| Research Article |
Open Access |
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| Pharmacokinetic Evaluation of Metolazone
Tablets using healthy Human Volunteers |
Basvan Babu*, Selvadurai Muralidharan, Subramaniya Nainar Meyyanathan and
Bhojraj Suresh |
| Department of Pharmaceutical Analysis, J.S.S.College of Pharmacy, (Off Campus of JSS University, Mysore) Ootacamund, Rocklands, Ooty |
| *Corresponding author: |
Dr. Basvan Babu,
Department of Pharmaceutical
Analysis,
J.S.S.College of pharmacy, Tamilnadu, India,
Tel: +91-423-
2443393,
Fax: +91-423-2442937,
E-mail: thammababu@yahoo.co.in. |
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| Received November 25, 2009; Accepted December 22, 2009; Published December 22, 2009 |
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| Citation: Babu B, Muralidharan S, Meyyanathan SN, Suresh B (2009) Pharmacokinetic Evaluation of Metolazone Tablets using healthy Human Volunteers. J Bioanal Biomed 1: 039-040. doi:10.4172/1948-593X.1000008 |
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| Copyright: © 2009 Babu B, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited. |
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| Abstract |
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| A sensitive and reproducible high performance liquid
chromatography (HPLC) method has been developed and
validated for the quantification of metolazone in human
plasma, after solid phase extraction (SPE). A Good resolution
was achieved on a reverse-phase LichroCART
Purospher® C18 column using the mobile phase acetonitrile–
0.5% triethylamine (35:65) in isocratic elution with
a total run time of 15 min. The analyte, metolazone, was
detected by using high performance liquid chromatography
with the support of photo diode array detector. Limit
of detection and Lower limit of quantification was found
to be 1 and 2.5 ng/mL. The present method was successfully
applied in the pharmacokinetic study of metolazone
in human plasma. |
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| Keywords |
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| Metolazone; Bioavai labi l ity studies;
Pharmacokinetic evaluvation |
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| Introduction |
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| This work deals with the studies carried out by the writer in
this laboratory for the past one year on the development of analytical
method used for the estimation of Metolazone in biological
fluids and Bioequivalence studies on Metolazone tablets.
Metolazone is a diuretic / saluretic / antihypertensive drug of the
quinazoline class. The chemical name is 7-chloro-1, 2, 3, 4-
tet r ah ydro-2-met hyl -3- (2-meth yl ph en yl )-4-oxo-6-
quinazolinesulfonamide. Metolazone is a quinazoline diuretic,
with properties generally similar to the thiazide diuretics. The
actions of Metolazone result from interference with the renal
tubular mechanism of electrolyte reabsorption. Metolazone acts
primarily to inhibit sodium reabsorption at the cortical diluting
site and to a lesser extent in the proximal convoluted tubule.
Sodium and chloride ions are excreted in approximately equivalent
amounts. The increased delivery of sodium to the distal tubular
exchange site results in increased potassium excretion.
Metolazone does not inhibit carbonic anhydrase. A proximal
action of Metolazone has been shown in humans by increased
excretion of phosphate and magnesium ions and by a markedly
increased fractional excretion of sodium in patients with severely
compromised glomerular filtration. This action has been demonstrated
in animals by micropuncture studies. The antihypertensive
mechanism of action of Metolazone is not fully understood
but is presumed to be related to its saluretic and diuretic
properties (wikipedia). Only limited methods have been reported
for HPLC and LC-MS (Brodie et al., 1981; Farthing et al., 1990; Farthing et al., 1994; Wei et al., 2007; Jadhav et al., 2009; Guidance
for Industry Bioanalytical Method Validation. U.S, 2001; Guidance for Industry, Statistical Approaches in Establishing
Bioequivalence. U.S, 2001). Our aim is to develop a new and
sensitive bioanalytical method. |
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| Experimental |
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| Reagents and materials |
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| Working standard of Metolazone with 99.86% purity was obtained
from German Remedies Ltd., Mumbai, India. Celecoxib
(purity 99.38%) working standard was obtained from Cadila
Health Care Ltd., Ahmedabad, India. Acetonitrile (HPLC grade),
obtained from Qualigens Fine Chemicals, Mumbai and potassium
dihydrogen ortho phosphate, ortho phosphoric acid, methanol,
and trichloroacetic acid (all analytical grade reagent) were
purchased from S.D. Fine Chem. Ltd., Mumbai. In house mill Q
water was used throughout the study. Fresh frozen human plasma
used in the method development was obtained from the Vijay
Hospital, Ooty, and was stored at -70 ± 2°C until required. |
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| Instrumentation |
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| The HPLC system consisted of a HPLC 10 AT-VP (Shimadzu
Ltd., Japan), Manual injector port with 100μL loop (Rheodyne,
USA) and UV detector (Shimadzu Ltd., Japan). The wavelength
of the detector was set at 225 nm. Detector output was quantified
on CLASS VP (Version 6.01) chromatography software.
Separation was carried out on a LichroCART Purospher® C18,
(250 mm × 4.6mm, 5μm), Japan, using mixture of 0.5% triethyl
amine (pH 3.5) and acetonitrile (65:35, v/v) as a mobile phase,
at a flow rate of 1 mL/min. Total analysis time was 15 min. All
analysis was performed at room temperature. |
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| Preparation of calibration standard and quality control standards |
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| Stock solutions of metolazone and celecoxib (I.S) (1 mg/ml)
were prepared in a mixture of water and acetonitrile (1:1 v/v)
and stored at 8°C. The stock solution of metolazone was further
diluted with the mixture of water and acetonitrile to give series
of standard solutions. Calibration standard of metolazone (2.5,
5.0, 10.0, 25.0, 50.0, 75.0, 100.0, 250.0 and 500.0 ng/ml) were
prepared by spiking appropriate amount of the standard solution
in blank plasma. Lowest quality control standards (LQC), median
quality control standards (MQC) & highest quality control
standards (HQC) were prepared by spiking drug free plasma with metolazone to give solutions containing 6.0, 50.0, and 500.0,
ng/mL, respectively. They were stored at -20 ± 2°C till analysed. |
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| Sample preparation with SPE |
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| To 0.5ml plasma sample containing metolazone (calibration
standard), 0.5ml of internal standard (1.0μg/ml) was added and
vortexed. Phenomenex Strata C18-E SPE cartridge was conditioned
with acetonitrile and water sequently. The above conditioned
cartridge 1 mL of sample solution was added.The cartridge
was washed with water. The drug and internal standard
was eluted from the cartridge using 1.0 mL of mobile phase.
None of the drug free plasma samples studied in this assay yield
endogenous interference at these retention times (Figure 1). |
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|
Figure 1: Sample Chromatogram. |
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| Results and Discussion |
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| Accuracy and precision |
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| The mean percent accuracy of the proposed method was found
to be 98.37%. Intra day precision for metolazone was 5.75 ±
0.38, 49.19 ± 0.17, 498.35 ± 0.87 for the spiked concentration
at 6.0, 50.0 and 500.0 ng/mL respectively. Inter day precision
for metolazone was 6.01 ± 0.21, 49.39 ± 0.34 and 499.97 ± 0.32
for the spiked concentration at 6.0, 50.0 and 500.0 ng/mL, respectively. |
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| Linearity |
|
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| The linearity study was carried out on seven different
concentration of metolazone were analysed (2.5, 5.0, 10.0, 25.0,
50.0, 75.0, 100.0, 250.0 and 500 ng/mL). The peak area response
was linear over the concentration range studied. Each experiment
at all concentration was repeated three times on three separate
days to obtain the calibration data. The coefficient of correlation
‘r2’ was found to be 0.9978. |
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| Recovery |
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| The mean extraction recoveries of metolazone determined over
the concentration of 6.0, 50.0 and 500.0 ng/ml were 98.87%,
99.03% and 99.94%. |
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| Stability study |
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| Short-term and long-term stock solution stability studies evaluated,
which proved no significant deviation from normal value
when stored at 4°C. The stability of metolazone in plasma was determined by measuring concentration change in quality control
samples over time. Stability was tested by subjecting the
quality controls to three freeze-thaw cycles and compared with
freshly prepared quality control samples. The mean concentration
of metolazone in quality control samples did not change
significantly within the time period under the indicated storage
conditions. Long-term stability studies results conclude that
metolazone is stable in plasma matrix at least for 30 days when
stored at -20 ± 2°C. |
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| Pharmacokinetic evaluation |
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| The Pharmacokinetic evaluation of Cmax, Tmax, Half Life, K
elimination, AUC0–t, and AUC0–∞ these parameters were calculated.
The Reference and Test formulation was compared and
this result is presented in Table 1. |
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|
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| Conclusion |
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| SPE of metolazone from plasma was found to be more precise
than the other extraction methods. The current method guarantees
a high precision, accuracy, recovery and a relatively short
analysis time and will be a useful tool in the pharmacokinetic
study of metolazone in humans. |
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| References |
| |
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- www.Free wikipedia.com.
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