Clinical Biochemistry 44 (2011) 907–915

Contents lists available at ScienceDirect

Clinical Biochemistry

j ourna l homepage: www.e lsev ie r.com/ locate /c l inb iochem

Determination of valganciclovir and ganciclovir in human plasma by liquidchromatography tandem mass spectrometric detection

Onkar Singh ⁎, Saurabh Saxena, Sanjeev Mishra, Arshad Khuroo, Tausif MonifClinical Pharmacology and Pharmacokinetics, Ranbaxy Research Laboratories, Plot GP-5, Sector 18, HSIDC, Gurgaon, Haryana-122015, India

⁎ Corresponding author. Fax: +91 124 2342017, +9E-mail address: onkar.singh@ranbaxy.com (O. Singh

0009-9120/$ – see front matter © 2011 The Canadian Sdoi:10.1016/j.clinbiochem.2011.04.003

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 29 November 2010Received in revised form 1 March 2011Accepted 3 April 2011Available online 16 April 2011

Keywords:ValganciclovirGanciclovirLC/MS/MSSolid phase extractionBioequivalence

Objectives: The aim of this work was to develop a simple, sensitive and selective LC/MS/MS method for theassay of valganciclovir and ganciclovir in human plasma.Design and methods: Sample preparation involved solid phase extraction on mix mode cation exchanger.Separation was performed on Chromolith RP18e column using water, trifluoroacetic acid (1 M, pH 4.4) andmethanol (29.9:0.1:70, v/v) as mobile phase. Both analytes were detected by electro spray ionization massspectrometry in positive ion multiple reaction monitoring mode.Results: CCs with good linearties having r≥0.9990 and ≥0.9992 were obtained in the range of 5–800 ng/mLand 70–11,200 ng/mL for valganciclovir and ganciclovir, respectively. The extraction recoveries were around85% for both the analytes.Conclusion: The method provided a simple and selective procedure that can be easily used for the evaluationof the pharmacokinetic profile of valganciclovir and ganciclovir in human plasma.

© 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Cytomegalovirus (CMV) is present in most of the adult populationand leads to lifelong viral persistence [1]. CMV is a viral genus of theHerpes viruses group commonly known as HCMV in humans orHuman Herpes Virus 5 (HHV-5). CMV is the most common viralcongenital infection, producing both sensorineural hearing loss andmental retardation. In AIDS patients, CMV retinitis is the mostcommon manifestation of infection and often leads to blindness [2].

The prophylactic antiviral therapy for the treatment of CMVinvolves use of oral ganciclovir, acyclovir and valaciclovir (prodrug foracyclovir) (Fig. 1). Intravenous administration of ganciclovir is choiceof drug treatment for CMV infections but it is associated with a risk ofintravenous line related infections. Oral ganciclovir is safe andeffective as maintenance therapy for CMV infections and is moreconvenient for patients to take than intravenous ganciclovir [3].Valganciclovir is an L-valyl ester prodrug of ganciclovir with muchimproved oral bioavailabilty (approximately 60%, 10 fold highercompared to ganciclovir) and provides drug exposure comparable tothat achieved with intravenous ganciclovir. Thus offers the perspec-tive of replacing suboptimal oral prophylactic ganciclovir, acyclovir,valaciclovir and intravenous treatment with ganciclovir [4].

There are various HPLC methods reporting the determination ofvalganciclovir (Fig. 1) or ganciclovir alone or simultaneously with someother anti retroviral drugs. Topal et al. [5] has reported determination of

1 124 2342030.).

ociety of Clinical Chemists. Publish

valganciclovir by RP-HPLCwith10 ng/mL limit of quantitation. Chu et al.[6] have reported aHPLCmethod for determination of ganciclovirwith alower limit of quantitation (LLOQ) 40 ng/mL and run time of 15 minMasahiko et al. [7] developed a fluorescence derivatization techniqueusingHPLC for determination of ganciclovir. HPLCpulsed amperometricdetection was used by Satoshi et al. [8] to determine ganciclovir alongwith acyclovir with 50 ng/mL and 10 ng/mL LLOQ, respectively. Areverse phase chromatographic method was developed by Anders et al.[9] to determine the therapeutic concentrations of ganciclovir in plasma,urine as well as dialysate (from continuous renal replacement therapy)from solid organ transplant recipient treated with either ganciclovir orits prodrug valganciclovir in combination with a wide variety of otherconcomitant drugs. The limit of quantitation in plasma was 100 ng/mL.Thesemethods either lack sensitivity or have longer run time.Mercus etal. [10] developed a method for simultaneous determination ofvalganciclovir and ganciclovir by LC/MS/MS and evaluated in a 6 yearold girl after oral valganciclovir. Another method was developed byHong et al. [11] for the simultaneous determination of both the drugs inhuman plasma. Both of these methods involve protein precipitationtechnique for the extraction of valganciclovir and ganciclovir fromhuman plasma, hence may not produce much cleaner sample foranalysis.

We in this communication are reporting a solid phase extraction(SPE) method for the simultaneous determination of valganciclovirand ganciclovir. The purpose of this study was to get a selectivemethod with much cleaner sample and LLOQ of 5 ng/mL and 70 ng/mL, for valganciclovir and ganciclovir, respectively to support abioequivalence study. The developed method is validated as per FDAguidelines [12].

ed by Elsevier Inc. All rights reserved.

N

NH

N

N

O

NH2

OO CH3

OH

O

NH2

CH3

Valganciclovir

N N

NHN

O

NH2

O

OH OH

Ganciclovir

N

N

N

NH

O

O

NH2NH2

OO

CH3

CH3

Valaciclovir

N N

NHN

O

NH2

O

OH

Acyclovir

Fig. 1. Molecular structure of valganciclovir, ganciclovir, valaciclovir and acyclovir.

908 O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

Materials and methods

Chemicals and reagents

Valganciclovir was procured from Varda Biotech (India), ganciclo-vir was a USP standard. Valacyclovir and acyclovir were provided byRanbaxy Research Laboratories (Gurgaon, India). HPLC grade meth-anol and formic acid were procured from s. d. fine chemicals(Mumbai, India) and trifluoroacetic acid was obtained from AcrosOrganics (New Jersey, USA). HPLC-grade water from MILLI Q system(Millipore, MA, USA) was used. Oasis MCX cartridges (30 mg/1 cc) forsample extraction were obtained from Waters corporation (Milford,Massachusetts, USA).

Instrument conditions

High performance liquid chromatography (HPLC) coupled withmass spectrometer was used for the entire analysis. HPLC instrumentwas supplied by Perkin Elmer equipped with PE200 series pump, autosampler and column oven. Mass spectrometer was triple Quadrupole(API-3000) manufactured by MDS Sciex (Toronto, Canada). Solidphase extractor speed disk 48 (Orochem Technologies, USA) andTurbovap LV concentrator (Caliper life science, USA) were used forextraction and eluent drying, respectively. Mass spectrometerequipped with turbo ion spray in positive ion mode was used foranalysis. Various MS operating parameters, parent and daughter ions(m/z) (Fig. 2) for all the four compounds are listed in Table 1.

Chromatographic conditions

Reverse phase chromatography was employed in this method.Pump was operated at 0.8 mL/min flow rate (0.5 mL/min wasintroduced to MS through a flow splitter), auto sampler temperaturewas set at 5 °C. Chromolith RP 18e (50 mm×4.6 mm) chromatographiccolumn (Merck) was used for separation and was maintained at 35 °C.Mobile phase consists of water, trifluoroacetic acid (1 M, pH 4.4) andmethanol (29.9:0.1:70, v/v). The total LC run time was 2.5 min with10 μL injection volume.

Preparation of stock solution, working solution, CC standards (CC) andquality control samples (QC)

Separate primary standard stock solutions of valganciclovir andganciclovir (1 mg/mL) were prepared in water whereas valaciclovirand acyclovir (1 mg/mL)were prepared in citric acid solution (0.01 M,pH 3.0). Working solution of both the analytes and their internalstandard (IS) (valaciclovir, 750 ng/mL and acyclovir, 1000 ng/mL)were prepared with methanol:water (50:50, v/v). Calibration stan-dards were prepared for both the analytes by serial dilution of stocksolution in methanol:water (50:50, v/v). Human plasma concentra-tions of valganciclovir (5, 15, 30, 75, 150, 300, 600 and 800 ng/mL)and ganciclovir (70, 210, 475, 1050, 2100, 4200, 8400 and 11,200 ng/mL)were prepared by spiking theworking solution into a pool of drugfree plasma.

Primary QC stock solution of valganciclovir and ganciclovir (1 mg/mL)was prepared froma separateweighing. QC sampleswere preparedby serial dilution of primary QC stock solution with methanol:water(50:50, v/v) and spiked in a pool of drug free plasma at four differentconcentration levels i.e. limit of quantitation QC (LOQQC) (5 ng/mLvalganciclovir and 70 ng/mL ganciclovir), low QC (LQC) (15 ng/mLvalganciclovir and 200 ng/mL ganciclovir), mediumQC (MQC) (300 ng/mL valganciclovir and 4200 ng/mL ganciclovir) and high QC (HQC)(600 ng/mL valganciclovir and 8400 ng/mL ganciclovir). Primary stocksolutionswere stored between 1 and 10 °C and protected from light. Allplasma samples were stored below −20 °C.

Data treatment

Standard CCs were plotted as the chromatographic peak area ratio ofanalyte/IS versus the corresponding nominal plasma concentrations. A1/concentration2 weighted regression analysis was used to determinethe slope, intercept and coefficient of correlation (r) using Analystsoftware version 1.4. The suitability of the calibration model wasconfirmed by back calculating the concentrations of the calibrationstandards.

Sample preparation

A solid phase extraction method was developed to isolatevalganciclovir and ganciclovir from plasma using valaciclovir and

+

(II)

(III)

(I)

(IV)

125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e59.7e5 152.1

175.1 226.3133.0

N N

N

O

OH

Acyclovir

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5152.0

325.2

174.1146.2 156.3

0.092232.1204.1

N

N

N

O

O

O

OO

Valaciclovir

Ganciclovir

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e58.7e5

Inte

nsity

, cps

Inte

nsity

, cps

Inte

nsity

, cps

152.1

256.3

N

N

NH

N

O

O

140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

140130 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

360 370

140 150 160 170 180 190 200 210 220 230 240 250 260 270

m/z, amu

m/z, amu

m/z, amu

m/z, amu

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

Inte

nsity

, cps

152.1

338.5

321.4303.3

149.5 355.2163.2 176.4 204.2 240.311.19 207.1 268.4226.4

H+

H+

H+

H+

N

NH

N

N

O

H2N

H2N

H2N

HN

HN

NH2

NH2

NH2

OO CH3

CH3

CH3

CH3

OH

HO

HO

O

Valganciclovir

Fig. 2. MS spectrum of valganciclovir (I), ganciclovir (II), valaciclovir (III) and acyclovir (IV).

909O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

acyclovir as IS, respectively. The thawed samples were vortexed toensure complete mixing of contents. 50 μL of IS working solution wasmixed with 200 μL aliquot of each plasma sample except for blank.The samples were diluted with 200 μL of 2% formic acid. Thepretreated samples were then transferred to Oasis MCX solid phaseextraction (SPE) cartridges, which were preconditioned usingmethanol, HPLC-grade water at 1–15 psi pressure. Loading of sampleson cartridges was followed by washing with 1 mL of 2% formic acidand then with 1 mL methanol. The analytes were eluted off thecartridges with 1 mL of 5% ammoniated methanol. Eluent wasevaporated to dryness at 15 psi and 50 °C under a stream of dry

nitrogen. The residue was reconstituted with 300 μL of mobile phase,transferred into vials for analysis.

Validation procedure

Eight point CC standards were designed at the nominal concen-tration of 5–800 ng/mL for valganciclovir and 70–11,200 ng/mL forganciclovir in human plasma. The acceptance criteria for these CCswere a correlation coefficient (r) of 0.9800 or better, and each backcalculated standard concentration must be within 15% deviation fromnominal value except for LLOQ, for which the maximum acceptable

Table 1Main working parameters of tandem mass spectrometer.

Parameters Valganciclovir Ganciclovir Valaciclovir Acyclovir

Declustering potential (V) 35 36 38 38Focusing potential (V) 95 95 100 110Entrance potential (V) 10 10 10 10Collision energy (V) 25 17 22 17Cell exit potential (V) 15 15 15 15Dwell time (millisecond) 200 200 200 200Molecular ion/daughterion transition (m/z)

355.2/152.1 256.3/152.1

325.2/152.0

226.3/152.1

Polarity PositiveNebulizer gasa 12Curtain gasa 14CAD gasa 6Ion source voltage (V) 5500Source temperature, °C 500

a These are typical values based on settings and scale defined in software applicationto control gas parameter.

910 O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

deviation was set at 20%. At least 67% of the non-zero standards wererequired to meet the acceptance criteria including acceptable LLOQand upper limit of quantification (ULOQ).

The following parameters were evaluated during validation of themethod; selectivity, linearity, precision and accuracy, extractionrecovery, stability viz. long term stability, freeze-thaw stability,bench top stability, stock solution stability, auto-sampler stability,and dilution integrity and matrix effect.

SelectivitySelectivity or specificity is assessed to demonstrate that the

intended analytes are measured and that their quantification is notaffected by the presence of the matrix components, known metab-olites, degradation products, or co-administered drugs. Test forselectivity was carried out using six different lots of blank plasma,two lots of hemolyzed plasma and two lots of lipemic plasmaprocessed and analyzed to determine the extent to which endogenoussubstances may contribute to the interferences to analytes and IS.LLOQ sample was spiked in the cleanest blank, processed andanalyzed by same method. The aim of performing the selectivitycheck with these different types of plasma samples is to ensure thequality of the results of study sample analysis. The peak area responseof interfering peak at the RT of analyte should be less than 20% ofmean analyte peak area in processed LLOQ samples. The interference

Table 2Selectivity test for valganciclovir, ganciclovir, valaciclovir and acyclovir in different lots of p

Sample name Valganciclovir Ganciclovir

Area Area (%) Area Are

LLOQ-1 3125 NAP 32,587 NALLOQ-2 3074 NAP 32,987 NALLOQ-3 3031 NAP 30,124 NALLOQ-4 2998 NAP 31,025 NALLOQ-5 3102 NAP 32,796 NALLOQ-6 3009 NAP 30,101 NAMean 3056.5 NAP 31,603.3 NABlank-1 72 2.4 54 0.1Blank-2 29 0.9 382 1.2Blank-3 112 3.7 469 1.4Blank-4 38 1.2 601 1.9Blank-5 94 3.1 272 0.8Blank-6 37 1.2 403 1.2Hemolyzed Blank-1 28 0.9 307 0.9Hemolyzed Blank-2 56 1.8 546 1.7Lipimic Blank-1 89 2.9 398 1.2Lipimic Blank-2 37 1.2 172 0.5

NAP = Not applicable.

is considered insignificant if the interfering peak area response at theRT of IS is less than 5% of area of mean peak area response in theprocessed LLOQ samples.

LinearityThe linearity evaluation was performed on three batches of spiked

samples. Each batch of spiked plasma samples included one completeset of CC standards and six replicate of QC samples at LOQQC, LQC,MQC and HQC levels. The linearity of bioanalytical method wasdetermined by plotting area ratio of analyte/IS against theoreticalconcentration of analyte. Calibrationmeasurements were subjected toleast squares regression analysis (1/x, 1/x2 and none) by computerprogram, to provide information on the slope, y-intercept, correlationcoefficient (r) and the back calculated concentrations. The coefficientof correlation should be 0.9800 or greater.

Within and between (intra and inter) assay precision and accuracyThe within and between (intra and inter) assay precision and

accuracy were determined by analyzing six replicates of QC samplesproduced at four different concentration levels i.e. LOQQC, LQC, MQCand HQC for valganciclovir and ganciclovir each in a batch and onthree different batches. Precision of the assay was measured by thepercent coefficient of variation over different concentration levels.The acceptance criteria for within and between assay precision were20% or better for LOQQC and 15% or better for other QCs. The accuracyof the assay was defined as the ratio of absolute value of the calculatedmean values of the QC samples to their respective nominal values,expressed as percentage and the criteria for accuracy was 100±20%or better for LOQQC and 100±15% or better for other concentrations.

RecoveryRecovery of the method was obtained by comparing the peak area

response of six replicates of both the drugs spiked in human plasmaproduced at three different concentration levels i.e. LQC, MQC andHQC with the peak area response of equivalent neat samples,respectively. Similarly, the recovery of IS was determined. Thevariation in recovery between all levels of QC's should be ≤20%.

StabilityVarious factors determine the stability of a drug viz. the chemical

properties, matrix, container system and storage conditions. Stabilityexperiments in stock solution and plasma were performed veryextensively to evaluate the stability of valganciclovir, ganciclovir and

lasma.

Valaciclovir Acyclovir

a (%) Area Area (%) Area Area (%)

P 102,456 NAP 102,321 NAPP 147,895 NAP 109,878 NAPP 154,381 NAP 102,498 NAPP 102,799 NAP 112,014 NAPP 101,212 NAP 100,013 NAPP 112,365 NAP 120,236 NAPP 120,184.7 NAP 107,826.7 NAP7 686 0.57 251 0.231 541 0.45 953 0.888 259 0.22 612 0.570 315 0.26 801 0.746 249 0.21 799 0.748 652 0.54 243 0.237 948 0.79 1198 1.113 725 0.60 351 0.336 643 0.54 381 0.354 525 0.44 978 0.91

0.2 0.4 0.6 0.8 1.21.0 1.4 1.6 1.8 2.0Time min

0.2 0.4 0.6 0.8 1.21.0 1.4 1.6 1.8 2.0

Time min

0.2 0.4 0.6 0.8 1.21.0 1.4 1.6 1.8 2.0

Time min

0.2 0.4 0.6 0.8 1.21.0 1.4 1.6 1.8 2.0

Time min

0.2 0.4 0.6 0.8 1.21.0 1.4 1.6 1.8 2.0Time min

0.2 0.4 0.6 0.8 1.21.0 1.4 1.6 1.8 2.0Time min

0

5

10

15

20

25

30

35

40

45

50

Inte

nsity

, cps

050

100150200250300350400450500550600650700

Inte

nsity

, cps

0.01000.02000.03000.04000.05000.06000.07000.08000.09000.01.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e42.1e4

Inte

nsity

, cps

0.96

(III)Valganciclovir

02468

10121416182022242628303234363840

Inte

nsity

, cps

1.63 2.110.30 1.21 1.32

1.951.46

0.71 1.810.441.06

0.18

0500

100015002000250030003500400045005000550060006500700075008000

Inte

nsity

, cps

0.94

0.01000.02000.03000.04000.05000.06000.07000.08000.09000.01.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e4

Inte

nsity

, cps

0.94(VI)

Ganciclovir

(I) (II)

(IV) (V)

Fig. 3. MRM LC/MS/MS chromatogram (I) and (IV) of blank human plasma; (II) and (V) human plasma spiked with LLOQ; and (III) and (VI) standard zero sample.

911O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

their respective IS. All the experimental conditions which the drugsactually encountered during sample analysis were simulated duringmethod validation to evaluate the various stabilities like long termstability at −20 °C and −50 °C, freeze thaw stability, bench topstability (room temperature stability of drug in plasma). Stocksolution stability of both the drugs and their IS and auto samplerstability (post preparative stability at auto sampler temperatureranging from 1 to 5 °C) were also evaluated.

To evaluate long term stability, a long period was defined as thetime elapsed between the start of sampling and at the end of sampleanalysis. The aliquots of QCs were first frozen at −20 °C and −50 °Cfor 86 days and then thawed to be extracted and tested against freshCC and QCs. The difference between the starting concentration and theconcentration after intended storage period will show whether thedrugs in plasma can degrade under these conditions. Evaluation offreeze-thaw stability involves estimation of analytes stability afterthree freeze-thaw cycles. Bench-top stability at room temperature(25 °C) was determined. The stock stability of both the drugs and theirinternal standards was evaluated at refrigerated temperature (be-tween 1 and 10 °C) by comparing the peak area response of stabilitystock and fresh stock. The stability of samples in auto sampler at (1–5 °C) was evaluated in order to check the stability for resident time ofprepared samples in auto sampler.

All stability exercises were carried out using four replicate QCsamples at two different concentration levels i.e. LQC and HQC againstfresh CC standards and the results were compared with the fresh QCsamples. The acceptance criterion is same as that for precision andaccuracy.

Dilution integrityA test for sample dilution with blank matrix during validation is

performed as some sample concentrations may exceed the ULOQ.Dilution integrity test was performed by preparing samples at aconcentration approximately two times the concentration of 90%ULOQ. These samples were diluted to two times and four times withblank matrix so as to bring the concentration within the CC range andthen analyzed against set of fresh CC standards. The acceptancecriteria for the diluted QC samples are the same as that of QC samplesin precision and accuracy batch.

Matrix effectMass spectrometric bioanalysis is greatly influenced by matrix

effect which may lead to suppression or enhancement of ionization ofanalytes. So quantitative measurement of matrix effect is necessary toget valuable information in LC/MS/MS based bioanalytical methods.The matrix effect is measured in terms of matrix factor which isdefined as a ratio of the analyte peak response in the presence ofmatrix components to the analyte peak response in the absence ofmatrix components.

Matrix Factor MFð Þ = Peak area response in presenceof matrix components = Peak

area response in absence of matrix components:

A MF greater than 1 may be due to ion enhancement and less than1 may be due to ion suppression. Similarly IS can also experience ionenhancement or ion suppression. To take into account the matrixeffects of IS, an IS-normalized matrix factor was calculated by

0 50 100 150 200 250 300 350 400 450

Analyte Conc. / IS Conc.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

(A)

(B)

0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4

Analyte Conc. / IS Conc.

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

An

alyt

e A

rea

/ IS

Are

aA

nal

yte

Are

a / I

S A

rea

500 550 600 650 700750 800

Fig. 4. Calibration curve with spiked human plasma samples. (A) — Linearity range forvalganciclovir (5–800 ng/mL) and (B) — linearity range for ganciclovir (70–11,200 ng/mL).

912 O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

substituting the peak area response with peak area ratio (analyte/IS)in the above equation for MF [13]. The IS-normalized matrix factorwas calculated in six different lots of matrix plasma at two differentconcentration levels i.e. LQC and HQC to determine the variability ofmatrix effect in samples from different individuals. The variability inmatrix effect as measured by the coefficient of variation should be lessthan 15%.

Application to clinical pharmacokinetic studyTo demonstrate that this LC/MS/MS method is applicable to

pharmacokinetic studies, it has been used to determine valganciclovir

Table 3Within (intra) assay precision and accuracy for valganciclovir and ganciclovir.

Batchnumber

Statistical parameters Valganciclovir

LOQQC LQC M

Nominal QC concentration(ng/mL)

5 10 3

1 Mean QC Concentration (ng/mL) 5.02 10.94 3SD (±) 0.101 0.189CV (%) 2.0 1.7% Nominal 100.4 109.4 1

2 Mean QC Concentration 4.95 9.87 2SD (±) 0.384 0.546CV (%) 7.8 5.5% Nominal 99.0 98.7

3 Mean QC concentration 5.21 10.68 3SD (±) 0.258 0.545CV (%) 5.0 5.1% Nominal 104.2 106.8 1

and ganciclovir concentrations in plasma from healthy humansubjects. Subjects were given valganciclovir orally under the specificprotocol used and developed at Ranbaxy Research Laboratories(India). The study was performed in accordance with guidelines laiddown by International Conference on Harmonization and USFDA [14].

Results and discussion

Method development

Selective and sensitive analytical methods for the quantitativeevaluation of drugs and their metabolites (analytes) are critical for thesuccessful conduct of preclinical and/or biopharmaceutics and clinicalpharmacological studies. In order to develop a method with desiredLLOQ, it was necessary to use tandem mass spectrometry (MS/MS)detection. MS/MS methods are capable of discriminating moreefficiently as compared to LC or LC/MS between the analyte andmatrix signals. Thus provides improved limit of detection for trace-mixture analysis. In this study, ESI was chosen as the ionizationsource. It was found that the signal intensity of both the analytes andtheir IS in human plasma was high using ESI source as compared toAPCI and moreover the regression curves were also linear. The electrospray ionization of valganciclovir and ganciclovir produced abundantmolecular ions at m/z (M+H+) 355.2 and 256.3 (Fig. 2), respectivelyunder positive ionization conditions. The M+H+ ions from valganci-clovir and ganciclovir were selected as the precursor ion andsubsequently fragmented in MS/MS mode to obtain the product ion.The fragment ions at m/z 152.1 (Fig. 2) were produced as theprominent product ions for both valganciclovir and ganciclovir. Thequantification of analytes was performed using the MRMmode due tothe high sensitivity of MRM data acquisition, where the precursor andthe product ions are monitored.

As we were aiming to achieve good resolution and symmetricpeak, various combinations of mobile phase and columns were triedkeeping in mind to get high response, a low retention time (RT), andbetter peak shape. Different combinations of acetonitrile, methanoland water with changed content of each component and buffer wereinvestigated to identify the optimal mobile phase that produced thebest sensitivity and peak shape. The acidic modifier, trifluroacetic acidalong with water and methanol could improve the peak shape,response and increase in the water content beyond 29.9% producesbroad peak shape. It was found that the combination of methanol,water and trifluoroacetic acid in the ratio of 70:29.9:0.1, v/v is anappropriate mobile phase. Good peak shape and resolution wereachieved using Chromolith RP18e (50 mm×4.6 mm) and abovementioned mobile phase. The flow rate was optimized to 0.8 mL/min (0.5 mL/min flow was introduced into MS with a split) which

Ganciclovir

QC HQC LOQQC LQC MQC HQC

00 600 70 190 4200 8400

15.89 591.25 74.2 181.7 4128.8 8361.23.457 6.143 3.14 7.21 162.31 201.21.1 1.0 4.2 4.0 3.9 2.4

05.3 98.5 105.9 95.6 98.3 99.584.58 618.63 68.1 196.8 4338.1 8213.812.01 18.3 2.01 4.44 81.23 102.364.2 3.0 3.0 2.3 1.9 1.2

94.9 103.1 97.3 103.6 103.3 97.812.84 589.21 78.1 202.6 4114.3 8256.311.1 18.54 5.52 9.65 125.61 110.193.5 3.1 7.1 4.8 3.1 1.3

04.3 98.2 111.6 106.6 98.0 98.3

Table 4Between (inter) batch precision and accuracy for valganciclovir and ganciclovir.

Statistical parameters Valganciclovir Ganciclovir

LOQQC LQC MQC HQC LOQQC LQC MQC HQC

Nominal QC concentration (ng/mL) 5 10 300 600 70 190 4200 8400Mean QC concentration (ng/mL) 5.24 11.13 318.69 589.61 66.20 196.50 4151.20 8562.10SD (±) 0.085 0.514 14.480 17.230 5.520 11.980 104.630 134.520CV (%) 1.6 4.6 4.5 2.9 8.3 6.1 2.5 1.6% nominal 104.8 111.3 106.2 98.3 94.6 103.4 98.8 101.9n 18 18 18 18 18 18 18 18

n=number of replicates.

913O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

permitted a RT of 0.9 min for both valganciclovir and ganciclovirmaking it possible to quantify in routine analysis and analyzing morethan 450 samples per day.

An ideal IS should be a structural analog, stable and labeledcompound, track the analyte during the extraction and compensatefor any analyte on the column and any inconsistent response.Valaciclovir, acyclovir and penciclovir were selected as IS for bothvalganciclovir and ganciclovir during method development. Due tostructural similarity and similar pKa values valaciclovir and acyclovirproved to be good IS for valganciclovir and ganciclovir, respectively.The stability of stock solutions of both the IS is more in 0.01 M citricacid solution (pH 3) compared to water when stored in refrigeratorbetween 1 and 10 °C for 15 days. Hence, the stock solutions wereprepared in 0.01 M citric acid solution (pH 3) which provides greaterstability.

Initially protein precipitation with methanol and acetonitrile wasevaluated followed by liquid–liquid extraction with ethyl acetate,dichloromethane and different combinations of dichloromethane andisopropyl alcohol during method development. Both protein precip-itation and liquid–liquid extraction resulted in low extractionrecovery as well as marked matrix effect. More selective solid phaseextraction using Oasis MCX cartridge resulted in better extractionrecovery and minimal matrix effect. Many efforts were made to makethe method more reproducible and robust during method develop-ment. The samples were diluted with formic acid solution (2%, v/v)which also helped in ionizing the basic analytes. This solution servedas first wash to remove the acidic impurities followed by organic washwith methanol to remove the neutral impurities. Ammoniatedmethanol with varying proportion of ammonia solution (0.25% to25%, v/v) is evaluated to get an optimal elution solution. Ammoniatedmethanol (5%, v/v) served as suitable elution solution. Elution solventand volatile impurities were evaporated by drying the eluent underdry nitrogen at 50 °C at about 15 psi. The dried extract wasreconstituted in 300 μL of mobile phase and its 10 μL was introducedto MS.

Method validation

SelectivityThe peak area response observed in all the lots of blank plasma at

RT of analytes were ≤3.7% of the mean LLOQ peak area response,whereas the peak area response observed at the RT of IS was ≤1.1%the mean peak area response of IS used in the sample preparation

Table 5% Recovery of valganciclovir, ganciclovir, valaciclovir and acyclovir.

S. no. QC Valganciclovir Ganciclovir Valaciclovir Acyclovir

1 LQC 88.9 82.6 79.5 86.32 MQC 92.4 89.6 80.4 74.13 HQC 85.3 84.2 74.3 80.7Mean 88.87 85.47 78.07 80.37SD (±) 5.891 2.857 5.128 6.213CV (%) 6.6 3.3 6.1 4.2

(Table 2). Fig. 3 demonstrates the selectivity results with thechromatograms of blank plasma, peak of valganciclovir and ganciclo-vir at lower limit of quantification (LLOQ) and zero standard (plasmasample with IS only). The solid phase extraction method employedgave a very good selectivity for the analysis of valganciclovir,ganciclovir and their IS in the blank human plasma. All thechromatograms were Gaussian in shape. No interferences werefound for valganciclovir, ganciclovir or IS. The retention time forboth the analytes was less than 1.5 min, which makes it suitable forroutine analysis.

LinearityThe eight point CCs were linear over the concentration range 5 to

800 ng/mL for valganciclovir and 70 to 11,200 ng/mL for ganciclovir asshown in Fig. 4. The calibration model was selected based on theanalysis of data. The best-fit and least squares residuals for CC wereachieved with a 1/x2 weighing factor. The coefficient of correlation (r)was consistently 0.9990 or greater during the course of validation forboth valganciclovir and ganciclovir.

Within and between (intra and inter) assay precision and accuracyTables 3 and 4 show a summary of within and between assay

precision and accuracy data for QC samples. Both intra and inter CVranged from 1.0% to 7.8% for valganciclovir and 1.2% to 8.3% forganciclovir. The accuracy for valganciclovir ranged from 94.9% to111.3% and 94.6% to 111.6% for ganciclovir.

RecoveryThe mean recovery (process efficiency) for valganciclovir from

plasma was 88.87% (±5.891 SD, n=3) and 85.47% (±2.857 SD,n=3) for ganciclovir. The mean recovery for both the IS i.e.,valaciclovir and acyclovir was 78.07% (±5.128 SD, n=3) and80.37% (±6.213 SD, n=3), respectively (Table 5).

StabilityThe results for all the stability exercises obtained were well within

the acceptable limits (%CV≤15%, accuracy 100±15%). The plasmasamples were found to be stable during storage at−20 °C and−50 °Cfor a period of 86 days (Table 6). The comparative freeze-thawstability calculated at the end of third freeze and thaw cycle rangedfrom 104.1% to 107.0% and 93.9% to 98.2% for valganciclovir andganciclovir, respectively (Table 6). Bench-top stability evaluationinvolved analysis of six replicates of LQC and HQC stability samples,which had been kept at room temperature for 16 h. The comparativestability ranged from 96.5% to 104.0% for valganciclovir and 90.9% to101.2% for ganciclovir (Table 6). Stock solution of valganciclovir andganciclovir stored at 1–10 °C was found stable for 15 days. Thepercent stability of stock solutions of valganciclovir, ganciclovir,valaciclovir and acyclovir was found out to be 99.9%, 104.2%, 94.5%,and 92.9%, respectively. The processed samples were found to bestable in auto injector for 54 h. Comparative auto sampler stability forvalganciclovir and ganciclovir ranged from 103.3% to 106.0% and101.5% to 107.8%, respectively (Table 6). Stock solution stability of

Table 6Different stability parameters of valganciclovir and ganciclovir.

Stability parameter Valganciclovir Ganciclovir

Sample concentration(ng/mL)

Concentration found(ng/mL)

Precision(%)

Accuracy(%)

Sample concentration(ng/mL)

Concentration found(ng/mL)

Precision(%)

Accuracy(%)

Long termstability

At −20 °C(86 days)

10.0 9.8 4.2 98.0 190.0 182.8 1.6 96.2600.0 574.6 3.8 95.8 8400.0 8341.9 1.2 99.3

At −50 °C(86 days)

10.0 9.1 6.5 91.0 190.0 204.2 2.5 107.5600.0 563.5 2.9 93.9 8400.0 8245.9 3.9 98.2

Freeze thaw stability 10.0 10.7 2.7 107.0 190.0 178.4 4.1 93.9600.0 624.6 5.8 104.1 8400.0 8248.6 2 98.2

Bench top stability (16 h) 10.0 10.4 3.4 104.0 190.0 172.8 1.7 90.9600.0 578.9 6.7 96.5 8400.0 8501.7 3.6 101.2

Auto sampler stability(54 h)

10.0 10.6 5.5 106.0 190.0 204.9 1.6 107.8600.0 619.5 7.2 103.3 8400.0 8525.6 1.9 101.5

Table 7Stability of valaciclovir and acyclovir.

S. no. Valacyclovir Acyclovir

Stability solution storedin 0.01 M citric acid

Stability solutionstored in water

Comparisonsolution

Stability solution storedin 0.01 M citric acid

Stability solutionstored in water

Comparisonsolution

1 501,240 451,421 550,147 963,470 841,478 996,7142 521,478 482,173 562,147 986,014 854,214 999,7853 563,248 455,003 552,013 963,214 843,697 990,2014 502,142 492,139 501,478 951,473 840,120 993,4705 500,012 490,210 567,418 920,147 841,475 973,2186 521,040 451,124 584,159 921,471 846,973 982,487Mean 518,193.3 470,345.0 552,893.7 950,964.8 844,659.5 989,312.5SD 24,184.45 19,861.72 28,021.39 25,902.70 5261.89 9880.79%CV 4.7 4.2 5.1 2.7 0.6 1.0% Stability 93.7 85.1 96.1 85.4

914 O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

valaciclovir and acyclovir when stored between 1 and 10 °C in 0.01 Mcitric acid solution (pH 3) for 15 days is shown in Table 7.

Dilution integrityDilution integrity of the samples met the acceptance criteria for

accuracy (100±15%) and %CV (b15%). Hence the real samples can bediluted, if required during clinical sample analysis.

Matrix effectThe %CV of IS-normalized matrix factor for both valganciclovir and

ganciclovir in different lots of plasmawas less than 8% as shown in Table 8.

Table 8IS-Normalized matrix factor for valganciclovir and valganciclovir in different lots ofplasma.

Matrixlot

Valganciclovir Ganciclovir

IS-normalized matrixfactor

IS-normalized matrixfactor

LQC HQC LQC HQC

1 0.991 0.991 1.085 1.0982 1.027 0.954 0.949 0.9543 1.008 1.015 1.093 0.9734 0.929 1.185 1.098 0.9885 0.975 1.072 1.078 1.0876 0.981 0.945 1.056 0.996Mean 0.986 1.043 1.061 1.020SD 0.05376 0.08216 0.07066 0.0475%CV 5.5 7.9 6.7 4.7

Clinical application

The method has been successfully used in pharmacokinetic studyof valganciclovir after oral dose (2 tablets containing 450 mg ofvalganciclovir, each, once a day) in healthy, adult, human, malesubjects under fasting condition. The study has been cleared by theethics review board for human studies and healthy volunteers havesigned an informed consent. Blood samples were collected from 35healthy volunteers at different time points in each period. A totalnumber of 1890 subject samples with 280 QC samples were analyzed.The mean Cmax for valganciclovir was around 325 ng/mL and4450 ng/mL for ganciclovir as can be seen from the mean valganci-clovir and ganciclovir pharmacokinetic curves (Figs. 5 and 6). The

0

50

100

150

200

250

300

350

0 5 10 15 20 25

Scheduled_Time (Hr)

RT

Mea

n (

ng

/ml)

Fig. 5. Mean plasma concentration–time plot of valganciclovir (fasting study) afteradministration of 900 mg (450 mg×2) dose of valganciclovir (n=35).

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 5 10 15 20 25

Scheduled_Time (Hr)

RT

Mea

n (

ng

/ml)

Fig. 6. Mean plasma concentration–time plot of ganciclovir (fasting study) afteradministration of 900 mg (450 mg×2) dose of valganciclovir (n=35).

915O. Singh et al. / Clinical Biochemistry 44 (2011) 907–915

AUC0–t obtained was 656.0 ng·h/mL and 22,649.9 ng·h/mL forvalganciclovir and ganciclovir, respectively.

Conclusion

In short, the method is developed for the quantitative estimationof valganciclovir and ganciclovir through LC/MS/MS in human plasmausing solid phase extraction, and fully validated on basis of selectivity,linearity, precision, accuracy, recovery, various stabilities parameters,dilution integrity and matrix effect. This method provides us withrapid, sensitive, reproducible quantification of valganciclovir andganciclovir. The advantage of this method is that it gives cleanersample using lesser plasma volume for analysis with minimum effectsof variations, with an increase in the selectivity of the method. Shortrun time is helpful in analyzingmore than 450 samples per day hence,useful to increase the productivity. The validated method can work onlinear range of 5 to 800 ng/mL for valganciclovir and 70 ng/mL to11,200 ng/mL for ganciclovir. Hence, the method is highly selectiveand suitable for pharmacokinetics.

Acknowledgments

The authors wish to acknowledge the support and facilitiesreceived from Ranbaxy Research Laboratories, Gurgaon, India tocarry out this work and permit for publication.

References

[1] Abu-Nader Patel R. Current management strategies for the management andtreatment of cytomegalovirus infection in solid organ transplant recipient.BioDrugs 2000;134:159–75.

[2] Ryan KJ, Ray CG, editors. Sherris medical microbiology. 4th ed. McGrawHill0838585299; 2004. p. 556. 566–9.

[3] Drew W Lawrence, Ives David, Lalezari Jacob P, Crumpacker Clyde, FollansbeeStephen E, Spector Stephen A, et al. Oral ganciclovir as maintenance treatment forcytomegalovirus retinitis in patients with AIDS. New Engl J Med 1995;333:615–20.

[4] Perrottet N, Beguin A, Meylan P, Pascual M, Manuel O, Buclin T, et al. J Chrm B2007;8552:420–9.

[5] DoganTopal B, Uslu B, Ozkan SA. Development and validation of an RP-HPLCmethod for determination of valganciclovir in human serum and tablets.Chromatographia September 2007;66(Supplement 1):97–101.

[6] Chu F, Kiang CH, Sung ML, Huang B, Reeve RL, Tarnowski T. 1J Pharm Biomed AnalNov 1999;21(3):657–67.

[7] Masahiko T, ShuujiH,MasahikoK,Megumi F, Nobufumi O,Masaaki K. Determinationof anti-virus drug, ganciclovir, in human serum by HPLC with precolumnfluorescence derivatization using phenylglyoxal. Anal Sci 2001;17(7):811.

[8] Satoshi K, Yoh T, Mitsuru S, Tsuyoshi S, Hiroyuki F, Satoru T, et al. Liquidchromatographic method for the determination of ganciclovir and/or acyclovir inhuman plasma using pulsed amperometric detection. J Chromatogr B November25 2002;780(2):289–94.

[9] Anders A, Christin NH, Leon R. Determination of ganciclovir in different matricesfrom solid organ transplanted patients treated with a wide range of concomitantdrugs. J Pharm Biomed Anal February 19 2007;43(3):1039–44.

[10] Mercus B, Hugh W, Christian K, George W, Christoph R. Oral valganciclovir inchildren: single dose pharmacokinetics in a six-year-old girl. Pediatr Infect Dis J2004;23:263.

[11] Hong RX, Xue NL, Wei LC, Gang YL, Nan NC, Chen Y. A sensitive assay forsimultaneous determination of plasma concentrations of valganciclovir and itsactive metabolite ganciclovir by LC/MS/MS. J Chromatogr B 2007;848:329.

[12] Guidance for industry: bioanalytical method validation. US Department of Healthand Human Services, Food and Drug Administration Centre for Drug Evaluationand Research (CDER), Centre for Veterinary Medicine (CBM); May 2001.

[13] Viswathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, et al. AAPS J2007;9(1):E30.

[14] FDA guidance for industry: bioavailability studies for orally administered drug-products— general considerations. US Department of Health and Human Services,Food and Drug Administration Centre for Drug Evaluation and Research (CDER);2000.