conditions

to obtain adequate response, sharp peak shape and a short run time. It has been

documented in an earlier report37 that stability of VCV is pH dependent, with maximum

stability at pH≤4 in aqueous media and gastrointestinal fluids. Thus, separation was

attempted using various combinations of methanol/acetonitrile, acidic buffers and

additives like formic acid on different reversed-phase columns [Chromolith RP18

(100mm×4.6mm, 5µm), Kromasil C18 (50/100mm×4.6mm, 5µm), Hypersil C18

(50/100mm×4.6mm, 5µm), Waters Acquity UPLC BEH C18 (2.1mm×50mm,1.7µm),

ACE C18 (50×2.1mm, 5µm) and Gemini C18 (50/150mm×4.6mm, 5µm)]. Best results

in terms of reproducibility, complete separation and peak shape were obtained with

Gemini C18 (150mm×4.6mm, 5µm) column compared to others and hence it was

selected for further study. The analytes showed poor reproducibility for proposed linear

range except for Gemini C-18 column which offered superior peak shape, efficient

separation, desired linearity and reproducibility for VCV, ACV and IS from endogenous

plasma matrix. This may be attributed to the large surface area (396m2/g) of Gemini C-

18 compared to other columns (300-187m2/g). A mobile phase (pH 3.5) consisting of

0.1% formic acid in water and methanol (30:70, v/v) was found most suitable for eluting

VCV, ACV and IS at 1.47, 2.11 and 2.51min respectively. A flow-rate of 0.8mL/min

with 70% flow splitting produced good peak shapes and permitted a run time of 3.0min

per analysis. Previous studies27,31 have reported longer run times (≥4min) for their

simultaneous separation.

Maria K et al.31 have reported a protein precipitation method to separate these polar

drugs from human plasma. Thus, the extraction was carried out initially via protein

precipitation with common solvents like acetonitrile, methanol and acetone, but the

sensitivity (especially for VCV) and reproducibility were poor in all the solvents. There

was frequent clogging of the column which required on-line flushing. Liquid-liquid

extraction technique was also tested to isolate the drugs from plasma using diethyl ether,

dichloromethane, methyl tert butyl ether and isopropyl alcohol (alone and in

combination) as extracting solvents.

However, the recovery was inconsistent with some ion suppression (greater than 15%

CV) for both the analytes. Finally, optimization of solid phase extraction process was

Chapter-1

Page 24

initiated on Waters Oasis HLB,Waters Oasis MCX and Phenomenex Strata cartridges.

Addition of strong acid like HCl during sample preparation helped in maintaining the

analyte in the ionized form with better retention on Waters Oasis HLB as compared to

other cartridges. Further, use of strong acid during processing enhanced the stability of

VCV and subsequently gave consistent recovery, especially at the LLOQ level with

minimum matrix interference. Initially, different washing solvents like water, dilute

formic acid, acetic acid, hydrochloric acid (alone and in combination) were tried to

achieve desired specificity and reproducibility. But due to polar nature of analytes (VCV

and ACV) none of the washing solvents worked satisfactorily.

Moreover, the recoveries were low when compared with neat samples. Thus, washing

step was avoided; instead, direct elution was done sequentially with water and methanol

respectively. This helped in getting desired recovery, sensitivity, reproducibility with

minimum matrix effect and fast turns around for analysis.

By virtue of its similarity in chromatographic behavior ketoconazole and fluconazole

hydrochloride were tested to minimize analytical variation due to solvent evaporation,

integrity of the column and ionization efficiency. The results found, were superior with

fluconazole hydrochloride as compared to ketoconazole in terms of consistency and

reproducibility and hence it was selected as the internal standard.

1.3.2 Method Validation

1.3.2.1 Selectivity

Possible interferences at the retention times of ACV, VCV and IS from endogenous

compounds were checked during the validation by testing six different batches of

K2EDTA human plasma, one lipemic blank plasma and one lot of haemolysed blank in

order to check the absence of signals for the retention times of each compound.

Selectivity was carried out by analyzing the six blank plasma samples spiked with ACV

and VCV (LLOQ level) and IS.

1.3.2.2 Linearity

Linearity of the method was evaluated using bulk spiked plasma samples in the

concentration range as mentioned above using the method of least squares. Six such

linearity curves were analyzed. Each calibration curve consisted of a blank sample, a

zero sample (blank + IS) and eight concentrations. The standard curves were linear over

the concentration ranges of 47.6 -10255.1 and 5.0 - 1075.2ng/mL for ACV and VCV

Chapter-1

Page 25

respectively. The mean correlation coefficient was 0.9966 for ACV and 0.9980 for VCV.

Samples were quantified using the ratio of peak area of analyte to that of IS. A weighting

factor linear regression (1/concentration) was performed with the nominal concentrations

of calibration levels. Peak area ratios were plotted against plasma concentrations. The

limits of quantitation were 47.6 and 5.0ng/mL for ACV and VCV respectively which

were better than the previously reported LC methods using gradient solution27.

1.3.2.3 Recovery

The recovery of drug and IS was evaluated at three concentration levels namely low,

medium and high quality control. Recovery was calculated by comparing its response in

replicate samples with that of neat standard solution responses. Analyte recovery from a

sample matrix (extraction efficiency) is a comparison of analytical response from an

amount of analyte added to that determined from sample matrix. The extraction

efficiency of ACV from human plasma at the concentrations of LQC, MQC and HQC

was found to be 50.1, 50.3 and 51.7% respectively. The extraction efficiency of VCV

from human plasma at the concentrations of LQC, MQC and HQC was found to be 46.0,

47.2 and 45.3%. The mean recovery for the internal standard was 79.0% (Table 1.1)

which was superior to the LC method published earlier using LLE27.

Table 1.1 Percentage Recovery of ACV, VCV and IS

Nominal concentrations (ng/mL) % Recovery

ACV VCV ACV VCV Internal Standard

140.3 14.7 50.12 45.96

78.98779.4 81.7 50.25 47.16

8856.7 928.6 51.69 45.28

1.3.2.4 Precision and Accuracy

Intra-day accuracy and precision were evaluated from replicate analyses (n = 6) of

quality-control samples containing ACV and VCV at different concentrations on the

same day. Inter-day accuracy and precision were also assessed from the analysis of the

same QC samples on separate occasions in replicate (n = 6). QC samples were analyzed

against calibration standards.

All calibration curves were found to be linear over the range of 47.6-10255.1 and

5.0 -1075.2ng/mL. The precision for six plasma samples spiked with ACV and

VCV at LLOQ concentration was 1.3 and 2.63% with a mean accuracy of 98.6

Chapter-1

Page 26

and 101.8% respectively (Table 1.2 and 1.3). The inter-batch assay accuracy for

acyclovir and valacyclovir ranged between 96.0 - 106.3 and 97.4 - 105.5%

respectively, whereas intra-batch accuracy ranged between 98.8 - 105.9 and 91.9

- 102.7%. The inter-batch precision for acyclovir and valacyclovir ranged

between 4.0 - 4.8 and 4.5 - 8.9% and intra batch precision ranged between 3.3 -

3.9 and 2.8 - 5.3%. The results are presented in Table 1.4 and 1.5. Representative

chromatograms of ACV and VCV are depicted in Fig.1.2 - 1.5. All the results

were found within the acceptable limit of precision not more than 15.0% and

accuracy 85.0 - 115.0%, except LLOQ for which precision was not more than

20% and accuracy was between 80.0 - 120.0%.

Table 1.2 Results of Six Calibration Curves for Determination of ACV in Human

Plasma

Conc.added

(ng/mL)

Conc. determined

(mean ± S.D) (ng/mL)

Precision

(%)

Accuracy

(%)

47.6 47.0 ± 0.6 1.3 98.6

95.3 96.1 ± 3.5 3.7 100.9

216.6 219.4 ± 11.0 5.0 101.3

492.2 524.0 ± 19.5 3.7 106.5

1230.6 1254.7 ± 28.3 2.3 102.0

2563.8 2573.1 ± 78.9 3.1 100.4

5127.6 4948.0 ± 210. 4 4.3 96.5

10255.1 9631.2 ± 148.7 1.5 93.9

Table 1.3 Results of Six Calibration Curves for Determination of VCV in Human

Plasma

Conc.added

(ng/mL)

Conc. determined

(mean ± S.D) (ng/mL)

Precision

(%)

Accuracy

(%)

5.0 5.1 ± 0.1 2.6 101.8

10.0 9.7 ± 0.6 6.5 96.7

22.7 22.3 ± 0.7 3.1 98.0

51.6 53.3 ± 1.7 3.2 103.4

129.0 128.0 ± 3.4 2.7 99.2

268.8 267.1 ± 4.2 1.6 99.4

537.6 538.8 ± 13.7 2.5 100.2

1075.2 1089.9 ± 28.8 2.6 101.4

Chapter-1

Page 27

Table 1.4 Results of Six Quality Control Batches for Determining ACV Concentrations in HumanPlasma Samples

Conc. added

(ng/mL)

Intra-batch (n=6) Inter-batch (n=36)

Conc. determined

(mean ± S.D)

(ng/mL)

Precision

(%)

Accuracy

(%)

Conc. determined

(mean ± S.D)

(ng/mL)

Precision

(%)

Accuracy

(%)

49.1 49.9 ± 1.7 3.3 101.6 49.9 ± 2.4 4.8 101.7

140.3 147.7 ± 5.0 3.4 105.3 149.1 ± 6.2 4.2 106.3

779.4 825.6 ± 31.3 3.8 105.9 824.2 ± 37.6 4.6 105.8

8856.7 8749.7± 340.8 3.9 98.8 8505.2 ± 342.4 4.0 96.0

Table 1.5 Results of Six Quality Control Batches for Determining VCV Concentrations in HumanPlasma Samples

Conc.

added

(ng/mL)

Intra-batch (n=6) Inter-batch (n=36)

Conc. determined

(mean ± S.D)

(ng/mL)

Precision

(%)

Accuracy

(%)

Conc. determined

(mean ±S.D )

(ng/mL)

Precision

(%)

Accuracy

(%)

5.1 4.7 ± 0.2 5.0 91.9 5.4 ± 0.5 9.0 105.5

14.7 14.2 ± 0.8 5.3 96.7 14.8 ± 1.1 7.1 100.7

81.7 84.0 ± 3.6 4.3 102.7 79.6 ±4.4 5.6 97.4

928.6 938.6 ± 26.6 2.8 101.1 921.5 ±41.7 4.5 99.2

1.3.2.5 Matrix Factor

The matrix of plasma constituents over the ionization of analyte was determined by

comparing the concentrations of the post-extracted plasma standard QC samples (n = 6)

with the concentration of analyte from neat samples at equivalent concentrations38-40.The

matrix effect intended method was assessed using chromatographically screened human

plasma. For VCV, the precision (%CV) at HQC and LQC levels was within 5.8 -2.5%.

For ACV, the precision (%CV) at HQC and LQC levels were between 11.5-11.8%

respectively (Table1.6 and 1.7).

1.3.2.6 Dilution Integrity

During the course of study, probability of encountering samples with concentrations

above the upper limit of quantitation (ULOQ) could not be ruled out and therefore

dilution with drug free plasma is necessary to bring them within the calibration range. To

establish the effect of dilution on the integrity of samples, six aliquots of 20,000 and

2,000ng/mL of ACV and VCV respectively were prepared. The samples were subjected

Chapter-1

Page 28

to fivefold dilution (n = 6) and ten -fold dilution (n = 6) with drug free human plasma to

bring them within the calibration range. The samples were processed, analyzed and the

concentrations obtained were compared with theoretical values.

The precision for dilution integrity standards at 1:5 and 1:10 for ACV were 4.24 and

5.5% and for VCV were 4.98 and 8.26 % respectively. The mean accuracy for dilution

integrity of 1:5 and 1:10 for ACV were 101.00 and 101.23% while for VCV they were

102.63 and 100.21% respectively, which are within the acceptance limits of 85.00 -

115.00% (Table1.8 and 1.9).

Table1.6 Matrix Effect of ACV

Description

HQC LQC

Nominal Concentration (ng/mL)

8856.709 140.290

Calculated Concentrations (ng/mL)

Aliquot 1 8457.707 154.874

Aliquot 2 9382.712 154.277

Aliquot 3 8551.045 148. 682

Aliquot 4 8060.566 160.700

Aliquot 5 8709.871 154.349

Aliquot 6 8047.147 152.812

Mean 8534.841 154.282

SD 493.662 3.874

% CV 5.78 2.51

Table1.7 Matrix Effect of VCV

Description

HQC LQC

Nominal concentration (ng/mL)

928.550 14.708

Calculated Concentration (ng/mL)

Aliquot 1 966.636 16.548

Aliquot 2 1111.782 16.747

Aliquot 3 784.317 13.418

Aliquot 4 942.246 16.860

Aliquot 5 936.199 15.173Aliquot 6 882.452 19.224

Mean 937.272 16.328

SD 107.463 1.933% CV 11.47 11.84

Chapter-1

Page 29

Table1.8 Dilution Integrity for ACV

Description

DI Spiked Concentration (23307.128ng/mL)

DI 1/5 sample (ng/mL) DI 1/10 sample (ng/mL)

Sample

conc.

With dilution

factor

Sample conc. With dilution factor

Aliquot 1 4644.496 23222.478 2244.179 22441.789

Aliquot 2 4986.045 24930.226 2518.017 25180.171

Aliquot 3 4448.933 22244.666 2374.490 23744.899

Aliquot 4 4570.042 22850.209 2179.428 21794.227

Aliquot 5 4886.601 24433.006 2474.476 24744.758

Aliquot 6 4713.307 23566.533 2365.341 23653.409

Mean 4708.2373 23541.1865 2359.3217 23593.217

SD 199.707 998.539 129.869 1298.697

% CV 4.24 4.24 5.50 5.50

% Mean Accuracy 101.000 101.230

Table1.9 Dilution Integrity for VCV

Description

DI Spiked Concentration (2443.554ng/mL)

DI 1/5 sample(ng/mL) DI 1/10 sample (ng/mL)

Sample conc. With dilution factor Sample conc. With dilution factor

Aliquot 1 501.515 2507.574 242.0497 2420.970

Aliquot 2 508.371 2541.853 252.905 2529.052

Aliquot 3 453.104 2265.521 256.159 2561.586

Aliquot 4 525.116 2625.580 205.566 2055.663

Aliquot 5 508.763 2543.813 260.737 2607.373

Aliquot 6 512.551 2543.813 260.737 2607.373

Mean 501.569 2507.849 244.872 2448.7233

SD 24.987 124.938 20.218 202.180

% CV 4.98 4.98 8.26 8.26

% Mean Accuracy 102.630 100.210

Chapter-1

Page 30

Fig.1.2. Representative Chromatograms of Acyclovir Extracted Blank Sample

Fig.1.3. Representative Chromatograms of Acyclovir LLOQ Sample

Chapter-1

Page 31

Fig.1.4. Representative Chromatograms of Valacyclovir Extracted Blank Sample

Fig.1.5. Representative Chromatograms of Valacyclovir LLOQ Sample

Chapter-1

Page 32

1.3.2.7 Stability Study

Evaluation of the stability of samples was based on the comparison of various samples

against freshly prepared sample of the same concentration. Percentage difference

between the back calculated concentrations obtained for the sample under investigation

and freshly prepared sample was evaluated. Four aliquots, each of LQC and HQC

concentrations were used for stability study.

The bench top stability (at 20-30°C) was determined for 1h 41min by comparing the

accuracy of the mean concentrations for the low and high QCs for ACV and VCV were

found to be 102.1 and 99.7%, 96.3 and 93.6 respectively. The freeze-thaw stability was

determined at -50°C for the low and high QCs of ACV and VCV, which underwent three

freeze thaw cycles. In each freeze thaw cycle, the frozen plasma samples were thawed at

room temperature for 2-3h and refrozen for 12-24h. The accuracy of the mean

concentrations for the low and high QCs of ACV and VCV were found to be 101.4 and

99.5%, 103.4 and 100.3% respectively. Auto sampler stability of the plasma samples

were over 14h 20min was established. All the stability results were tabulated in Table

1.10, were found within the acceptable.

Table 1.10 Stability Results for ACV and VCV

Stability of ACV Stability of VCV

Sampleconcentrations(ng/mL) (n=6)

(nominal)

Conc. found(mean, n=6)

(ng/mL)

Precision(%)

Accuracy(%)

Sampleconcentrations(ng/mL) (n=6)

(nominal)

Conc. found(mean, n=6)

(ng/mL)

Precision(%)

Accuracy(%)

Bench top stability (01 hour 41 minutes)

141.9 (140.2) 144.8 2.9 102.1 14.5 (14.6) 14.4 2.7 96.3

8548.6(8851.8) 8518.3 2.5 99.7 953.6 (924.7) 892.5 2.7 93.6

Freeze thaw stability (after 3 cycles below -500c)

141.9 (140.2) 142.4 7.0 101.4 14.6 (14.6) 15.1 10.0 103.4

9185.6(8851.8) 9142.5 6.1 99.5 975.7 (924.7) 978.2 3.9 100.3

Auto sampler stability ( 14 hours 20 minutes at 50c)

153.1 (140.3) 146.0 6.4 95.4 15.6 (14.7) 16.1 4.1 103.3

8340.5(8856.7) 8016.1 2.9 96.1 961.7 (928.6) 962.7 2.1 100.1

Chapter-1

Page 33

1.4 CONCLUSION

The LC-MS-MS assay described here is simple, selective, precise and accurate for

quantification of acyclovir and valacyclovir in human plasma; fully validated according

to FDA guidelines. The developed assay method was validated and found to be precise

(inter batch 4.0-4.8% , 4.5-8.9% for ACV and VCV respectively; intra batch 3.3-3.9%,

2.8-5.3% for ACV and VCV respectively) and accurate (inter batch 96.0-106.3%, 97.4-

105% for ACV and VCV respectively; intra batch 98.8-105.9% ; 91.9-102.7% for CV

and VCV respectively) over a wide concentration range with no interference by

endogenous compounds. This method is highly sensitive (LLOQ 47.0ng/mL for ACV,

1089.9 for VCV) and produced consistent recovery even with less injection volume

(2µL). This method could be used for the routine therapeutic monitoring of the drug and

pharmacokinetic studies.

Chapter-1

Page 34

REFERENCES

1 Perry C.M., and Faulds D. Valaciclovir: A review of its antiviral activity,

pharmacokinetic properties and therapeutic efficacy in herpes virus infections. Drugs

1996; 52: 754-772.

2 Burnette T.C., and De Miranda P. Metabolic disposition of the acyclovir prodrug

valaciclovir in the rat. Drug Metab Dispos 1994; 22:60-64.

3 Beutner K.R. Valacyclovir: A review of its antiviral activity, pharmacokinetic

properties and clinical efficacy. Antiviral Res 1995; 28:281– 286.

4 Acosta E.P., and Fletcher CV. Valacyclovir. Ann Pharmacother, 1997; 31:185-188.

5 Lycke J., Andersen O., Svenerholm B., Appelgren L., and Dahlof C. Acyclovir

concentrations in serum and cerebrospinal fluid at steady state. J Antimicrob

Chemother 1989; 24:947 - 954.

6 Tadepalli S.M., Quinn R.P., and Averett D.R. A competitive enzyme-linked

immunosorbent assay to quantitate acyclovir and BW B759U in human plasma and

urine. Antimicrob Chemother 1986; 29:93 - 98.

7 Mascher H., Kikuta C., Metz R., and Vergin H. New, high-sensitivity high-

performance liquid chromatographic method for the determination of acyclovir in

human plasma, using fluorometric detection. J Chromatogr B 1992; 583:122-128.

8 Peh K.K., and Yuen K.H. Simple high-performance liquid chromatographic method

for the determination of acyclovir in human plasma using fluorescence detection.

J Chromatogr B 1997; 693:241-244.

9 Bangaru R.A., Bansal Y.K., Rao A.R.M., and Gandhi T.P. Rapid, simple and

sensitive high-performance liquid chromatographic method for detection and

determination of acyclovir in human plasma and its use in bioavailability studies.

J Chromatogr B 2000; 739:231-237.

10 Fernandez M., Sepulveda J., Teobaldo A., and Plessing C.V. Technique validation by

liquid chromatography for the determination of acyclovir in plasma. J Chromatogr B

2003; 791:357-363.

11 Schenkel F., Rudaz S., Daali Y., Oestreicher M.K., Veuthey J.L., and Dayer P.

Development and validation of a new reversed-phase ion pairing liquid

chromatographic method with fluorescence detection for penciclovir analysis in

plasma and aqueous humor. J Chromatogr B 2005; 826:1-7.

Chapter-1

Page 35

12 Land G., and Bye A. Simple high-performance liquid chromatographic method for

the analysis of 9-(2-hydroxyethoxymethyl)guanine (acyclovir) in human plasma and

urine. J Chromatogr A 1981; 224:51-58.

13 Salamoun J., Sprta V., Sladek V., and Smrz M. Determination of acyclovir in plasma

by column liquid chromatography with fluorescence detection. J Chromatogr A

1987; 420:197-202.

14 Sommadossi J.P., and Bevan R. High-performance liquid chromatographic method

for the determination of 9-(1,3-dihydroxy-2-prooxymethyl) guanine in human

plasma. J Chromatogr A 1987; 414:429-434.

15 Cronqvist J., and Nilsson-Ehle I. Determination of acyclovir inhuman serum by high-

performance liquid chromatography. J Chromatogr A 1988; 11:2593-2598.

16 Molokhia A.M., Niazy E.M., El-Hoofy S.A., and El-Dardari M.E. Improved liquid

chromatographic method for acyclovir determination in plasma. J Chromatogr A

1990; 13:981-986.

17 Nebinger P., Koel M. Determination of acyclovir by ultrafiltration and high-

performance liquid chromatography. J Chromatogr A 1993; 619:342-346.

18 Swart K.J., Hundt H.K.L., Groenewald A.M. Automated high-performance liquid

chromatographic method for the determination of acyclovir in plasma. J Chromatogr

A 1994; 663:65 -70.

19 Boulieu R., Gallant C., and Silberstein N. Determination of acyclovir in human

plasma by high-performance liquid chromatography. J Chromatogr B 1997; 693:233-

236.

20 Quinn R.P., De Miranda P., Gerald L., and Good S.S. A sensitive radioimmunoassay

for the antiviral agent BW248U [9-(2-hydroxyethoxymethyl)guanine]. Anal Biochem

1979; 98:319-324.

21 Svensson J.O, Barkholt L., and Sawe J. Determination of acyclovir and its metabolite

9-carboxymethoxymethylguanine in serum and urine using solid-phase extraction

and high-performance liquid chromatography. J Chromatogr B 1997; 690:363-366.

22 Brown S.D., White C.A., Chu C.K., and Bartlett M.G. Determination of acyclovir in

maternal plasma, amniotic fluid, fetal and placental tissues by high-performance

liquid chromatography. J Chromatogr B 2002; 772:327-334.

Chapter-1

Page 36

23 Caamano M.M., Garcia L.V., Elorza B., and Chantres J.R. Improved RPLC

determination of acyclovir using hexylamine as silanol masking agent. J Pharm

Biomed Anal 1999; 21:619-624.

24 Tzanavaras P.D., and Themelis D.G., High-throughput HPLC assay of acyclovir and

its major impurity guanine using a monolithic column and a flow gradient approach.

J Pharm Biomed Anal 2007; 43:1526-1530.

25 Bahrami G., Mirzaeei S., and Kiani A. Determination of acyclovir in human serum

by high-performance liquid chromatography using liquid–liquid extraction and its

application in pharmacokinetic studies. J Chromatogr B 2005; 816:327-331.

26 Fernandez M., Sepulveda J., Aranguiz T., and Von Plessing C. Technique validation

by liquid chromatography for the determination of acyclovir in plasma. J Chromatogr

B 2003; 791:357-363.

27 Pham-Huy C., Stathoulopoulou F., Sandouk P., Scherrmann J.M., Palombo S., and

Girre C. Rapid determination of valaciclovir and acyclovir in human biological fluids

by high-performance liquid chromatography using isocratic elution. J Chromatogr B

1999; 732:47-53.

28 Weller S., Blum M.R., Doucette M., Burnette T., Cederberg D.M., De Miranda P.,

and Smiley M.L. Pharmacokinetics of the acyclovir pro-drug valaciclovir after

escalating single and multiple-dose administration to normal volunteers. Clin

Pharmacol Ther 1993; 54:595-600.

29 Liang J, Gang W., Wei-Yue L., Ling-Jie Xu., and Jun P., Quantitative determination

of acyclovir in aqueous humor by LC-MS. Chromatographia 2006; 63: 239-242.

30 Zeng L., Nath C. E., Shaw P.J., Earl J.W., and McLachlan A.J. HPLC-fluorescence

assay for acyclovir in children. Biomed Chromatogr 2008; 22: 879-887.

31 Maria K., Evagelos G., Sofia G., Michael K., and Irene P. Selective and rapid liquid

chromatography/negative-ion electrospray ionization mass spectrometry method for

the quantification of valacyclovir and its metabolite in human plasma. J of

Chromatogra B 2008; 864; 78-86.

32 Schaeffer H.J., Beauchamp L., De Miranda P., Elion G.B., Bauer D.J., and Collins P.

9-(2-Hydroxyethoxymethyl) guanine activity against viruses of the herpes group.

Nature 1978; 272:583-585.

33 Blum M.R., Liao S.H., and De Miranda P. Overview of acyclovir pharmacokinetic

disposition in adults and children. Am J Med 1982; 73:186-192.

Chapter-1

Page 37

34 O’Brien J.J., and Campoli-Richards D.M. Acyclovir: An updated review of its

antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs 1989;

37:233-309.

35 Phan D.D., Chin-Hong P., Lin E.T., Anderle P., Sadee W., and Guglielmo B.J. Intra-

and Interindividual Variabilities of Valacyclovir Oral Bioavailability and Effect of

Coadministration of an hPEPT1 Inhibitor. Antimicrob Chemother 2003; 47: 2351-

2353.

36 Bleyzac N., Barou P., Massenavette B., Contamin B., Maire P., and Berthier J.C.,

Aulagner G. Assessment of acyclovir intraindividual pharmacokinetic variability

during continuous hemofiltration, continuous hemodiafiltration, and continuous

hemodialysis. Ther Drug Monit 1999; 21:520-525.

37 Granero G.E., and Amidon G.L., Stability of valacyclovir: implications for its oral

bioavailability. Int J Pharm 2006; 317; 1, 14-18.

38 Matuszewski B.K., Constanzer M.L., and Chavez-Eng C.M. Strategies for the

assessment of matrix effect in quantitative bioanalytical methods based on HPLC-

MS/MS. Analytical Chemistry 2003; 75: 3019-3030.

39 Dams R, Huestis M.A., Lambert W.E., and Murphy C.M. Matrix effect in bioanalysis

of illicit drugs with LC-MS/MS: influence of ionization type, sample preparation,

and biofluid. J Ameri Soci Mass Spec 2003; 14: 1290-1294.

40 Viswanathan C.T., Bansal S., Booth B., DeStefano A.J., Rose M.J., Sailstad J., Shah

V.P., Skelly J.P., Swann P.G., and Weiner R. Quantitative bioanalytical methods

validation and implementation: best practices for chromatographic and ligand

binding assays. AAPS Journal 2007; 9(1): E30-E42.

41 Guidance for Industry: Bioanalytical method validation. US food and drug

administration, center for drug evaluation and research, 2001. Available from

http://www.fda.gov/cder/guidance/4252fnl.htm.