ww.sciencedirect.com

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6

Available online at w

journal homepage: www.elsevier .com/locate/ jopr

Original Article

Quantification of Acamprosate in human plasma by LC-ESI-MS/MS with solid phase extraction: Application to abioequivalence study

Kanchana Mala Kanala a,b,*, Babu Rao Chandu c, Nagiat T. Hwisa c, Mukkanti Khagga d,Prakash Katakam c, B.R. Challa e

a Jawaharlal Nehru Technological University, Research and Development Center, Anantapur, Andhra Pradesh 515002, IndiabRatnam Institute of Pharmacy, Department of Pharmaceutics, Pidathapolur, Muthukur, Nellore, Andhra Pradesh 524346, Indiac Faculty of Pharmacy, Department of Pharmaceutics, University of Al-Zawiya, 13, Libyad Jawaharlal Nehru Technological University, Department of Chemistry, Hyderabad, Andhra Pradesh 500072, IndiaeNirmala College of Pharmacy, Department of Pharmaceutical Chemistry, Kadapa, Andhra Pradesh 560012, India

a r t i c l e i n f o

Article history:

Received 27 March 2013

Accepted 16 May 2013

Available online 18 June 2013

Keywords:

Acamprosate

Human plasma

LC-ESI-MS/MS

Pharmacokinetics

Pharmacokinetic study

* Corresponding author. Jawaharlal Nehru T515002, India. Tel.: þ91 8088259567.

E-mail address: kanchanareddy1@gmail.c0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.05.010

a b s t r a c t

Background: The purpose of this investigation was to explore high selective, sensitive, rapid,

stable, reproducible extraction method in long run with broader linear range. At the same

time, it could be expected that, this method would be efficient in analyzing large numbers

of plasma samples obtained for pharmacokinetic, bioavailability or bioequivalence studies.

Methods: A simple, sensitive, selective and rapid high-performance liquid chromatography

coupled with tandem mass spectrometry was developed and validated for quantification of

Acamprosate in human plasma. The chromatography was performed by using waters

atlantis HILIC, (2.1 mm � 50 mm, 3.0 mm) column connected with guard column waters

atlantis HILIC, (2.1 mm � 10 mm, 3.0 mm). Acamprosate-d12 calcium trihydrate used as an

IS. The extraction of drug and internal standard were obtained by solid phase extraction.

The linearity was proved with concentration range 1.00e250.00 ng/ml for Acamprosate in

human plasma.

Results and discussion: The LOQ was demonstrated at 1.00 ng/ml. The within-batch, be-

tween-batch precision was found to be 2.21e4.07% and 2.00e3.20%. The within-batch,

between-batch accuracy was found to be 96.26e102.00% and 98.27e102.00% for Acam-

prosate. Drug and IS were eluted within 3.0 min.

Conclusion: The developed LC-MS/MS assay for Acamprosate is rapid, simple, sensitive,

selective and suitable for routine measurement of sample analysis. The validated method

was successfully applied in pharmacokinetic study of human plasma.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

echnological University, Research and Development Center, Anantapur Andhra Pradesh

om (K.M. Kanala).2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6390

1. Introduction acetonitrile, were purchased from J.T. Baker Inc. (Phillipsburg,

Acamprosate is the calcium salt of acetylhomotaurine and is

chemically known as calcium 3-acetamidopropane-1-sulfo-

nate. Acamprosate is a psychotropic drug used in the treat-

ment of alcohol dependence. The mechanism of action is

believed to be through inhibition of glutaminergic N-methyl-

D-aspartate receptors and activation GABA-grgic receptors.1e3

Acamprosate calcium, C10H20O8N2S2Ca, has a molecular

weight of 400.48 and three free acid molecular weight of

181.21. It is a white odorless powder and is freely soluble in

water and practically insoluble in ethanol and dichloro-

methane.4 Literature survey reveals that only a few methods

are reported previously to determine Acamprosate by using

proton emission tomography,5 LC-MS,6e9 HPLC,10 Capillary

zone electrophorsis,11 LC-fluremetric electrochemical detec-

tion12 in a variety of matrices like human plasma5e8 and dog

urine,9 dog plasma,10 pharmaceutical.11,12 Among all reported

methods, LC-MS6e9 methods attain best results. Ghosh C, et

al6 explained more about matrix effect of Acamprosate in

biological matrices and they developed the method by using

precipitation extractionmethod. Same authors (GhoshC, et al)

reported7 for quantification Acamprosate with the linearity

range between 7.04 and 702.20 ng/ml with Precipitation

extraction method by using LC-MS/MS in human plasma.

Hammarberg et al8 reported the method, both in human

plasma and CSF (Ceribrospinal fluid) by using LC-MS/MS and

they quantified the drug with the linearity range between 9

and 33 ng/ml in CSF and 25 times higher than CSF in human

plasma. Rhee et.al9 reported the method in dog plasma by

precipitation extraction method with LC-MS/MS with the

linearity range between 200 and 10,000 ng/mL. Chabenat et

al,10 reported themethod in dog urine by usingHPLC. As of our

knowledge, the reported methods does not provide stable,

reproducible extractionmethods interms ofmatrix effect, and

with high sensitive method.

The purpose of this investigation was to explore high se-

lective, sensitive, rapid, stable, reproducible extraction

method in long run with broader linear range. At the same

time, it could be expected that, this method would be efficient

in analyzing large numbers of plasma samples obtained for

pharmacokinetic, bioavailability or bioequivalence studies.

2. Experimental

Acamprosate obtained from Emcure Pharmaceuticals, Pune,

India and Acamprosate D12 was obtained from Vivan life

sciences, Mumbai, India (Fig. 1A and B). LC grade methanol,

Fig. 1 e Molecular structures of Acamprosate calcium (A)

and Acamprosate D12 calcium trihydrate (B).

NJ, USA). Reagent grade formic acid and ammonium formate

were procured from Merck (Mumbai, India). Human plasma

(K2EDTA) was obtained from Doctors Pathological Lab,

Hyderabad. The CAMPRAL� enteric coated tablets containing

333 mg Acamprosate per tablet, were obtained from Forest

pharmaceuticals, INC, USA.

2.1. Instrumentation

The 1200 Series HPLC system (Agilent Technologies, Wald-

bronn, Germany), Mass spectrometry API 4200 triple quadru-

pole instrument (ABI-SCIEX, Toronto, Canada) and data

processing was performed on Analyst 1.5.1 software package

(SCIEX).

2.2. Detection

The mass spectrometer was operated in the multiple reaction

monitoring (MRM) mode. Sample introduction and ionization

were electrospray ionization in the negative ion mode. Sour-

ces dependent parameters optimized were as follows: nebu-

lizer gas flow: 20 psi; Heatergas flow 40 psi; curtain gas flow: 8

psi; ion spray voltage (ISV): 5500 V; temperature (TEM): 650 �C.The compound dependent parameters such as the decluster-

ing potential (DP), focusing potential (FP), entrance potential

(EP), collision energy (CE), cell exit potential (CXP) were opti-

mized during tuning as 55, 30, 10, 18, 12 eV for Acamprosate

and Acamprosate D12, respectively. The collision activated

dissociation (CAD) gas was set at 5 psi using nitrogen gas.

Quadrupole 1 and quadrupole 3 were both maintained at a

unit resolution and dwell time was set at 600 ms for Acam-

prosate and Acamprosate D12. The mass transitions were

selected asm/z 180.0/ 79.9 for Acamprosate andm/z 186.1/

79.9 for Acamprosate D12. The parent and product ion spectra

for Acamprosate and Acamprosate D12 are represented in

Figs. 2a and b, 3a and b respectively. The data acquisition was

ascertained by Analyst 1.5.4software.

2.3. Chromatography

Waters Atlantis, HILIC, 50 � 2.1 mm, 3 mm, was selected as the

analytical column connected with Guard column Waters

Atlantis, HILIC, 10 � 2.1 mm, 3 mm. Column temperature was

set at 40 �C. Mobile phase composition was 10 mM Ammo-

nium formate pH 3.5: Acetonitrile (10:90 v/v). Source flow rate

250 mL/min without split. Injection volume of 10 mL. Acam-

prosate and Acamprosate D12 were eluted at 2.1 � 0.2 min,

with a total run time of 3.0 min for each sample.

2.4. Preparation of stock solutions and standards

Acamprosate and Acamprosate D12 standard stock solutions

100 mg/mL each were prepared by dissolving the appropriate

standard in methanol. From the Acamprosate stock solution

calibration and quality control standards were prepared by

using screened human blank plasma as diluent. Calibration

standards were prepared at concentration levels of 1.00, 2.00,

5.00, 25.00, 50.00, 100.00, 150.00, 200.00 and 250.00 ng/mL.

Fig. 2 e (a) Parent ion mass spectrum of Acamprosate, (b) product ion mass spectrum of Acamprosate.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6 391

Quality control standards were prepared at concentration

levels of 1.00, 3.00, 125.00 and 175.00 ng/mL for Acamprosate.

Internal standard spiking solution at 50 ng/mL concentration

was prepared by using 50% methanol solution from Acam-

prosate D12 standard stock solution. Calibration and quality

control standards were prepared from two separate stock

solutions of Acamprosate and stored at �30 �C. Internal

standard spiking solution was stored in refrigerator condi-

tions at 2e8 �C until analysis.

2.5. Sample preparation

Solid phase extraction procedure was used for isolation of

Acamprosate from the plasma samples. For this purpose,

50 mL of Acamprosate D12 ((IS) concentration of 50 ng/mL)

250 mL plasma (respective concentration of plasma sample)

was added into riavials then vortexed approximately. Fol-

lowed by 1000 ml of water was added and vortexed for 2 min.

These samples were added into SPE Catridges (Agilent poly-

mer SAX, 3Ml, 60mg, 60 mm)whichwere pre conditioned with

1mlmethanol, followed by 1mlwater. After that, the samples

which were in SPE, were washed with 1 ml water, followed by

1 ml Methanol. Elute the cartridges with 2 ml of 20% formic

acid solution into separate glass cultured tubes and evaporate

at 70 �C. Then these sampleswere reconstituted with 100 mL of

20% formic acid solution PH-3.5 and vortexed. Finally, 900 mL

of acetonitrile was added to each sample and vortexed for

2 min. At last, these samples were centrifuged at 4000 rpm at

20 �C for 5min. Then transferred the sample into auto sampler

vials with caps and 20 mL of sample from each autosampler

was allowed to instrument at optimized chromatographic

conditions.

2.6. Method validation

2.6.1. SelectivitySix different screened lots of human plasma samples were

selected from different donors for selectivity. These screened

lots were used for validation experiments to test for interfer-

ence at the retention time of analyte internal standard.

2.6.2. Matrix effectThe matrix effect due to the plasma matrix was used to

evaluate the ion suppression/enhancement in a signal when

comparing the absolute response of QC samples after pre-

treatment (SPE) with the reconstitution samples extracted

blank plasma sample spiking with analyte. Experiments were

performed at LQC and HQC levels in triplicate with six

different plasma lots with the acceptable precision (%CV) of

�15%.

2.6.3. Precision and accuracyIt was determined by replicate analysis of quality control

samples (n ¼ 6) at LLOQ (lower limit of quantification), LQC

(low quality control), MQC (medium quality control), HQC

(high quality control) and ULOQ (upper limit of quantification)

levels. Precision and accuracy should be within 15% for all the

standards except LLOQ. For LLOQ it should be within 20%.

Fig. 3 e (a) Parent ion mass spectrum of Acamprosate D12, (b) Product ion mass spectrum of Acamprosate D12.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6392

2.6.4. RecoveryThe recovery was carried out between extracted area to non

extracted area of each concentration. For Acamprosate re-

covery was proved at LQC, MQC, HQC level and for Acam-

prosate D12 recovery was proved at single concentration at

respective standards.

2.6.5. Dilution integrityDuring real subject sample analysis, some unknown sample

concentrationsmay fall above ULOQ and belowMQC Level. To

evaluate the actual concentration of those unknown samples,

dilution integrity test was performed at 1.5 times of ULOQ

concentrations were prepared and performed at six replicates

from each level (½, ¼ of ULOQ) and calculated by applying

dilution factor 2 and 4 with freshly prepared standards.

2.6.6. StabilityStability of the drug was proved in stock solution, and in

plasma samples. Stability of internal standard was proved in

stock solution. Stability of the drug and internal standard

stock solution was performed by comparing the area of sta-

bility samples to its freshly prepared stock solutions. Stock

solution stability was proved for 9 days and evaluated. Sta-

bility of the drug in plasma samples was proved at LQC, HQC

levels using six replicates each with its freshly prepared

samples of same concentration. Reinjection reproducibility

stability, benchtop stability, autosampler stability, freeze-

ethaw stability and long term stability was proved for drug in

plasma samples. The reinjection reproducibility was evalu-

ated by comparing the extracted plasma samples that were

injected immediately (time 0 h), with the samples that were

re-injected after storing in the autosampler at 4 �C for 26 h.

Stability samples were kept on bench (Benchtop stability) for

25 h and processed along with freshly prepared standards and

proved the stability for 25 h. The stability of spiked human

plasma samples prepared and stored at 4 �C in autosampler

(autosampler stability) was evaluated for 79 h. Freezeethaw

stability at �30 �C at 4th cycle was performed and proved for 3

cycles by comparing with freshly prepared samples. Long

term stability was proved for 34 days with its freshly prepared

standards at respective concentrations. All these stability

samples % Accuracy was less than 15%. The stability was

proved as per USFDA guidelines.13

2.7. Application of method

2.7.1. Analysis of plasma samplesThe bioanalytical method described above was applied to

determine acamprosate concentrations in plasma following

oral administration of healthy human volunteers. These vol-

unteers were contracted in APL Research centre, Hyderabad,

India and to each one of the 14 healthy volunteers were

administered a 333 mg dose (one 333 mg tablet) via oral with

240 ml of drinking water. The reference product CAMPRAL�

tablets, Manufactured by Forest pharmaceuticals, INC. USA.

333 mg, and test product Acamprosate tablet (test tablet)

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6 393

333 mg were used. Study protocol was approved by IEC

(Institutional Ethical committee) and by DCGI (Drug Control

General of India). Blood samples were collected as pre-dose (0)

hr 5 min prior to dosing followed by further samples at 0, 2,

2.5, 3, 3.5, 4, 4.5, 5, 5.75, 6.5, 7.25, 8, 9.5, 12, 14, 18, 24, 30, 36, 48,

56, 60, 72, 84 and 96 h.

After dosing, 5 ml blood sample was collected each pre-

established time in vacutainers containing K2EDTA. A total

of 50 (25 time points for reference, and 25 for test) time points

were collected and centrifuged at 3200 rpm, 10 �C, 10 min.

Then they were kept frozen at �30 �C until sample analysis.

Test and reference were administered to same human vol-

unteers under fasting conditions separately and these volun-

teers were washed minimum 9 days intervals as per protocol

approved by IEC.

2.7.2. Pharmacokinetics and statistical analysisPharmacokinetics parameters from human plasma samples

were calculated by a non-compartmental statistics model

using WinNon-Lin5.0 software (Pharsight, USA). Blood sam-

ples were taken for a period of 3e5 times the terminal elimi-

nation half-life (t1/2) and it was considered as the area under

the concentration time curve (AUC) ratio higher than 80% as

per the FDA guidelines. Plasma Acamprosate concentration-

time profiles were visually inspected and Cmax and Tmax

values were determined. The AUC0et was obtained by the

trapezoidal method. AUC0eN was calculated up to the last

measureable concentration and extrapolations were obtained

using the last measureable concentration and the terminal

elimination rate constant (Ke). The terminal elimination rate

constant (Ke), was estimated from the slope of the terminal

exponential phase of the plasma of Acamprosate

concentration-time curve (by means of the linear regression

method). The terminal elimination half-life t1/2 was then

calculated as 0.693/Ke. Regarding AUC0et, AUC0eN and Cmax

bioequivalence was assessed bymeans of analysis of variance

(ANOVA) and calculating the standard 90% confidence in-

tervals (90% CIs) of the ratios test/reference (logarithmically

transformed data). The bioequivalence was considered when

the ratio of averages of log transformed data was within

80e125% for AUC0et, AUC0eN and Cmax.14,15

3. Results and discussion

3.1. Method development

Mass parameters optimization, chromatography optimiza-

tion, suitable extraction method optimization to be optimized

during method development, prior to validate the method.

3.1.1. Mass spectrometry detection parameters optimizationDuring mass parameters optimization, type of ionization is

important to get the respective parent and product ions. In our

case, Electrospray ionization (ESI) was chosen as ionization

technique. In ESI mode, compound dependent parameters

(DP, EP, FP, CE, CXP) and source dependent parameters

(CUR,CAD, Heatergas, nebulizer gas) temperature, voltage

conditions were optimized to get better signal and response of

the analyte and internal standard. Acamprosate gave more

response in negative ion mode as compare to the positive ion

mode. The predominant peaks in the primary ESI spectra of

Acamprosate and Acamprosate D12 corresponds to the MH-

ions at m/z 180.0 and 186.1 respectively (Figs. 2a, 3a). Product

ions of Acamprosate and Acamprosate D12 scanned in

quadrupole 3 after a collision with nitrogen in quadrupole 2

had an m/z of 79.91 and 79.9 respectively [Figs. 2b, 3b].

3.1.2. Chromatography optimizationDuring chromatographic optimization, selection of suitable

mobile phase and suitable column are the primary aspects.

Mobile phase containing ammonium acetate and acetonitrile

in varying combinations was tried, but a low response was

observed. Further, it was changed to acetic acid: acetonitrile

(20:80 v/v) and acetic acid: methanol (20:80 v/v) observed bad

peak shape. After that, mobile phase containing 0.1% formic

acid in water in combination with methanol and acetonitrile

with varying combinations were tried. Using a mobile phase

containing 10 mM ammonium formate (Ph: 3.5): acetonitrile

(10:90 v/v), the best signal along with a marked improvement

in the peak shape was observed for Acamprosate and

Acamprosate D12. Different columns like, symmetry shield

RP18 (50 � 2.1 mm, 3.5 mm), Inertsil ODS-2V (50 � 4.6 mm,

5 mm), Hypurity C18 (50 � 4.6 mm, 5 mm) and Hypurity

Advance (50 � 4.0 mm, 5 mm) were used in the method

development. Symmetry shield RP18 column gave a relatively

good peak shape, but the response was low. Using Hypurity

C18 column poor chromatography was observed. Good

response was observed with waters Atlantis, HILIC,

50 � 2.1 mm, 3 mm, was selected as the analytical column

connected with Guard column Waters Atlantis, HILIC,

10 � 2.1 mm, 3 mm. It gave satisfactory peak shapes for both

Acamprosate and Acamprosate D12. Flow rate of 0.25 mL/min

without splitter was utilized and reduced the run time to

3.0 min. Both Drug and IS were eluted with shorter time at

2.1 min. For an LC-MS/MS analysis, utilization of stable

isotope-labeled or suitable analog drugs as an internal stan-

dard proves helpful when a significant matrix effect is

possible. In our case, Acamprosate D12 was found to be best

for the present purpose. The column temperature was

adjusted to 40 �C. Injection volume of 20 mL sample is

adjusted for better ionization and chromatography.

3.1.3. Extraction optimizationDuring extraction stage different extraction procedures like

PPT (protein precipitation), LLE (liquideliquid extraction), and

SPE (solid phase extraction). We found ion suppression effect

in protein precipitation method for drug and internal stan-

dard. Further, we tried with SPE and LLE. Out of all, we

observed that SPE is suitable for extraction of drug and IS.

Autosampler wash is optimized as 80% methanol. Several

compounds were investigated to find a suitable IS, and finally

Acamprosate D12 found the most appropriate internal stan-

dard for the present purpose. There was no significant effect

of IS on analyte recovery, sensitivity or ion suppression. High

recovery and selectivity was observed in the solid phase

extraction method. These optimized detection parameters,

chromatographic conditions and extraction procedure resul-

ted in reduced analysis time with accurate and precise

detection of Acamprosate in human plasma.

Fig. 5 e LOQ chromatogram of Acamprosate and

Acamprosate D12 in human plasma.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6394

3.2. Method validation

A thorough and complete method validation of Acamprosate

in human plasma was done following USFDA guidelines.13

The method was validated for selectivity, sensitivity, matrix

effect, linearity, precision and accuracy, recovery, dilution

integrity, reinjection reproducibility and stability.

3.2.1. Selectivity and sensitivityThere is no interference observed for Acamprosate and

Acamprosate D12 at their retention time in blank plasma

(Fig. 4) and LOQ (Fig. 5). These interferences are within the

acceptance criteria for all six lots of blank samples. The LLOQ

for Acamprosate was 1.00 ng/mL. The intra-run, inter-run

precision and accuracy of the LLOQ plasma samples con-

taining Acamprosate was 3.56 and 102.00% and 2.0 and

102.21%, respectively. All the values obtained below 1.00 ng/

mL for Acamprosate were excluded from statistical analysis

as they were below the LLOQ values validated for Acam-

prosate.

3.2.2. Matrix effectThe CV % of ion suppression/enhancement in the signal was

found to be 1.0% at MQC level for Acamprosate indicating that

the matrix effect on the ionization of analyte is within the

acceptable range under these conditions.

3.2.3. LinearityCalibration curves were plotted as the peak area ratio

(Acamprosate/Acamprosate d12) versus (Acamprosate) con-

centration. Calibration was found to be linear over the con-

centration range of 1.00e250.00 ng/mL. The precision was less

than 5.30% and the accuracy ranged from 98.00% to 101.20%.

The determination coefficients (r2) were greater than 0.9985

Fig. 4 e Blank plasma chromatogram of Acamprosate and

Acamprosate D12.

for all curves (Table 1). The deviations of the back calculated

values from the nominal standard concentrations were less

than 15%.

3.2.4. Precision and accuracyPrecision and accuracy for this method was controlled by

calculating the intra and inter-batch variations at four con-

centrations (1.00, 3.00, 125.00 and 175.00 ng/mL) of QC samples

in six replicates. As shown in Table 2, the intra-day precision

was less than 4.07% and the accuracy ranged from 96.26% to

102.00%. Inter-day precision was less than 3.20% and the ac-

curacy ranged from 98.27% to 102.00%. The inter-run, intra-

run precision (% CV) was �15% and inter-run, intra-run ac-

curacy was in between 85 and 115 for Acamprosate. All these

results (Table 2) indicate the adequate reliability and repro-

ducibility of this method within the analytical curve range.

Table 1 e Calibration curve details.

Concentration(ng/ml)

Mean � SD %CV Accuracy

1.00 0.99 � 0.02 2.00 99.00

2.00 2.01 � 0.05 2.50 100.50

5.00 5.06 � 0.27 5.30 101.20

25.00 24.88 � 0.62 2.50 99.52

50.00 49.00 � 1.73 3.50 98.00

100.00 100.99 � 1.93 1.90 100.99

150.00 151.14 � 3.93 2.60 100.76

200.00 198.24 � 5.44 2.70 99.12

250.00 250.79 � 2.61 1.00 100.32

SD: Standard deviation, CV ¼ Coefficient of variation.

Table 2 e Within-run and between-run precision and accuracy.

Nominal addedconcentration (ng/mL)

Within-run (n ¼ 6) Between-run (n ¼ 36)

Mean � S.D. Precision (CV %) Accuracy Mean � S.D. Precision (CV %) Accuracy

1.00 1.02 � 0.04 3.56 102.00 1.02 � 0.02 2.00 102.00

3.00 2.90 � 0.08 2.76 96.67 3.01 � 0.09 3.00 100.33

125.00 122.23 � 4.97 4.07 97.78 123.38 � 3.99 3.20 98.70

175.00 168.46 � 3.72 2.21 96.26 171.98 � 4.79 2.80 98.27

SD: Standard deviation, CV ¼ Coefficient of variation.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6 395

3.2.5. RecoveryThe recovery following the sample preparation using Solid

Phase extraction method was calculated by comparing the

peak area of Acamprosate in plasma samples with the peak

area of solvent samples. The recovery of Acamprosate was

determined at three different concentrations 3.00, 125.00 and

175.00 ng/mL and found to be 89.19%, 101.72% and 99.48%

respectively. The overall average recovery of Acamprosate

and Acamprosate d12 and found to be 96.80% and 87.40%

respectively.

3.2.6. Dilution integrityThe mean back calculated concentrations for 1/4 and 1/2

dilution samples were within 85e115% of their nominal. The

% CV for 1/4 and 1/2 dilution samples were 3.4% and 3.5%

respectively.

3.2.7. StabilitiesQuantification of Acamprosate in plasma subjected to 3

freezeethaw (�30 �C to room temperature) cycles showed the

stability of the analyte. No significant degradation of Acam-

prosate was observed even after 73 h storage period in the

autosampler tray, and the final concentrations of Acampro-

sate was between 99.33% and 100.84% of the theoretical

values. In addition, the long term stability of Acamprosate in

QC samples after 65 days of storage at �30 �C was also eval-

uated. The concentrations ranged from 99.67% to 99.96% of

the theoretical values. These results confirmed the stability of

Table 3 e Stability of the drug in plasma samples.

Stability Spiked plasmaconcentration

(ng/mL)

Concentrationmeasured (ng/mL)(mean � S.D; n ¼ 6)

CV(%)

(n ¼ 6)

Benchtop

stability

(41 h)

3.00 2.99 � 0.10 3.45

175.00 178.38 � 5.78 3.24

Autosampler

stability

(73 h)

3.00 2.98 � 0.10 3.2

175.00 176.47 � 3.89 2.21

Long term

stability

(65 days)

3.00 2.99 � 0.09 3.16

175.00 174.93 � 9.23 5.27

Freeze & thaw

stability

(cycle 3,

48 h)

3.00 3.16 � 0.10 3.21

175.00 179.29 � 1.06 0.59

Acamprosate human plasma for at least 65 days at �30 �C(Table 3). Acamprosate and Acamprosate D12 stability in stock

solution was performed against freshly prepared stock solu-

tions for 13 days. The % change for Acamprosate and Acam-

prosate D12 were �0.01% and 0.01%.

3.3. Pharmacokinetic study

The proposed method was applied to the determination of

Acamprosate in plasma samples for the purpose of estab-

lishing the bioequivalence of a single 333 mg dose (one

333 mg Tablet) in 14 healthy volunteers. Typical plasma

concentrations versus time profiles are shown in Fig. 6.

Plasma concentrations of Acamprosate were in the standard

curve range and remained above the 1.00 ng/mL quantitation

limit for the entire sampling period. The pharmacokinetic

parameters for test and reference products were shown in

Table 4, Table 5. The mean ratio of AUC0et/AUC0eN was

higher than 90% with following the Food and Drug Admin-

istration Bioequivalence Guideline.14,15 The ratio test/refer-

ence (T/R) and 90% confidence intervals (90 CIs) for overall

analysis were comprised within the previously stipulated

range (80e125%). Therefore, it can be concluded that the two

Acamprosate formulations (reference and test) analyzed are

bioequivalent interms of rate and extent of absorption at

fasting conditions.

Fig. 6 e Mean plasma concentrations of test vs. reference

after 333 mg dose (1 3 333 mg tablet) in 14 healthy

humans.

Table 4 e Mean Pharmacokinetic parameters ofAcamprosate in 14 healthy humans after oraladministration of 333 mg test and reference productsunder fasting conditions.

Pharmacokinetic parameter Acamprosate

Test Reference

AUC0et (ng h/mL) 1909.49 2307.12

Cmax (ng/mL) 110.09 119.83

AUC0eN (ng h/mL) 1911.23 2308.41

Kel (h_1) 0.07069 0.09274

Tmax (h) 8 8

AUC0eN: area under the curve extrapolated to infinity.

AUC0et: area under the curve up to the last sampling time.

Cmax: the maximum plasma concentration.

Tmax: the time to reach peak concentration.

Kel: the apparent elimination rate constant.

Table 5 e Test/reference pharmacokinetic parameters ofAcamprosate after administration of 333 mg of test andreference products in 14 healthy humans under fastingconditions.

Pharmacokinetic parameters AUC0et AUC0eN Cmax

Test/ref 82.77 82.79 91.87

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 3 8 9e3 9 6396

4. Conclusion

The developed method is high selective, sensitive, rapid, sta-

ble and reproducible. Analyte was compared its respective

deuterated internal standard. Solid phase extractionwas used

to extract the drug and internal standard from plasma sam-

ples. This method was validated over the concentration range

of 1.00e250.00 ng/ml as per regulatory guidelines. Finally, This

method was applied to pharmacokinetic study in healthy

human volunteers under fed conditions.

Conflicts of interest

All authors have none to declare.

Acknowledgments

The authors are grateful to the Indian Institute of Chemical

Technology, Hyderabad for literature survey and Manipal

Acunova, Manipal, India for their Lab facility for this research

work.

r e f e r e n c e s

1. Zornoza T, Cano MJ, Polache A, Granero L. Pharmacology ofacamprosate: an overview. CNS Drug Rev. 2003Winter;9(4):359e374. Review.

2. Saivin S, Hulot T, Chabac S, Potgieter A, Durbin P, Houin G.Clinical pharmacokinetics of acamprosate. Clin Pharmacokinet.1998 Nov;35(5):331e345. Review.

3. Rhee YS, Park S, Lee TW, et al. Investigation of therelationship between in vitro and in vivo release behaviors ofacamprosate from enteric-coated tablets. Arch Pharm Res.2008 Jun;31(6):798e804.

4. Zornoza T, Cano-Cebrian MJ, Hipolito L, Granero L, Polache A.Evidence of a flip-flop phenomenon in acamprosatepharmacokinetics: an in vivo study in rats. Biopharm DrugDispos. 2006 Oct;27(7):305e311.

5. Courtyn J, Cornelissen B, Oltenfreiter R, Vandecapelle M,Slegers G, Strijckmans K. Synthesis and assessment of [11C]acetylhomotaurine as an imaging agent for the study of thepharmacodynamic properties of acamprosate by positronemission tomography. Nucl Med Biol. 2004;31(5):649e654.

6. Ghosh C, Shinde CP, Chakraborty BS. Influence of ionizationsource design onmatrix effects during LC-ESI-MS/MS analysis. JChromatogrBAnalytTechnolBiomedLifeSci. 2012;893e894:193e200.

7. Ghosh C, Jha V, Shinde CP, Chakraborty BS. A LC-MS analysisof acamprosate from human plasma: pharmacokineticapplication. Drug Test Anal. 2011;3(10):735e742.

8. Hammarberg A, Beck O, Eksborg S, et al. Acamprosatedeterminations in plasma and cerebrospinal fluid aftermultiple dosing measured by liquid chromatography-massspectroscopy: a pharmacokinetic study in healthy volunteers.Ther Drug Monit. 2010;32(4):489e496.

9. Rhee YS, Park JH, Park S, et al. Analysis of acamprosate inbeagle dog plasma by LC-MS-MS. Arch Pharm Res.2008;31(8):1035e1039.

10. Chabenat C, Ladure P, Moore N, Boucly P, Boismare F.Application of an analytical method to calciumacetylhomotaurinate determination in urine.Arzneimittelforschung. 1989;39(11):1413e1414.

11. Fabre H, Perrin C, Bosc N. Determination of homotaurine asimpurity in calcium acamprosate by capillary zoneelectrophoresis. J Chromatogr A. 1999;853(1e2):421e430.

12. Chabenat C, Ladure P, Blanc-Continsouza D, Boismare F,Boucly P. Determination of calcium acetylhomotaurinate byliquid chromatography with fluorimetric and electrochemicaldetection. J Chromatogr. 1987;414(2):417e422.

13. Guidance for Industry: Bioanalytical Method Validation. U.S.Department of Health and Human Services, Food and DrugAdministration,Center forDrugEvaluationandResearch (CDER),Center for Biologics Evaluation and Research (CBER); May 2001.

14. Chandu Babu Rao, Nama Sreekanth, Kanala Kanchanamala,Challa Balasekhara Reddy, Shaik Rihana Parveen,Mukkanti Khagga. Quantitative estimation of riluzole inhumanplasma by LC- ESI- MS/MS and its application to abioequivalencestudy.AnalBioanalChem. 2010;398(3):1367e1374.

15. Guidance for Industry Bio Availability and Fed Bio EquivalenceStudies for Orally Administered Drug Products-GeneralConsiderations. U. S. Department of Health and HumanServices Food and Drug Administration Centre for DrugEvaluation and Research (CDER); March 2003.