Simultaneous Spectrophotometric Estimation of the
Amlodipine Besylate and Hydrochlorothiazide in Pharmaceutical Preparations and
Biological Samples
Neela Manish Bhatia, Snehal
Jawaharlal Deshmane, Harinath Nivrutti More, Prafulla Balkrishna Choudhari
Bharati Vidyapeeth college of Pharmacy, Near
Chitranagari , Morewadi , Kolhapur, Maharashtra, 416013, India,
Author E-mail: neela.bhatia08@rediffmail.com
ABSTRACT
Two new simple, selective economical and reproducible
UV spectrophotometeric method simultaneous estimation of two component drug
mixture of Amlodipine besylate (AB)) and hydrochlorothiazide (HCT) in
pharmaceutical preparation, blood sample and urine has been developed. The first method depends on first derivative method at 245
and 238 nm for AB and HCT also in plasma and
urine respectively. The linearity over the concentration ranges 2.5-17.5µg/ml
for both the drugs AB and HCT. The second method is based on two wavelength method, which uses the difference of
absorbance value at 271 nm and 395 nm for estimation of Hydrochlorothiazide
(HCT) and absorbance at 363 nm for amlodipine besylate (AB). In formulation, plasma and urine Methanol: water (50:50v/v) was used as solvent, in which
Amlodipine besylate and Hydrochlorothiazide shows linearity in the range of
2.5-17.5 µg/ml for both the drugs respectively. Standard deviation was <1.5
in the assay of tablets. This method validated and the result was compared
statically. The %RSD value was satisfactorily low and recovery was close to
100% indicating reproducibility and accuracy of this method. Both the drug
obeys Beer’s law in the concentration range employed for analysis. Full
analytical validation of this method performed according to International
Conference on Harmonization Q2 (R1) and USFDA Bioanalytical methods guidelines.
These proposed methods were found to be simple, accurate, rapid, specific,
economic and advantageous in having lower limit of detection
KEYWORDS: simultaneous determination; derivative
spectrophotometry and two wavelength method, Amlodipine besylate,
Hydrochlorothiazide, plasma, urine.
INTRODUCTION:
Amlodipine besylate (AB),
2-[(2-aminoethoxy)-methyl]-4-(2-Chorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine
dicarboxylic acid 3-ethyl-5-methyl ester, benzenesulfonate, is a potent dihydro
pyridine calcium channel blocker. Many analytical methods like HPLC1,2, HPTLC3,
and micellar electro kinetic chromatograph4 have been reported. Hydrochlorothiazide
(HCT) 6-chloro -3,4 dihydro -2H,-1,2,4 Benzothiadiazine-7- Sulfonamide,1,1-dioxide, is a It is
used as diuretic. Combination therapy of AB and HCT
is recently used for the treatment of hypertension and diuretic. It is
official in USP5,BP6, IP7. Many analytical methods like HPLC8,9, HPTLC10,
and micellar electro kinetic chromatography11 were reported for
determination of HCT alone or in combination with other antihypertensive drugs.
However, no method is reported till date
for simultaneous determination of these two drugs from their binary mixture.
In this communication we report a new
UV-derivative spectrophotometric method and Two-wavelength method for
simultaneous determination of both the drugs in formulation, plasma and urine..
MATERIAL AND METHODS:
1.
PC based Jasco V-530 UV-Visible
double beam Spectrophotometer with 1 cm Matched pair quartz cells and spectral
bandwidth of 2 nm.
2.
Revolutionary laboratory
centrifuge, Make-Remi, Model- R-8C.
3.
Ultrasonicator, Make-Spectra lab,
Model- UCB 70D.
4.
Hot air oven, Make-Metalab
5.
Refrigerator, Make-LG, Model-
29500Ms
6.
AY 120 (max 1209 d = 0.1 mg)
Shimadzu Balance.
Reagents:
a) Amlodipine besylate salt,
b) Hydrochlorothiazide,
c) Excipent, Titanium dioxide
d) Spectroscopic grade methanol,
e) Glass distilled water,
f) Lead nitrate,
g) 0.1M acetic acid,
Pharmaceutical preparation:
A commercial Pharmaceutical preparation, (Amlokind-H
tablet Mankind pharma Ltd., New Delhi) was assayed. Its declared content was as
follows:
Amlodipine besylate 5mg
Hydrochlorothiazide 12.5mg
Derivative spectrophotometric method:
Standard Solution and calibration graphs for Derivative
spectrophotometric and two wavelength method.
Preparation
of sample solutions:
Standard stock solutions were freshly
prepared by dissolving amlodipine besylate and Hydrochlorothiazide in methanol: glass
distilled water (50:50v/v) to obtain a concentration of 1.0mg/ml for each
compound. Appropriate volume aliquots of the stock solution were transferred to
100ml calibration flasks. Accurate volumes were transferred into two sets of
100ml calibration flasks, and sonicated for 10 min and finally volume was made
up to the mark with the solvent. For selection of analytical wavelengths for
derivative method and two wavelength method (Method 1and 2), standard solution
of 100 μg/ ml of AB and HCTZ were scanned in the wavelength range of
200-400 nm. In first method wavelengths 245 nm for AB and 238nm for HCT in
derivative method. In second method two wavelength method AB direct
estimated at 363nm and HCT-Absorbance difference at 271nm and 395nm.
Preparation of standard calibration
curve:
The standard solutions (100 µg/ml) of both the drugs
were prepared in methanol: glass distilled water (50:50v/v). From the standard
drug solutions, eight working mixed standard solutions containing AB and HCTZ
with concentration of 0, 2.5, 5, 7.5, 10, 12.5, 15, and 17.5 μg/ml for AB
and 17.5, 15, 12.5, 10, 7.5, 5, 2.5 and 0 μg/ml of HCTZ were prepared.
The absorbance of above solutions was measured at the selected wavelengths and
the calibration curve for both drugs was constructed. Calibration curve for HCTZ
was plotted by recording absorbance difference at the two selected wavelengths
i. e. 271 nm and 396 nm, against the concentration of drug in eight mixed
standards. Similarly calibration curve for AB was plotted by recording
absorbance at 361 nm for two wavelength method and for derivative method 238nm
for HCT and 245nm for AB, against the concentration of drug in eight mixed
standards. Both the drugs obeyed Beer’s law in the concentration range of
2.5-17.5 μg/ml for AB and HCTZ.
Fig. 1: Overlain spectra of Amlodipine
besylate (AB) and Hydrochlorothiazide (HCTZ) in formulation.
Method 1: UV derivative
spectrophotometric method:
The absorption spectra thus obtained were
derivarized from first to forth order. First derivative spectra of the two
drugs were recorded and it was observed that AB and HCT showed zero absorbance
at 245nm and 238nm, respectively. As at the zero crossing point on the first
derivative spectra of on drug, the other drug showed significant absorbance,
these two wavelengths were employed for the estimation of AB and HCT
respectively without any interference. The calibration curves were plotted at
these two wavelengths (245 and 238 nm) using different concentrations of AB and
HCT against absorbances which showed linearity. The AB and HCT showed linearity
with absorbance in the range of 0-17.5µg/ml each at their selected wavelengths.
The derivative method was used for determination of intercept which was AB
-.0.0006 HCT 0.0023, the slope 0.0012and 0.0049 the coefficient of correlation
for AB and HCT 0.9995 and 0.9991 respectively in formulation , In plasma
intercept which was AB 0.0009 HCT 0.0004, the slope 0.0011and 0.0007 the
coefficient of correlation for AB and HCT 0.9995 and 0.9995 formulation but in
urine intercept which was AB 0.0005 HCT 0.0005, the slope 0.0023and 0.0008
the coefficient of correlation for AB and HCT 0.9995 and 0.9995 respectively.
The concentration of AB and HCT present in mixed standards were determined
against calibration curve of each drug in the quantitation mode. The overlain
spectra of AB and HCTZ was give in Fig.No1
Method 2: Two wavelength method:
The
utility of Two-wavelength data processing programme is to calculate the unknown
concentration of component of interest present in a mixture containing both a
component of interest and an unwanted interfering component by the mechanism of
the absorbance difference between two points on the mixture spectra is directly
proportional to the concentration of the components of interest, independent of
the interfering components. It involves the estimation of two components, each
time considering the second one to
be an
interfering one. An important criterion for this method is the presence of some
absorbance in the spectrum of interfering component, where the component of
interest has its λ max. With such flexibility, the interference
among the components can be dramatically reduced. The measuring wavelength can
be spaced either equally of arbitrarily, depending on the measuring purpose.
The number of measuring wavelengths ranges from the number of components
(applicable when pure component samples are used) to the number of standard
samples minus 1 (applicable when mixed standard samples are used).
Procedure for tablet analysis form
formulation:
Twenty tablet Amlokind-H (Mankind Pharma
Ltd, label claim Amlodipine besylate equivalent to 5 mg of Amlodipine base and
12.5 mg of HCT). Were accurately weighted and finely powdered. Quantities of
the powered tablet equivalent to 5 mg of Amlodipine base and 12.5mg
hydrochlorothiazide (one tablet)were weighted accurately, taken and dissolved
in methanol: water (50:50v/v) in 100ml volumetric flasks, After 30 min of
mechanically shaking, the solution was filtered through watmann filter paper
(No. 41). The residue was washed three times with 10ml of solvent then volume
was completed to 100ml with solvent. The filtrate was further diluted to get
final concentrations of both drugs in the linearity range. Absorbance was noted
at the selected wavelengths and concentrations were determined. The analysis
was carried out in set of nine. Replicate and mean % label claim was determined
and the results were statistically validated (Table 3and4).
Procedure for tablet analysis form urine:
Tablet estimation was done, Amlokind-H
(Mankind pharma Ltd, label claim Amlodipine besylate equivalent to 5 mg of
Amlodipine base and 12.5 mg of HCTZ). Twenty tablets were weighed and finely
powered amount equivalent to 5 mg of AB and 1.5mg HCTZ was taken in 10 ml
volumetric flask. This was then dissolved in 5 ml methanol: glass distilled
water (50:50) add 5 ml urine sample by sonication for about 5 min To this added
0.5 g lead nirate to precipated out the chloride present. The solution was
filtered and excess of lead present in the filtrate.
Filtered through Watmann filter paper
(No.41). The filtrate was further diluted to get final concentrations of both
drugs in the linearity range. Absorbance was noted at the selected wavelengths
and concentrations were determined. The analysis was carried out in set of
nine. Replicate and mean % label claim was determined and the results were
statistically validated (Table 1).
Procedure for tablet analysis for in
plasma:
Tablet estimation was done, Amlokind-H
(Mankind pharma Ltd, label claim Amlodipine besylate equivalent to 5 mg of
Amlodipine base and 12.5 mg of HCTZ). Twenty tablets were weighed and finely
powered amount equivalent to 5 mg of AB and 1.5mg HCTZ was taken in 10 ml
volumetric flask. This was then dissolved in 5 ml methanol by sonication for
about 5 min. The volume was made up to the mark by water and was
Filtered through Watmann filter paper
(No. 41). The filtrate was further diluted to get final concentrations of both
drugs in the linearity range. Absorbance was noted at the selected wavelengths
and concentrations were determined. The analysis was carried out in set of
nine. Replicate and mean % label claim was determined and the results were
statistically validated (Table 1).
Method validation:
As per the ICH and USFDA Guidelines the method validation parameters checked were
precision, accuracy, linearity limit of detection, limit of quantitation and
robustness.
Recovery studies:
To study the recovery of AB and HCT,
recovery experiments were performed using the method of addition. A fixed
amount of pure drug sample solution was added to one drug solution. The total
amount of the drug was then determined using the proposed methods and the
amount of the added drug was calculated by difference.
Fig. 2: Overlain (first order derivative)
spectra of Amlodipine besylate (AB) and Hydrochlorothiazide(HCTZ) in
formulation.
RESULTS:
In
the present work, two methods, namely, UV-derivative method, two wavelength
spectroscopy were developed for the simultaneous spectroscopic estimation of AB
and HCT in commercially available tablet dosage form. From the overlain spectra
of the two drugs, 271 nm and 395 nm for AB and 361nm for HCT were selected at a
concentration of 10 μg/ml and AB at a concentration of 10 μg/ml,
respectively (fig. 1).
The use of eight mixed standards and three
sampling wavelengths of 271 nm (λ max of HCT) 395 nm, and 363
nm (λ max of AB) gave optimum accuracy, precision, time,
economy, and sensitivity. The wavelengths used for UV derivative method is
238nm for (λ max HCT) and 245 nm (λ max of AB)
(fig. 2). The proposed procedures were successfully applied to the
determination of AB and HCT in the commercially available tablets dosage form,
and the results are presented in Tables 2 and 3 The values of standard
deviation for the validation parameters (linearity, accuracy, repeatability,
intermediate precision, and reproducibility) of AB and HCT in tablet
formulation were found to be between 0.08 and 0.68 for method A, 0.069 and
1.230 for method B,
A
critical evaluation of the proposed method was performed by the statistical
analysis of the experimental data. In order to demonstrate the validity and
applicability of the proposed methods, recovery studies were performed by
analyzing synthetic mixtures of AB and HCT with different composition ratios.
The percentage recoveries of AB and HCT from excipients.
The
percentage recoveries were found to be 99.5-101%, 98-103% in formulation, In
plasma 99.34%, 99,67% and in urine 98.10% , 98.21% for methods A, and For
method B was found to be 98.5-11%, 98-16% in formulation, In plasma 99.40%,
98,67% and in urine 99.10% , 98.89% respectively, and the results presented in
Tables 3 and 4 agreed with the labelled content. The relative standard
deviations were found to be within the limit, indicating good accuracy,
precision, and repeatability of the proposed methods.
The LOD and LOQ values for AB were found to
be least by the two wavelength method – 0.31 μg/ml and 0.91 μg/ml in
formulation in plasma0.34 μg/ml and 0.94 μg/ml and in urine 0.22
μg/ml and 0.75 μg/ml , respectively; and in derivative method 0.38
μg/ml and 0.89 μg/ml, respectively In derivative method LOD and LOQ
was found to be the two wavelength method – 0.22 μg/ml and 0.54 μg/ml
in formulation in plasma0.34 μg/ml and 0.94 μg/ml and in urine 0.22
μg/ml and 0.76 μg/ml , respectively; and in derivative method 0.27
μg/ml and 0.78 μg/ml, respectively With respect to the validation
results obtained from the above developed methods, two wavelength method is
more accurate, precise, and simple, with limited errors. The derivative methods
are accurate and precise but require mathematical calculations, which make the
work complicated. It can be said that the two methods described here are
simple, accurate and could be used for rapid and reliable determination of AB
and HCT in routine laboratory analysis.
CONCLUSION:
This is for the first time that UV-derivative
spectrophotometric and Two wavelength methods are being reported for the
determination of AB and HCT .The described methods give accurate and precise
results for determination of AB-HCT mixtures in tablets without prior
separation and are easily applied for routine analysis. The most striking
feature of the derivative method is its simplicity and rapidity,
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