6.1 Drug profile 6.1.1 Olmesartanshodhganga.inflibnet.ac.in/bitstream/10603/12765/14/14...6.1.1...
Transcript of 6.1 Drug profile 6.1.1 Olmesartanshodhganga.inflibnet.ac.in/bitstream/10603/12765/14/14...6.1.1...
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6.1 Drug profile
6.1.1 Olmesartan
Olmesartan is indicated for the treatment of hypertension. It may be used
alone or in combination with other antihypertensive agents [1]. It is used for the
treatment of patients with high blood pressure according to the drug label [2].
Olmesartan is in a class of medications called angiotensin II receptor antagonists.
Fig: 6.a. Structure of Olmesartan
IUPAC name : 4-(2-hydroxypropan-2-yl)-2-propyl-1-({4-[2-
(1H-1, 2,3,4-tetrazol-5-yl) phenyl] phenyl}
methyl)-1H-imidazole-5-carboxylic acid
Molecular Formula : C29H30N6O6
Molecular mass : 558.585 g/mol
Drug Bank accession number : DB00275 (APRD00223)
CAS number : 144689-63-4
Half life : 13 hrs
Therapeutic category : Anti hypertensive drug
Solubility : Soluble in Methanol, Slightly in Water
Route : Oral
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Mechanism of action:
It works by blocking the action of certain natural substances that
tighten the blood vessels, allowing the blood to flow more smoothly and the heart to
pump more efficiently. Angiotensin II is formed from angiotensin I in a reaction
catalyzed by angiotensin converting enzyme. Angiotensin II is the principle presser
agent of the renin-angiotensin system, with effects that include vasoconstriction,
stimulation of synthesis and release of aldosterone, cardiac stimulation and renal
reabsorption of sodium. Olmesartan blocks the vasoconstrictor effects of angiotensin
II by selectively blocking the binding of angiotensin II to the AT1 receptor in vascular
smooth muscle. Its action is, therefore, independent of the pathways for angiotensin II
synthesis. Olmesartan has more than a 12,500-fold greater affinity for the
AT1 receptor than for the AT2 receptor.
Adverse effects:
General side effects of Olmesartan are hives, difficulty breathing, swelling of
your face, lips, tongue/ throat. Several severe side effects are feeling like might pass
out, urinating less than usual or not at all, chest pain, fast heart rate, swelling in hands,
serious side effects of Olmesartan may include: dizziness, joint or muscle pain, back
pain, stomach pain, nausea, diarrhea, mild itching or skin rash, or weakness [3].
6.1.2 Hydrochlorothiazide:
Hydrochlorothiazide is a prototype drug of thiazide diuretics [4]. .it is
antihypertensive agent it increases the urination and reduces the amount of water and
sodium retained by the body. The drug is in the class of benzothiazide.
Hydrochlorothiazide is a 'water pill’ used to treat high blood pressure and fluid
retention caused by various conditions, including heart disease [5]. It causes the
kidneys to get rid of unneeded water and salt from the body into the urine. This
reduces the volume of the blood, decreasing blood return to the heart and thus cardiac
output and, by other mechanisms, is believed to lower peripheral vascular resistance.
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Fig: 6.b. Structure of Hydrochlorothiazide
IUPAC name : 6-chloro-1,1-dioxo-3,4-dihydro-2H-1$l^{6},2,4-
benzothiadiazine-7-sulfonamide
Molecular formula : C7H8ClN3O4S2
Molecular mass : 297.74 g/mol
Drug Bank accession number : DB00999 (APRD00092)
CAS number : 58-93-5
Half life : 5.6 – 14.8 hrs
Therapeutic category : Anti hypertensive drug
Solubility : Soluble in Methanol, Slightly in Water
Route : Oral
Excretion : Primarily excreted unchanged in urine
Mechanism of action:
Hydrochlorothiazide belongs to the thiazide class of diuretics. It reduces blood
volume by acting on the kidneys to reduce sodium reabsorption in the distal
convoluted tubule. The major site of action is in the nephron appears on an electro
neutral Na+, Cl- co-transporter by competing the chloride site on the transporter [6.7]. It
is also used to treat the hypertension, congestive heart failure, symptomatic edema,
diabetes insipidus, renal tubular acidosis, and the prevention of kidney stones,
hypercalciuria, Dent's disease and Méniere's disease, osteoporosis [8, 9].
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Adverse effects:
The drug dosage is increases; the following serious side effects occurs they are,
kidney disease, liver disease, glaucoma, asthma, allergies, diabetes, an allergy to sulfa
drugs, penicillin. The drug hydrochlorothiazide is more toxic in nature. The most
common side effects are muscle weakness, dizziness, cramps, thirst, stomach pain,
nausea, vomiting, diarrhea, loss of appetite, headache, hair loss, sore throat with fever,
unusual bleeding or bruising, severe skin rash with peeling skin, difficulty breathing
or swallowing[10].
The list of available brand names, label claim details and Manufacturer
company names of Olmesartan, Hydrochlorothiazide are shown in the table 6.1.
S.NO Brand Name Available form Label claim
In mg
Manufacturer
Olmesartan formulations:
1 OLMAT Tablet 20, 40 Micro Carsyon
2 OLMEZEST Tablet 10, 20 Sun
3 OLMY Tablet 10, 20, 40 Zydus
Hydrochlorothiazide formulations:
1 AQUAZIDE Tablet 12.5, 25mg SUN
2 BPZIDE Tablet 12.5, 25mg Stadmed
3 HYDRIDE Tablet 12.5, 25mg East west
4 HYZIDE Tablet 12.5, 25mg Zydus
Olmesartan and Hydrochlorothiazide combined formulations:
1 OLMAT-H Tablet OL-20, H-12.5 Micro carsyon
2 OLMETOR-H Tablet OL-20, H-12.5 Torrent
3 OLMY-H Tablet OL-40, H-12.5 Zydus
Table: 6.1: Formulations of Olmesartan and Hydrochlorothiazide
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6.2 Review of Literature
Chaitanya prasad MK et al [11] described a Reversed phase high performance
liquid chromatographic method (RP-HPLC) and validated for the estimation of
Olmesartan medoxomil in bulk and formulation. Selected mobile phase was a
combination of phosphate buffer with pH adjusted at 2.8 and acetonitrile (35:65%
v/v) and wavelength selected was 250 nm. Retention time of Olmesartan medoxomil
was 2.591 min. Linearity of the method was found to be 50-150 µg/ml, with the
regression coefficient of 0.9993. Quantification was done by calculating area of the
peak and the limit of detection and limit of quantitation were 0.02µg/ml and
0.09µg/ml, respectively. There was no significant difference in the intraday and inter
day analysis of Olmesartan medoxomil determined for three different concentrations
using this method. This method can be applied for the determination of Olmesartan
medoxomil in quality control of formulation without interference of the excipients.
Sharma RN et al [12] developed and validated a simple, sensitive and precise
RP-HPLC-DAD method for the determination of olmesartan medoxomil (AT-II
receptor blocker) in the presence of its degradation products. Olmesartan medoxomil
and all the degradation products were resolved on a C -18 column with the mobile
phase composed of methanol, acetonitrile and water (60:15:25, V/V/V, pH 3.5 by
ortho phosphoric acid) at 260 nm using a photodiode array detector. The method was
linear over the concentration range of 1-18 µg/ml and precise with RSD < 1 % in
intraday and interday study. Excellent recoveries of 99.3 ± 0.9 to 100.8 ± 1.2% proved
the accuracy of the method. Developed method was specific, as indicated by
chromatographic resolution > 2.0 for each peak and sensitive with LOD 0.03 µg/ml
and LOQ 0.1 µg/ml. The method was used to study the drug degradation behavior
under forced conditions. Four degradation products (DP-I, II, III, IV) were formed
during the degradation study in 0.1 mol/lit Hcl solution, whereas only DP-I, II and III
were formed in water, 0.01 mol/lit NaoH solution and 3% H2O2 solution. No
significant thermal or photolytic degradation was observed in solid drug. The method
was applied successfully for the assay of Olmesartan medoxomil in the tablet dosage
form.
Kumanan Raghunathan et al [13] developed an accurate, precise, specific, and
reproducible and stability indicating HPLC method for the estimation of Olmesartan
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medoxomil (OLM) in presence of its degradation products and related impurities for
assessment of purity of bulk drug and stability of its dosage forms. The
chromatographic separation was achieved on a column Luna C18 (Alkyl reversed
phases) using acetonitrile: 0.05M KH2PO4 (50: 50 v/v), pH-4.5 mobile phase, flow
rate was 1.0 ml/ min. The method was validated according to the regulatory
guidelines with respect to precision, accuracy, linearity and limit of detection (LOD)
and limit of quantification (LOQ). Results of estimation of OLM in tablet formulation
were accurate and precise with standard deviation < 2. All the validation parameters
were within acceptance range.
B. Raja et al [14] developed a simple reversed phase HPLC method for the
simultaneous determination of Olmesartan medoxomil in combination with
hydrochlorothiazide. The method was based on reversed phase liquid chromatography
using axterra symmetry C18 column (150 × 4.6 mm, 5μ) with UV detection at 230
nm. The mobile phase consisting of acetonitrile and potassium dihydrogen phosphate
buffer adjusted to pH 2.5 in a ratio of (45:55, v/v) and at a flow rate of 0.7 ml/min.
The method was linear over the concentration range for Olmesartan medoxomil 20-60
μg/ml and for hydrochlorothiazide 20-60 μg/ml. The recoveries of Olmesartan
medoxomil and hydrochlorothiazide were found to be in the range of 98.0-102.0%
and 98.0-102.0% respectively. The method was validated and was successfully
employed for the analysis of pharmaceutical formulations containing Olmesartan
medoxomil and hydrochlorothiazide in combined tablet dosage form.
D. J. Kalena et al [15] described a simple, precise, rapid, efficient and
reproducible reverse Phase high performance liquid chromatography (RP-HPLC)
method for the simultaneous estimation of AT and OLM present in its tablet dosage
forms. Chromatographic separations were carried out isocratically at 30°C ± 0.5°C on
a Kromasil C18 Column (5 μm, 250mm x 4.60mm) with a mobile phase composed of
Methanol: Acetonitrile: Water (pH 3.65) in the ratio of 50:27:23 % v/v at a flow
rate of 1.0 ml/min. Detection was carried out using a UV detector at 260 nm. The
retention times for AT and OLM were 5.3 ± 0.5 min and 3.4 ± 0.5 min respectively.
The linearity range for AT and OLM were found to be 10-60 μg/ml and 20-120μg/ml
with correlation coefficient of 0.996 and 0.999 respectively. The %recovery of the
proposed method was found in the range of 98.36-100.91 for AT and 99.27-100.99
for OLM. The relative standard deviations for three replicate measurements in three
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concentrations of standard solution were always less than 2%. The results of the study
showed that the proposed RP- HPLC method was simple, rapid, precise and accurate,
which may be useful for the routine estimation of AT and OM in bulk drug and in its
pharmaceutical dosage form.
Bahiamoussa et al [16] described reversed phase high performance liquid
chromatography (RP-HPLC) and high performance thin layer chromatography
(HPTLC), densitometry methods as a stability indicating assays of olmesartan
medoxomil in presence of its acid or alkaline induced degradation products.
Olmesartan medoxomil and its degradation products were analyzed by HPLC
equipped with UV-Variable wave length detector at 257 nm where quantitation was
achieved by isocratic elution on Agilent, Exclipse XDB- C18 column with mobile
phase composed of acetonitrile: methanol: water: glacial acetic acid (40:35:25:0.1
v/v/v/v) at flow rate 1ml/min. HPTLC was performed on aluminum packed Nano
silica gel 60 F254TLC plates as stationary phase with significant difference in Rƒ
values between olmesartan medoxomil and its degradates using chloroform:
methanol: formic acid (8:1.5:0.5 v/v/v) as mobile phase. Densitometric evaluation of
intact drug was carried out at 260 nm. The calibration curve of olmesartan medoxomil
in bulk form was linear from 0.5- 10 µg/ ml and 0.05- 1 mg/ml with mean percentage
accuracy 99.97 ± 1.085 % and 100.35 ± 1.060 % for HPLC and HPTLC methods,
respectively. The two proposed methods were successfully applied for the
determination of olmesartan medoxomil in drug substance and in drug product.
Methods validation was tested for linearity; accuracy; precision; selectivity and
robustness, according to USP guidelines.
Md. Arif Hossen et al [17] developed a simple, sensitive and specific liquid
chromatography (RP-HPLC) method and validated for the quantification of
hydrochlorothiazide and losartan potassium in tablet dosage form. A shim-pack CLC-
ODS column (250 mm X 4.6 mm, 5µ and a mobile phase constituting 0.025 M
phosphoric acid solution: acetonitrile (60:40 v/v, pH 3.0 adjusted with 80%
phosphoric acid) were used. The flow rate was 1.5 ml/min and detection was carried
by using ultraviolet (UV) detector at a wavelength of 254 nm. The retention times of
hydrochlorothiazide and losartan potassium were 3.748 min and 8.790 min,
respectively. The peaks of hydrochlorothiazide and losartan potassium were well
separated (resolution 22.17). The calibration curves were linear over the concentration
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range of 80% to 120% (r2 > 0.999 for both the drugs). The proposed method was
accurate with 100.165% recovery for hydrochlorothiazide and 100.422% recovery for
losartan potassium and precise (% RSD < 0.5). The proposed method was
successfully applied for the estimation of hydrochlorothiazide and losartan potassium
in market products (three brands) and potency was found within limit. Therefore, this
method can be a convenient and efficient option for the analysis of
hydrochlorothiazide and losartan potassium in tablet dosage form.
Kullai Reddy Ulavapally et al [18] developed a simple and accurate RP-HPLC
method for the simultaneous estimation of Hydrochlorothiazide, Amlodipine besylate
and Valsartan by using C18 column 150 x 4.6 mm, 5μm with a simple gradient
elution(0-4min, sol-A:80-55; 4-8min- sol-A:55-40; 8-11min- sol-A:40-30; 11-13min-
sol-A:30-80 and 13-16min- sol-A:80-80). Mobile phase comprising of sol-A (pH
3.00±0.05 of 0.01M Potassium dihydrogen phosphate) and sol-B (Acetonitrile).
Flow rate was 1.00 ml/min and the detection was monitored out by UV detector at
237nm. The retention times for Hydrochlorothiazide, Amlodipine besylate and
Valsartan were found 4.5, 6.0 and 10.6 minutes. The proposed method has permitted
the quantification of Hydrochlorothiazide, Amlodipine besylate and Valsartan over
linearity in the range of 5-75 µg/ml and applicable for bulk and all type of
pharmaceutical dosage forms.
S. S. Qutab et al [19] described a simple, sensitive, and inexpensive high-
performance liquid-chromatographic method for simultaneous determination of
hydrochlorothiazide and candesartan cilexetil in pharmaceutical formulations.
Chromatographic separation was achieved on a Phenyl-2 column with a 25:75:0.2
mixture of 0.02 M potassium dihydrogen phosphate, methanol, and triethylamine,
final pH 6.0 ± 0.1, as mobile phase. Detection was at 271 nm. Response was a linear
function of concentration in the range 5–45 µg/ml for hydrochlorothiazide and 12–56
µg/ml for candesartan cilexetil; the correlation coefficients were 0.9993 and 0.9991,
respectively. Total elution time for the two components was less than 5 min.
Meyyanathan sn, rajan s et al [20] developed a simple, selective, rapid, precise
and economical reverse phase high pressure liquid chromatographic method for the
simultaneous estimation of nebivolol and hydrochlorthiazide from pharmaceutical
formulation. Phenomenexgeminic (18) (25 cm×4.6 mm i.d., 5 μ) column with a
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mobile phase consisting of acetonitrile: 50mm ammonium acetate (adjusted to pH 3.5
using orthophosphoric acid) (70:30 v/v) at a flow rate of 1.0 ml/min was used.
Detection was carried out at 254 nm. Probenecid was used as an internal standard.
The retention times of probenecid, nebivolol and hydrochlorthiazide were 13.05, 3.32
and 4.25 min, respectively. The developed method was validated in terms of accuracy,
precision, linearity, limit of detection, limit of quantification and solution stability.
The proposed method can be used for the estimation of these drugs in combined
dosage forms.
Gandhimathi M et al [21] described a simple, precise and rapid HPLC method
and validated for the estimation of quinapril and hydrochlorothiazide simultaneously
in combined dosage form. The mobile phase used was a mixture of 0.1% v/v
triethylamine (pH 3.5), containing 1 mm of hexane sulphonic acid: acetonitrile
(30:70% v/v). The detection of quinapril and hydrochlorothiazide was carried out on
photo diode array detector at 220 nm. Results of the analysis were validated
statistically and by recovery studies. The proposed method can be successfully used to
determine the drug contents of marketed formulation.
Gong Q et al [22] described a HPLC method on C18 column using methanol-
0.1% phosphoric acid (20: 80) as mobile phase, and the detection wave length was
327 nm, the flow rate was 1.0 ml/min and the temperature of column was 400C.In the
HPLC method, the calibration curve for chlorogenic acid, hydrochlorothiazide were
linear in the range of 0.049 6-0.496 (r2 = 0.999 5) and 1.002-10.02 (r2 = 0.999 8). The
average recovery for chlorogenic acid, hydrochlorothiazide was 101.0% and 100.1%.
RSD were 2.0% and 1.4% (n=9), respectively. The method was convenient, precise
and reliable for determining the content of chlorogenic acid and hydrochlorothiazide.
Jain ps et al [23] developed and validated a simple, specific, accurate and
precise stability-indicating reversed-phase high-performance liquid chromatographic
method for simultaneous estimation of olmesartan medoxomile (olme), amlodipine
besylate (amlo) and hydrochlorothiazide (hctz) in tablet dosage form. The method was
developed using an RP C18 base deactivated silica column (250 × 4.6 mm, 5 µm)
with a mobile phase consisting of triethylamine (pH 3.0) adjusted with
orthophosphoric acid (a) and acetonitrile (b), with a timed gradient program of t/%b:
0/30, 7/70, 8/30, 10/30 with a flow rate of 1.4 ml/min. Ultraviolet detection was used
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at 236 nm. The retention times for olme, amlo and hctz were found to be 6.72, 4.28
and 2.30 min respectively. The proposed method was validated for precision,
accuracy, linearity, range, robustness, ruggedness and force degradation study. The
calibration curves of olme, amlo and hctz were linear over the range of 50-150, 12.5-
37.5 and 31-93 µg/ml, respectively. The method was found to be sensitive. The limits
of detection of olme, amlo and hctz were determined 0.19, 0.16 and 0.22 µg/ml and
limits of quantification of olme, amlo and hctz were determined 0.57, 0.49 and
0.66µg/ml, respectively. Forced degradation study was performed according to
international conference on harmonization guidelines.
Carlucci g et al [24] developed an HPLC method with dad detection and
validated for the simultaneous determination of zofenopril and hydrochlorothiazide in
tablets. The separation was carried out through a gradient elution using an agilent
lichrospher C18 column (250x4.0 mm id, 5 microm) and a mobile phase consisting of
(a) water-tfa (99.9:0.1 v/v) and (b) acetonitrile-tfa (99.1:0.1 v/v) delivered at a flow-
rate of 1.0 ml/min. 8-chlorotheophylline was used as internal standard. Calibration
curves were found to be linear for the two drugs over the concentration ranges of 5.0-
40 and 1.0-20 µg/ml for zofenopril and hydrochlorothiazide, respectively. Linearity,
precision, accuracy, specificity and robustness were determined in order to validate
the proposed method, which was further applied to the analysis of commercial tablets.
The proposed method was simple and rapid, and gives accurate and precise results.
Hplc-dad method for the simultaneous determination of zofenopril and
hydrochlorothiazide in oral pharmaceutical formulations.
Singh Brijesh et al [25] described a simple, sensitive and rapid reverse phase
HPLC method for the simultaneous analysis of metoprolol succinate and
hydrochlorothiazide in a solid dosage form. The drugs were analysed by a reverse
phase C18 column using 50mm di-sodium hydrogen phosphate: methanol: acetonitrile
in a ratio of 52.5:22.5:25.0 as mobile phase. The flow rate was 1 ml/min and the
compounds were detected by a UV-detector at 222 nm at a column temperature of 24
± 2 ºc. The method was statistically validated for linearity and accuracy. The
retention time and drug content of metoprolol succinate and hydrochlorothiazide were
5.38 min, 96.05 % and 3.04 min., 97.64 %, respectively. The study shows that the
developed method was simple and accurate and that it would be suitable for the
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simultaneous determination of metoprolol succinate and hydrochlorothiazide in
pharmaceutical formulations.
Psrchnp et al [26] developed a simple, fast and precise reverse phase, isocratic
HPLC method for the separation and quantification of telmisartan and
hydrochlorothiazide in pharmaceutical dosage form. The quantification was carried
out using prontosil C18-EPS 4.6x150mm, 3µm enhanced polar selectivity column and
mobile phase comprised of potassium dihydrogen phosphate buffer pH adjusted to 3.2
± 0.5 with orthophosphoric acid and acetonitrile in proportion of ratio 55:45 and
degassed under ultrasonication. The flow rate was 0.8ml/min and the effluent was
monitored at 271nm. The retention time of telmisartan and hydrochlorothiazide were
5.01±0.5 and 2.94±0.5 respectively. The method was validated in terms of linearity,
precision, accuracy, specificity, limit of detection and limit of quantification.
Linearity of telmisartan and hydrochlorothiazide were in the range of 15.01 to
75.05µg/ml and 5.02 to 25.10µg/ml respectively. The percentage recoveries of both
the drugs were 100.8% and 99.5% for telmisartan and hydrochlorothiazide
respectively from the tablet formulation. The proposed method was suitable for
simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical
dosage form.
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6.3. Material and Methods
6.3.1. Instrumentation:
To develop a High Pressure Liquid Chromatographic method for quantitative
estimation of Olmesartan and Hydrochlorothiazide, an isocratic PEAK HPLC
instrument with Chromosil C18 column (250 mm x 4.6 mm, 5μ) was used. The
instrument was equipped with, Rheodyne manual sample injector with a 20 μl loop
for the injection of sample. PEAK LC software was used. UV 2301
Spectrophotometer was used to determine the wavelength of maximum absorbance.
Standard and sample drugs were weighed by using Denver electronic analytical
balance (SI-234) and pH of the mobile phase was adjusted by using Systronics digital
pH meter.
6.3.2 Chemicals and Solvents:
The drug samples, Olmesartan and Hydrochlorothiazide working standards were
obtained as gift sample by Ranbaxy Pvt. Ltd, Hyderabad, AP, India. The
pharmaceutical formulation was procured from local market. Methanol, Acetonitrile
TEA and water used were HPLC grade and were purchased from Merck Specialties
Private Limited, Mumbai, India. Orthophosphoric acid and remaining buffer solutions
used were AR Grade and purchased from Merck Specialties Private Limited,
Mumbai, India.
6.3.3 Preparation of standard stock solution:
Olmesartan and Hydrochlorothiazide (1mg/ml) standard stock solutions were
prepared using methanol as a solvent. Aliquots of mixed standard solutions of
Olmesartan and Hydrochlorothiazide were diluted in mobile phase to get a final
concentration of 40-100µg/ml.
6.3.4 Preparation of sample solution:
Pharmaceutical dosage form containing 8 mg of Olmesartan and 12.5 mg of
Hydrochlorothiazide was weighed and dissolved in 25 ml of methanol and sonicated
for 15 min. Using methanol the volume was made up to 50 ml and filtered through
0.45μ membrane filter. The final mixed sample solution corresponding to 40µg/ml of
Olmesartan and 60.25µg/ml of Hydrochlorothiazide was prepared.
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6.4. Method Development
For developing the method, a systematic study of the effect of various factors
was undertaken by varying one parameter at a time and keeping all other conditions
constant. Method development consists of selecting the appropriate wave length and
choice of stationary and mobile phases. The following studies were conducted for this
purpose.
6.4.1 Detection wavelength:
The spectrum of diluted solutions of the Olmesartan and Hydrochlorothiazide
in methanol was recorded. The absorption spectrum of Olmesartan and
Hydrochlorothiazide obtained by scanning the sample separately on UV
spectrophotometer in UV region (200-400nm) in spectrum mode showed that the drug
has maximum absorbance at 259nm. Analysis was carried out by adjusting the UV
detector of the HPLC system at 259nm.
6.4.2 Choice of stationary phase:
Preliminary development trials have performed with octadecyl columns with
different types, configurations and from different manufacturers. Finally the expected
separation and shapes of peak was succeeded Analytical column Inertsil ODS C-18
column with 250 x 4.6mm internal diameter and 5µm particle size.
6.4.3 Selection of the mobile phase:
Several systematic trials were performed to optimize the mobile phase.
Different solvents like Methanol, Water and Acetonitrile in different ratios and
different pH values of the mobile phase ratios by using different buffer solutions in
order to get sharp peak and base line separation of the components and without
interference of the excipients. Satisfactory peak symmetry, resolved and free from
tailing was obtained in mobile phase Methanol: Acetonitrile: TEA (46:50:04 V/V/) in
isocratic condition.
6.4.4 Selection of the mobile phase flow rate:
Flow rates of the mobile phase were changed from 0.5 – 1.2 ml/min for
optimum separation. A minimum flow rate as well as minimum run time gives the
maximum saving on the usage of solvents. It was found from the experiments that
1ml/min flow rate was ideal for the successful elution of the analyte.
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6.4.5. Optimization of HPLC Method:
All drugs were subjected to chromatographic analysis using mobile phases of
differing pH, flow rate using under mentioned chromatographic conditions. The
changes in the retention time of all drugs were noted as a function of changing mobile
phase, pH, flow rate, strength and selectivity. Initially the mobile phase taken for this
combination analysis was methanol: Acetonitrile: TEA (46:50:04 V/V/) and the flow
rate is 1.0ml/min, the wavelength was fixed at 259nm, 20 micro liters of the sample
was injected. The percentage RSD for precision and accuracy of the method was
found to be less than 2%. The method was validated as per the ICH guidelines.
API Concentration Olmesartan - 70µg/ml Hydrochlorothiazide- 70µg/ml
Mobile Phase Methanol: Acetonitrile: TEA(46:50:04 V/V/)
Wavelength 259nm
Column C18 Column
Pump mode Isocratic
Diluents Mobile phase
Injection volume 20µl
Mobile phase pH 6.8
Concentration 70µg/ml
Retention Time Olmesartan -1.5 Hydrochlorothiazide - 3.5
Run Time 10min
Area Olmesartan -317192 Hydrochlorothiazide - 350243
Theoretical Plates Olmesartan -6017 Hydrochlorothiazide - 32262
Tailing Factor Olmesartan -1.41 Hydrochlorothiazide - 1.39
Pump Pressure Ambient
Table 6.2: Optimized chromatographic conditions for Olmesartan and
Hydrochlorothiazide
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Figure 6.c: chromatogram of blank solution
Figure 6.d: standard chromatogram of Olmesartan and Hydrochlorothiazide
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Figure 6.e: standard chromatogram of Hydrochlorothiazide
Figure 6.f: standard chromatogram of Olmesartan
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6.5. Validation of the Proposed Method
The proposed method was validated as per ICH guidelines. The parameters studied
for validation were specificity, linearity, precision, accuracy, robustness, system
suitability, limit of detection and limit of quantification.
6.5.1 Linearity and range:
The mixed standard stock solution 100µg/ml of Olmesartan and 100µg/ml of
hydrochlorothiazide were further diluted to get Olmesartan and Hydrochlorothiazide
concentration in the range of 40 to 100 µg/ml. Linearity of the method was studied by
injecting six concentrations of the drug prepared in mobile phase into the LC system
keeping the injection volume constant. The peak areas were plotted against the
corresponding concentrations to obtain the calibration Curves indicates that the
response was linear over the concentration range studied with correlation coefficient
(r2) value, slope and intercept values were also shown in the given table 6.3.
S.NO Concentration
µg/ml
Olmesartan Hydrochlorothiazide
1 40 127605 146522
2 50 163545 180158
3 60 191473 215992
4 70 221862 246402
5 80 256579 276930
6 90 286424 313269
7 100 317192 350243
Calibration
range:
40-100
µg/ml
Slope:
Intercept:
C.C:
3172.797
1251.173
0.9998
3459.117
4318.556
0.9995
Table: 6.3 Linearity results of Olmesartan and hydrochlorothiazide
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Fig 6.g: Calibration curve of Olmesartan
\
Fig 6.h: Calibration curve of Hydrochlorothiazide
6.5.2 Precision:
The precision of the method was verified by repeatability and intermediate precision
studies. Repeatability was calculated from six replicate injections of freshly prepared
Olmesartan and Hydrochlorothiazide combined test solutions at a concentration range
of 70µg/ml on the same day. The experiment was repeated by assaying freshly
prepared solution at the same concentration additionally on two consecutive days to
determine intermediate precision. Peak areas were determined and % of RSD was
calculated.
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Intraday precision:
Injection No. Concentration Olmesartan Hydrochlorothiazide 1
70µg/ml
221884 246202 2 221015 246589 3 221328 246397 4 221492 246571 5 221030 246056 6 221563 246959
Result: SD:
Mean: % RSD
334.1578 221385.3
0.15
319.8998 246462.3
0.154
Table 6.4: Intraday precision results of Olmesartan and hydrochlorothiazide
Interday precision:
Injection No Concentration Olmesartan Hydrochlorothiazide
1
70µg/ml
221386 246176
2 221934 246372
3 221058 246087
4 221943 246905
5 221683 246712
6 221708 246534
Result:
SD: Mean:
% RSD:
342.1191 21618.7
0.154
314.4587 246464.3
0.127
Table 6.5: Interday precision results of Olmesartan and hydrochlorothiazide
6.5.3 Limit of Detection and Limit of Quantification:
To determine the Limit of Detection (LOD) sample was dissolved by using
mobile phase and injected until peak was disappeared. After 0.05µg/ml and 0.08
µg/ml dilution Peaks were not clearly observed, based on which 0.05µg/ml and
0.08µg/ml was considered as Limit of Detection for olmesartan and
hydrochlorothiazide, respectively. Limit of Quantification was 0.15µg/ml and
0.02µg/ml for Olmesartan and hydrochlorothiazide, respectively.
155
Parameter Olmesartan (µg/ml) Hydrochlorothiazide (µg/ml)
Limit of
Quantification 0.15
0.02
Limit of Detection 0.05 0.08
Table 6.6: LOD and LOQ results of Olmesartan and hydrochlorothiazide
6.5.4 Robustness of the method:
To evaluate robustness of a HPLC method, few parameters were deliberately
varied. The parameters included variation of flow rate, percentage of mobile phase
changes, and slight variations of the wave length changes. At standard concentration
was analyzed under these experimental conditions. It was observed that there were no
marked changes in chromatograms, which demonstrated that the developed method
was robust in nature. The robustness acceptance criteria set in the validation were the
same established on system suitability test. Robustness results were shown in table
6.7.
S.NO Parameter Change Olmesartan Hydrochlorothiazide
Area %Change Area %Change
1 standard No change 221386 0 246176 0
2
Mobile
phase -1
Mobile
phase -2
Methanol,
Acetonitrile,
TEA
(40:50:10)
(35:55:10)
221453
221467
0.03
0.04
246871
246951
0.28
0.31
4 pH 6.6
7.2
221867
221553
0.22
0.075
246374
246428
0.08
0.10
6 Wave
Length
261nm
257nm 221069
221047
0.143
-0.15 246985
246854
0.33
0.275
Table 6.7: Robustness results of Olmesartan and hydrochlorothiazide
156
6.5.6 Specificity:
By comparing the results obtained by injecting standard and blank specific of
the developed method was established. It was found that no peak was observed on
blank injection and standard solution showed two sharp and well resolved peaks,
indicates that the proposed method was specific. Specificity results were shown in
table 6.8.
Condition Olmesartan Hydrochlorothiazide
Standard Blank Standard Blank
Retention Time 1.5 No peak 3.5 No peak
Theoretical
plates
6017 No peak 32282 No peak
Tailing factor 1.41 No peak 1.39 No peak
Table 6.8: Specificity results of Olmesartan and hydrochlorothiazide
6.5.7 Accuracy:
The accuracy of the method was determined by standard addition method. A
known amount of standard drug was added to the fixed amount of pre-analyzed tablet
solution. Percent recovery was calculated by comparing the area before and after the
addition of the standard drug. Recovery test was performed at 3 different
concentrations i.e. 60µg/ml, 80µg/ml, 100µg/ml. The percent recovery was calculated
and results were presented in Table. Satisfactory recoveries ranging from 98.1 to
100.2 for Olmesartan and 98.5-101.3 for hydrochlorothiazide were obtained by the
proposed method. This indicates that the proposed method was accurate. Results of
the recovery were shown in table 6.9 for Olmesartan and 6.10 for
hydrochlorothiazide.
157
%
Recovery
Olmesartan
Target
(µg/ml)
Spiked
(µg/ml)
Total
(µg/ml)
Conc.,
Obtained
% of
Recovery
Conc.,
Obtained
50% 40 20 60 59.8 99.6 Average: 98.83
%RSD: 0.76 50% 40 20 60 59.3 98.8
50% 40 20 60 58.9 98.1
100% 40 40 80 79.2 99.0 Average: 99.52
%RSD: 0.45 100% 40 40 80 79.8 99.75
100% 40 40 80 79.9 99.8
150% 40 60 100 100.2 100.2 Average: 99.47
%RSD:0.71 150% 40 60 100 98.8 98.8
150% 40 60 100 99.4 99.4
Table 6.9: Accuracy results of Olmesartan
%
Recovery
Hydrochlorothiazide
Target
(µg/ml)
Spiked
(µg/ml)
Total
(µg/ml)
Conc.,
Obtained
% of
Recovery
Conc.,
Obtained
50% 40 20 60 59.5 99.1 Average: 98.97
%RSD: 0.42 50% 40 20 60 59.6 99.3
50% 40 20 60 59.1 98.5
100% 40 40 80 79.1 98.8 Average: 99.22
%RSD: 0.404 100% 40 40 80 79.4 99.25
100% 40 40 80 79.7 99.6
150% 40 60 100 99.7 99.7 Average: 99.90
%RSD:1.31 150% 40 60 100 101.3 101.3
150% 40 60 100 98.7 98.7
Table 6.10: Accuracy results of hydrochlorothiazide
158
6.5.8 Formulation:
From the prepared formulation solution, 20µl of the sample was injected into
HPLC system. Peak area response was compared with the standard values and the %
assay was calculated. Formulation estimation was carried out at market available
tablet of Olmesartan and Hydrochlorothiazide (CAMRI-4). Formulation result was
found to be 99.8% for Olmesartan and 99.93% for Hydrochlorothiazide. This
indicates that the proposed method can be successfully applicable for the estimation
of Olmesartan and Hydrochlorothiazide in formulations. Formulation chromatogram
was shown in figure 6.i. and table was shown in table 6.11.
Figure 6.i: Chromatogram of Olmesartan and hydrochlorothiazide
formulation
S.NO Formulation Available
form
Label
claim
Sample
concentration
Sample
estimated
%
Assay
1 Olmesartan Tablet 8mg 40 µg/ml 39.92µg/ml 99.8
2 Hydrochlorothiazide Tablet 12.5mg 60.25µg/ml 60.21µg/ml 99.93
Table 6.11: Formulation results of Olmesartan and hydrochlorothiazide
159
6.6 Results & Discussion
The results of validation studies on simultaneous estimation method developed
for Olmesartan and Hydrochlorothiazide in the current study involving Methanol:
Acetonitrile: Tri ethyl amine (TEA) (46:50:04 V/V/) were given below. Showed good
correlation coefficient (r2= 0.9998 for Olmesartan and r2 = 0.9995 for
Hydrochlorothiazide) in given concentration range 40-100 µg/ml Olmesartan and
hydrochlorothiazide. The mean values of the slope and intercept were 3172.797,
1251.173 for Olmesartan, and 3459.117, 4318.556 for Hydrochlorothiazide
respectively. The results of the repeatability and intermediate precision experiments
were above. The developed method was found to be precise as the % RSD values for
repeatability and intermediate precision studies were< 2 %, respectively as
recommended by ICH guidelines.
The recovery technique was performed to study the accuracy and
reproducibility of the proposed methods. For this, known quantities of the 40 µg/ml
solution were mixed with definite amounts of pre-analyzed formulations and the
mixtures were analyzed. The total amount of drug was determined by using the
proposed methods and the amount of added drug was calculated by the difference.
Satisfactory recoveries ranging from 98.1 to 100.2% for Olmesartan and 98.5-101.3%
for hydrochlorothiazide were obtained by the proposed method. This indicates that the
proposed method was accurate. Results of the recovery were shown in table 6.9 for
Olmesartan and 6.10 for hydrochlorothiazide. This showed that the recoveries of
Olmesartan and hydrochlorothiazide by the proposed methods were satisfactory.
Robustness of the method was confirmed by change in the optimized
chromatographic conditions and % change in each changed condition was calculated.
% change in all changed condition was found to be well acceptance criteria of less
than 2. This indicates that the proposed method is Robust.
The Limit of Detection (LOD) and Limit of Quantification (LOQ) of the
developed method were determined by injecting progressively low
concentrations of the standard solutions using the developed RP-HPLC method.
The LOD was the smallest concentration of the analyte that gives a measurable
response (signal-to-noise ratio of 3). The LOQ was the smallest concentration of
the analyte, which gives response that can be accurately quantified (signal-to-noise
160
ratio of 10). LOD and LOQ for Olmesartan and hydrochlorothiazide were found to
be 0.05 & 0.15 µg/ml, and 0.02 & 0.08µg/ml respectively. Formulation study was
carried out by using marketed formulation tablet of Olmesartan and
hydrochlorothiazide. Standard concentration was prepared and the area of the peak
response was used for the calculation of the % assay. It was found that up to 99%
accurately estimate Olmesartan and hydrochlorothiazide in pharmaceutical dosage
forms.
Thus the method developed in the present investigation is simple, sensitive,
accurate, rugged, robust, rapid and precise. Hence, the above said method can be
successfully applied for the estimation of Olmesartan and hydrochlorothiazide in
tablet dosage forms.
161
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