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CHAPTER 3

STUDIES ON SELECTED DRUGS AND THEIR METHOD

DEVELOPMENT AND VALIDATION BY RP-HPLC IN SOLID

DOSAGE FORM

3.1. DRUG PROFILE:

LISINOPRIL29-30:

Dosage Form : Lisinopril Tablets (Zestril) – 2.5mg, 5 mg, 10 mg, 20mg, 30mg

and 40mg.

Chemical Name : N2-[(1S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline

Chemical Structure :

Figure 3.1.1: Structure of Lisinopril

Molecular Formula : C21H31N3O5

Molecular Weight : 405.488g/mol

Physical Form : White crystalline powder

Storage : Store at controlled room temperature, 20-25°C (68-77°F). Protect

from moisture, freezing and excessive heat. Dispense in a tight container.

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Solubility : soluble in water & methanol, partially insoluble in acetone & ethanol

Drug Category:

Antihypertensive drug

Angiotensin converting enzyme (ACE) inhibitor

Clinical Pharmacology:

Mechanism of Action:

Lisinopril inhibits angiotensin-converting enzyme (ACE) in human subjects and

animals. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin I to

the vasoconstrictor substance, angiotensin II. Angiotensin II also stimulates aldosterone

secretion by the adrenal cortex. The beneficial effects of lisinopril in hypertension and

heart failure appear to result primarily from suppression of the renin-angiotensin-

aldosterone system. Inhibition of ACE results in decreased plasma angiotensin II which

leads to decreased vasopressor activity and to decreased aldosterone secretion. The latter

decrease may result in a small increase of serum potassium. In hypertensive patients with

normal renal function treated with ZESTRIL alone for up to 24 weeks, the mean increase

in serum potassium was approximately 0.1 mEq/L; however, approximately 15% of

patients had increases greater than 0.5 mEq/L and approximately 6% had a decrease

greater than 0.5 mEq/L. In the same study, patients treated with ZESTRIL and

hydrochlorothiazide for up to 24 weeks had a mean decrease in serum potassium of 0.1

mEq/L; approximately 4% of patients had increases greater than 0.5 mEq/L and

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approximately 12% had a decrease greater than 0.5 mEq/L. Removal of angiotensin II

negative feedback on renin secretion leads to increased plasma renin activity.

Pharmacokinetics:

Absorption:

Oral administration of lisinopril, peak serum concentrations occur within about 7

hours, although there was a trend to a small delay in time taken to reach peak serum

concentrations in acute myocardial infarction patients. Based on urinary recovery, the

mean extent of absorption of lisinopril is approximately 25% with inter-patient variability

of 6-60% over the dose range studied (5-80mg). The absolute bioavailability is reduced

approximately 16% in patients with heart failure. Lisinopril absorption is not affected by

the presence of food.

Distribution:

Lisinopril does not appear to be bound to serum proteins other than to circulating

angiotensin converting enzyme (ACE). Studies in rats indicate that lisinopril crosses the

blood-brain barrier poorly.

Metabolism: Does not undergo metabolism, excreted unchanged in urine

Elimination:

Lisinopril does not undergo metabolism and is excreted entirely unchanged into

the urine. On multiple dosing lisinopril has an effective half-life of accumulation of 12.6

hours. The clearance of lisinopril in healthy subjects is approximately 50 ml/min.

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Declining serum concentrations exhibit a prolonged terminal phase, which does not

contribute to drug accumulation. This terminal phase probably represents saturable

binding to ACE and is not proportional to dose.

Indication:

Lisinopril is indicated for the treatment of hypertension, to lower blood pressure.

Lowering blood pressure lowers the risk of fatal and non-fatal cardiovascular events,

primarily strokes and myocardial infarctions. These benefits have been seen in controlled

trials of antihypertensive drugs from a wide variety of pharmacologic classes including

lisinopril.

Adverse Effects:

Signs of an allergic reaction: hives; severe stomach pain, difficult breathing;

swelling of your face, lips, tongue, or throat. feeling like you might pass out.

urinating more or less than usual, or not at all.

fever, chills, body aches, flu symptoms.

tired feeling, muscle weakness, and pounding or uneven heartbeats.

chest pain.

Swelling, rapid weight gain.

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3.2 DRUG PROFILE:

AMLODIPINE BESYLATE29-33:

Dosage Form: NORVASC (amlodipine besylate) Tablets are formulated as white tablets

equivalent to 2.5, 5, and 10 mg of amlodipine for oral administration.

Chemical Name: 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-

methyl-1,4-dihydropyridine-3,5-dicarboxylate.

Chemical Structure:

Figure 3.2.1: Structure of Amlodipine besylate

Molecular Formula : C20H25ClN2O5.C6H6O3S

Molecular Weight : 567.1g/mol.

Physical Form : It is white crystalline powder.

Solubility : It is slightly soluble in water and sparingly soluble in ethanol.

Storage : Store bottles at controlled room temperature, 59° to 86°F (15° to

30°C) and dispense in tight, light-resistant containers.

Drug Category : NORVASC is the besylate salt of amlodipine, a long-acting

calcium channel blocker and Antihypertensive.

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Clinical Pharmacology:

Mechanism of action:

Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or

slow-channel blocker) that inhibits the transmembrane influx of calcium ions into

vascular smooth muscle and cardiac muscle. Experimental data suggest that amlodipine

binds to both dihydropyridine and nondihydropyridine binding sites. The contractile

processes of cardiac muscle and vascular smooth muscle are dependent upon the

movement of extracellular calcium ions into these cells through specific ion channels.

Amlodipine inhibits calcium ion influx across cell membranes selectively, with a greater

effect on vascular smooth muscle cells than on cardiac muscle cells. Negative inotropic

effects can be detected in vitro but such effects have not been seen in intact animals at

therapeutic doses. Serum calcium concentration is not affected by amlodipine. Within the

physiologic pH range, amlodipine is an ionized compound (pKa=8.6), and its kinetic

interaction with the calcium channel receptor is characterized by a gradual rate of

association and dissociation with the receptor binding site, resulting in a gradual onset of

effect.

Amlodipine is a peripheral arterial vasodilator that acts directly on vascular

smooth muscle to cause a reduction in peripheral vascular resistance and reduction in

blood pressure.

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Pharmacokinetics:

Absorption

Peak plasma amlodipine concentrations attained 6–12 hours after oral

administration. Absolute bioavailability ranges from 64–90%. Duration OF

Antihypertensive effects of amlodipine persist for at least 24 hours after administration.

Food does not affect bioavailability of amlodipine besylate tablets.

Distribution

Not known whether amlodipine is distributed into milk. Plasma Protein Binding

of amlodipine: Approximately 93%.

Metabolism

Amlodipine is extensively metabolized to inactive metabolites in the liver.

Elimination

Amlodipine is excreted in urine as metabolites (60%) and unchanged drug (10%).

Half-life

Terminal elimination half-life of amlodipine is 30–50 hours.

Indication:

Amlodipine besylate is indicated for the treatment of hypertension, chronic stable

angina and confirmed or suspected vasospastic angina.

Side Effects:

Generally mild and reversible and include headache and swelling (edema) of the

lower extremities.

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3.3. LITERATURE REVIEW:

LISINOPRIL:

Alaa El-Gindy34 et al., (2001) have described a three methods for the

determination of lisinopril in the pharmaceutical tablets. The spectrophotometric method

depends on the reaction of the lisinopril with sodium hypochlorite and phenyl hydrazine

to form a condensation product measured at 362 nm. The spectrophotometric method was

extended to develop a stability indicating method. The spectrofluorimetric method

depends on reaction of the lisinopril with o-phthalaldehyde in the presence of 2-

mercaptoethanol in borate buffer pH 9.5. The fluorescence of the reaction product was

measured upon excitation at a maximum of 340 nm with emission wavelength at 455 nm.

The HPLC method depends on using Hypersil silica column with a mobile phase

consisting of methanol–water–triethylamine (50:50:0.1 v/v) and the pH was adjusted to

2.6 with 0.1 N perchloric acid. Quantitation was achieved with UV detection at 210 nm

based on peak area.

Alaa El-Gindy35 et al., (2001) have presented a different spectrophotometric and

HPTLC-densitometric methods for the simultaneous determination of lisinopril and

hydrochlorothiazide in pharmaceutical tablets. The spectrophotometric methods include

third derivative (3D) ultraviolet spectrophotometry with zero crossing measurement at

217.4 and 233.4 nm, second derivative of the ratio spectra with measurement at 214.3 and

228.0 nm; both classical least squares and principal component regression were applied

to the UV absorption and first derivative spectra of the mixture. The HPTLC method was

based on separation of both drugs followed by densitometric measurements of their spots

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at 210 and 275 nm for lisinopril and hydrochlorothiazide, respectively. The separation

was carried out on Merck HPTLC aluminum plates of silica gel 60 F254, using

chloroform–ethylacetate–acetic acid (10:3:2 by vol.) as mobile phase. The linear and

second order polynomial were used for the regression equation of lisinopril and

hydrochlorothiazide, respectively.

Politou J A36 et al., (2002) have described a spectrophotometric method for the

determination of lisinopril (LN) in single and multicomponent tablets also containing

hydrochlorothiazide (HCT), based on the derivatization reaction with 1-fluoro-2,4-

dinitrobenzene (FDNB, Sanger reagent). Aqueous solutions of LN (4.5–27.2×10−5 M)

react with FDNB (in acetonitrile) at pH 8.2 (borate buffer) in dark at 60 °C for 45 min.

After acidification with HCl to decolourize 2,4–dinitrophenolate (the alkaline hydrolysis

product of FDNB), the LN-derivative is measured at 356.5 or 405.5 nm (only at 405.5 nm

if HCT is present). The calibration curves are linear (r>0.996 at both wavelengths) with a

between days precision of slopes of 1.8 and 2.3% at 405.5 and 356.5 nm, respectively.

The quantification limit is 3.49×10−5 M (0.014 mg) at 405.5 nm and 5.69×10−5 M (0.023

mg) at 356.5 nm. The accuracy and precision of the method were evaluated with the

analysis of synthetic mixtures (Er%: 0.30–0.60 and 0.27–1.00 at 405.5 and 356.5 nm,

respectively; RSD%: 0.48–0.92 and 0.35–0.51 at 405.5 and 356.5 nm, correspondingly;

recovery%: 99.2–100.4 at 405.5 nm and 97.9–104.3 at 356.5 nm). Results obtained from

the analysis of commercial preparations with the proposed method are in good agreement

with those obtained with the official HPLC method (% relative difference 0.2–2.5%).

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Ersoy L37 et al., (2003) have described an accurate and precise

spectrofluorimetric method is presented for the determination of lisinopril based on the

formation of a derivative formed with 7-chloro-4-nitrobenzofurazan. The derivatization

reaction proceeds quantitatively at pH 8.5–9.0 and 60 °C in 70 min when the molar ratio

of reagent to the drug is 170. After the extraction with ethyl acetate the fluorescence

intensity of the derivative was measured at 528 nm with excitation at 465 nm. Calibration

graph is rectilinear over the range of 50–1000 ng/ml with detection and determination

limits of 20 and 50 ng/ml, respectively. The regression equation is If=0.198C−0.299

(r=0.9999).

Abdel Razak O38 et al., (2003) have described the reaction of enalapril maleate

and lisinopril with 2,4-dinitrofluorobenzene has been used to form colored products and

polarographically active derivatives. The different experimental conditions have been

optimized. The proposed methods have been validated and applied to the determination

of both drugs in their commercial tablets. The results have been statistically compared

with those obtained using the official HPLC methods.

Tsakalof A39 et al., (2003) have developed and validated a sensitive, specific,

precise and accurate method for lisinopril quantitative determination in human serum.

The method comprises lisinopril isolation from serum by means of solid-phase extraction

followed by its quantification by liquid chromatography–mass spectrometry.

Chromatographic separation was performed at 55 °C on Kromasil C18 5 μm 250×3.2 mm

HPLC column with mobile phase composed of 50 mM ammonium formate buffer (pH

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3)–acetonirile–methanol (72:7:21, v/v/v). A Finnigan AQA benchtop mass spectrometer

with a pneumatically assisted electrospray (ES) interface and a single quadrupole mass

filter was used to detect and quantify lisinopril in column effluent. Ion signals were

acquired by selected ion monitoring of the protonated lisinopril ion m/z=406.5 (M+1).

The detector response was linear with r>0.9993 in the investigated concentration range

6–150 ng/ml. The mean recovery of lisinopril from serum samples was 88%. The limit of

quantitation for lisinopril was 6 ng/ml with a signal-to-noise ratio at this concentration

level S/N=34.75±3.9 (n=4).

Ali A El-Emam40 et al., (2004) have described a Two sensitive, simple and

specific methods based on spectrophotometry and reversed-phase HPLC with

fluorimetric detection for the determination of lisinopril in dosage forms as well as in

spiked human plasma using solid phase extraction (SPE) procedures. Both methods are

based on the derivatization of lisinopril with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

(NBD-Cl) in borate buffer of pH 9 to yield a yellow, fluorescent product. The

spectrophotometric method depends on measuring the formed yellow color at 470 nm

after optimization of the reaction conditions. The HPLC method is based on measurement

of the derivatized product using fluorescence detection at 540 nm (excitation at 470 nm).

The separation of the derivatized drug, the excess reagent and the internal standard

(bumetanide) was performed on a reversed-phase ODS column using isocratic elution

with methanol–0.02 M sodium dihydrogen phosphate, pH 3.0 (55:45, v/v) at a flow rate

of 1.0 ml/min. The calibration graphs were linear over the concentration ranges 2–20 or

0.02–3.2 μg/ml of lisinopril with minimum detectability of 0.3 and 0.008 μg/ml (6.1×10−7

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and 1.7×10−8 M) for the spectrophotometric and the HPLC methods, respectively. The

proposed methods were applied without any interference from the tablet excipients for

the determination of lisinopril in dosage forms, either alone or co-formulated with

hydrochlorothiazide. Interference from endogenous amino acids has been overcomed by

using the solid phase extraction technique, the percentage recovery (n=6) was

101.6±3.35.

Sagirli O41 et al., (2004) have developed a selective, sensitive and precise HPLC

method with fluorimetric detection for the assay of lisinopril in human plasma and urine.

The clean up of the sample was carried out by solid-phase extraction, firstly with C18-

cartridge and secondly with a silica-cartridge. After a pre-column derivatization with

fluorescamine, the reaction mixture was chromatographed on C18-column with gradient

elution, using methanol and 0.02 M phosphate buffer (pH = 3.2). The fluorescamine–

lisinopril derivative was detected fluorimetrically by monitoring the emission at 477 nm,

with excitation at 383 nm. Linear quantitative response curve was generated over a

concentration range of 5–200 ng/ml and 25–1000 ng/ml for plasma and urine samples,

respectively. The mean recovery of lisinopril from plasma and urine was 63.41 and

74.08%, respectively. Intra-day and inter-day R.S.D. and R.M.E. values at three different

concentrations were assessed.

Gilberto De Nucci42 et al., (2004) have developed an analytical method based on

liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem

mass spectrometry detection for the determination of Lisinopril in human plasma using

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Enalaprilat as internal standard. The analyte and internal standard were extracted from

the plasma samples by solid-phase extraction using Waters HLB Oasis® SPE cartridges

and chromatographed on a C8 analytical column. The mobile phase consisted of

acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The

method had a chromatographic total run-time of 6.5 min and was linear within the range

2.00–200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem

mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and

accuracy, calculated from limit of quantification (LOQ) samples (n=8), were 8.9 and

98.9%, respectively.

Rahman N43 et al., (2005) have proposed a two simple, rapid and sensitive

spectrophotometric methods for the determination of lisinopril in pure form and

pharmaceutical formulations. The methods are based on the charge transfer complexation

reaction of the drug with 7, 7, 8, 8, tetracyanoquinodimethane (TCNQ) and p-chloranilic

acid (pCA) in polar media. The lisinopril–TCNQ and lisinopril–pCA charge transfer

complexes dissociate in acetone and methanol, respectively, and yield coloured TCNQ

and pCA radical anions which are measured spectrophotometrically at 743 and 525 nm.

Under optimised experimental conditions, Beer's law is obeyed in the concentration range

of 2–26 and 25–300 μg ml–1 with molar absorptivity of 1.432 × 104 and 1.192 ×

104 l mol–1 cm–1 for TCNQ and pCA methods, respectively. Results of analysis are

validated statistically.

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Christopher A. Beasley44 et al., (2005) have developed and validate a stability-

indicating HPLC method for lisinopril, lisinopril degradation product (DKP), methyl

paraben and propyl paraben in a lisinopril extemporaneous formulation. The method

developed in this report is selective for the components listed above, in the presence of

the complex and chromatographically rich matrix presented by the Bicitra® and Ora-

Sweet SF™ formulation diluents. The method was also shown to have adequate

sensitivity with a detection limit of 0.0075 μg/mL (0.03% of lisinopril method

concentration). The validation elements investigated showed that the method has

acceptable specificity, recovery, linearity, solution stability, and method precision.

Yannis L. Loukas45 et al., (2005) have developed a rapid liquid

chromatography/tandem mass spectrometry (LC/MS-MS) method for the determination

of lisinopril in human plasma. Lisinopril and the internal standard enalaprilat (IS) were

extracted from human plasma by semi-automated solid phase extraction (SPE) using a

96-well format extraction plate. Initially, a 1:1 plasma: acetonitrile (ACN) mixture was

prepared and vortexed to achieve protein precipitation. After centrifugation, an aliquot of

the supernatant water/ACN solvent mixture was evaporated. The residue was dissolved in

a certain volume of a reconstitution solution, which was passed through the extraction

plate and the eluent was analyzed by combined reversed phase liquid chromatography

tandem mass spectrometry with positive ion electrospray ionization using multiple

reactions monitoring (MRM). The method was proved to be sensitive and specific for

both drugs and its statistical evaluation revealed excellent linearity for the range of

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concentrations 2.0–200.0 ng mL−1 and very good accuracy, and inter- and intraday

precisions.

Rajeev C46 et al., (2007) have described a LC–UV scan of lisinopril revealed the

presence of an unknown impurity (~0.14%) at a relative retention time of 3.26 employing

phosphate buffer-acetonitrile as binary gradient system while LC–MS analysis with

binary gradient system comprising of a ammonia–ammonium acetate buffer (pH 5.0) and

acetonitrile indicated it to be C31H41N3O7. The impurity was isolated by preparative

HPLC utilizing a linear gradient of water and acetonitrile. The structural analysis of the

isolated product by 1H, 13C NMR, mass spectroscopy and FT-IR revealed it to be a 4-

phenyl butanoic acid derivative (CL) of lisinopril.

Medenica M47 et al., (2007) have discussed a chromatographic method for

simultaneous determination of hydrochlorothiazide (HCTZ), lisinopril (L), and their

impurities in pharma-ceuticals. Chlorothiazide (CTZ) and disulfonamide (DSA), as

potential impurities in hydrochlorothiazide, and diketopiperazine (DKP), as an impurity

of lisinopril, were analyzed. The chromatographic behaviour of these substances on

different columns was studied using mobile phases of different polarity. The optimum

separations were achieved by gradient elution on a 4.6 mm × 20 mm, 3.5 μm particle

size, C18

column. The mobile phase was a gradient prepared by mixing 7:93 (v/v)

acetonitrile–25 mM potassium dihydrogen phosphate, pH 5, and 50:50 (v/v) acetonitrile–

25 mM potassium dihydrogen phosphate pH 5 in different ratios. The flow rate was 1.0

mL min-1. UV detection was performed at 215 nm. Methylparaben was used as internal

113

standard. The method was validated for selectivity, linearity, precision, and accuracy. The

limits of detection and quantification were determined experimentally.

Naveed S48 et al., (2011) were designed for the simultaneous determination of

lisinopril in presence of pravastatin, atorvastatin, and rosuvastatin using RP-HPLC

method. A Purospher star C18 (5 μm, 25×0.46 cm) column was used with mobile phase

consisting of acetonitrile:water (60:40 V/V, pH 3.0) with flow rate of 1.0 mL·min−1 and

the quantitative evaluation was performed at 225 nm. The retention time of lisinopril was

2.0 min and for pravastatin, rosuvastatin and atorvastatin was found to be 3.1, 4.5 and 8.3

min respectively. Suitability of this method for the quantitative determination of the

drugs was proved by validation in accordance with the requirements laid down by

International Conference on Harmonization (ICH) guidelines.

Prashant Singh49 et al., (2012) have developed and validated a new isocratic

reversed-phase high performance liquid chromatographic (HPLC) method with diode-

array UV detection for the determination of lisinopril in pharmaceutical formulation. The

method validation of lisinopril was performed by using Nucleosil (125 x 4.0mm, 5μm) as

stationary phase with mobile phase consisting of buffer solution, isopropyl alcohol and

triethylamine (95:5:0.1) at a flow rate of 1.0 ml/min. The column temperature and

spectrometric detection were monitored at 50°C and 215 nm, respectively. The 20 μl of

sample was injected for the run time of 7 min. The statistical analysis of data showed that

the validated method is within limits in all respective parameters.

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Venkat T50 et al., (2012) have developed a simple, precise, specific and accurate

Reverse phase HPLC method for the determination of Hydrochlorothiazide and Lisinopril

in bulk and pharmaceutical dosage forms. Chromatography was performed on a Sunfire

column C-18 column. (4.6 x 150mm, 5μm) , with potassium dihydrogen phosphate buffer

(pH 7.51) and methanol in the ratio of 40:60 v/v as a mobile phase at a flow rate of 1.0

ml/ min. Detection was performed at 280nm. The retention time of Hydrochlorothiazide

and Lisinopril was found to be 2.397 min and5.148. By adoption of this procedure is

Hydrochlorothiazide and Lisinopril eluted completely. Linear calibration plots for

Hydrochlorothiazide and Lisinopril were obtained between 250-750μg/ml and 100-

300μg/ml. The method of analysis was used for quantification Hydrochlorothiazide and

Lisinopril for in pharmaceutical preparations with a coefficient of variation < 2%. Results

of analysis were validated statistically and by recovery studies. The method was validated

according to the ICH guidelines with respect to specificity, linearity, accuracy, precision,

ruggedness and robustness.

Ahmed E. M. Saeed51 et al., (2012) have developed and validated a simple,

accurate, precise and sensitive reverse phase high performance liquid chromatography

(RP-HPLC) method for the determination of lisinopril dehydrate. Drug was resolved on a

C18 column (waters spherisorb 25 cm × 4.6 mm, 5μm), utilizing mobile phase of tetra

butyl ammonium hydroxide solution (0.03M aqueous) pH adjusted to 6.5 with diluted

orthophospharic acid (10 % aqueous) and acetonitrile in a ratio of 4:1 respectively.

Mobile phase was delivered at the flow rate of 1.0 ml/min. Ultra violet detection was

carried out at 210 nm. Separation was completed within 3.49 minutes. Calibration curve

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was linear with correlation coefficient (r2) = 0.9991over a concentration range 10-

50μg/ml. Recovery was between 99.29 and 100.70 percentage. Method was found to be

reproducible with relative standard deviation (RSD) for intra and interday precision of <

1.0 over the said concentration range.

Sultana N52 et al., (2012) have developed and validated a High performance

liquid chromatographic method and applied for the simultaneous determination of

lisinopril and NSAIDs in bulk, pharmaceuticals formulations and human serum. A

Purospher star C18 (5 μm, 25 × 0.46 cm) column was used with mobile phase consisting

of methanol: water: acetonitrile (80:17.5:2.5 v/v, pH 3.0) and quantitative evaluation was

performed at 225 nm with a flow rate of 1.0 mL·min–1. The retention time of lisinopril

was 2.2 min while naproxen, flurbiprofen, diclofenac sodium and mefenamic acid were

found to be 4.0, 4.5, 5.0 and 6.7 min respectively. Suitability of this method for the

quantitative determination of the drugs was proved by validation in accordance with the

requirements laid down by International Conference on Harmonization (ICH) guidelines.

Lakshmana Rao A53 et al., (2012) have developed an accurate and precise HPLC

method for the determination of lisinopril. Separation of the drug was achieved on a

reverse phase C8 column using a mobile phase consisting of phosphate buffer and

methanol in the ratio of 35:65v/v. The flow rate was 0.8 mL/min and the detection

wavelength was 215 nm. The linearity was observed in the range of 20-60 μg/mL with a

correlation coefficient of 0.9992. The proposed method was validated for its linearity,

accuracy, precision and robustness.

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Wael Abu Dayyih54 et al., (2013) have developed and validated a simple,

economical, precise, accurate, and rapid HPLC method for assay determination of

captoprill, lisinopril and imidapril simultaneously in their raw material and tablet dosage

forms. The chromatographic condition was performed on a mixture of acetonitrile and

phosphate buffer (25:75 v/v) ratio. The detection of Prills drugs was carried out at 210

nm with a flow rate 1.0ml/min. The retention times for lisinopril, captopril and imidapril

were 3.6, 4.4, and 7.4 min respectively. Results of the analysis were validated

statistically, and by recovery studies. The proposed method was successfully employed

for the estimation of the drug contents in marketed formulations according to ICH

guidelines and found to be suitable for simultaneous determination of Prills.

Naveed S55 et al., (2013) have developed a high performance liquid

chromatography (HPLC)-UV method for the simultaneous determination of lisinopril,

enalapril, captopril and fosinopril in human plasma is proposed. Good separation of the

analytes was achieved by gradient RP-HPLC with the mobile phase composed as

acetonitrile: water (60:40 v/v) adjusted to pH 3.0 by ortho phosphoric acid. Lisinopril,

enalapril, captopril and fosinopril were eluted from a Purospher STAR RP-and Hypersil

ODS column in 1.8, 2.9, 3.2 and 5.4 min respectively. Good linear relationships were

observed for all of the analytes (R2 higher than 0.995). Intra and inter-day precision and

accuracy results were 98.0 to 102%. Application of the suggested procedure was

successfully applied to the determination of these compounds in active pharmaceutical

preparations, dosage formulations and human serum with high percentage of recovery,

good accuracy and precision (no interference of excepients).

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Farhan Ahmed Siddiqui56 et al., (2013) have developed and validated a simple

high performance liquid chromatographic method for the simultaneous determination of

metformin hydrochloride and angiotensin-converting enzyme inhibitors (captopril,

lisinopril, and enalapril) in bulk dosage form and application in pharmacokinetics studies.

The separation was achieved on a Purospher® Star RP-18 endcapped (250 mm x 4.6 mm

id) column as stationary phase using methanol-water 50:50 (v/v) as mobile phase,

adjusted to pH 3.1 with phosphoric acid. Effluent was monitored at a flow rate of 1

mLmin-1 at room temperature (25oC), detector was set at 218 nm. The method was

validated according to ICH guidelines. The linearity was studied over the concentration

range of 10–10000 ngmL-1 for metformin and 30–10000 ngmL-1 for captopril, lisinopril,

and enalapril, demonstrating good linearity with minimum r = 0.9964, respectively.

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3.4 LITERATURE REVIEW:

AMLODIPINE BESSYLATE:

Argekar A. P57 et al., (2000) have developed a new simple, precise, rapid and

selective high-performance thin-layer chromatographic (HPTLC) method for the

simultaneous determination of atenolol (ATL) and amlodipine (AMLO) in tablets, using

methylene chloride: methanol: ammonia solution (25% NH3) (8.8:1.3:0.1; v:v) as the

mobile phase and Merck HPTLC plates (0.2 mm thickness) precoated with 60F254 silica

gel on aluminium sheet as the stationary phase. Detection was carried out

densitometrically using a UV detector at 230 nm. The retention factors of ATL and

AMLO were 0.33 and 0.75, respectively. Calibration curves were linear in the range 10–

500 mg ml/1 for both. Assays of ATL and AMLO were 49.87 mg per tablet (relative

standard deviation (R.S.D.), 1.3%) and 4.90 mg per tablet (R.S.D., 1.38%) for brand I,

and 49.27 mg per tablet (R.S.D., 1.12%) and 4.98 mg per tablet (R.S.D., 1.42%) for

brand II, respectively. The percentage recoveries for ATL and AMLO for brands I and II

were 99.06 and 99.30%, and 99.27 and 99.15%, respectively.

Sedef Atmaca58 et al., (2001) have developed a sensitive and specific HPLC

method for the assay of amlodipine in human plasma. The assay involves derivatization

with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column

and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline

hydrochloride was used as an internal standard. The assay was linear over the

concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day

reproducibility and accuracy were less than 11.80% and 12.00%, respectively.

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Rahman N59 et al., (2001) have developed a spectrophotometric method for the

determination of amlodipine besylate in pure form and in pharmaceutical preparations.

The method is based on the reaction of the primary amino group of the drug with

ninhydrin in N,N′-dimethylformamide (DMF) medium producing a coloured complex

which absorbs maximally at 595 nm. Beer's law is obeyed in the concentration range of

10–60 μg ml−1 with RSD of 0.66% and molar absorptivity of 6.52×103 l mol−1 cm−1. All

variables were studied in order to optimize the reaction conditions. No interference was

observed from common pharmaceutical adjuvants. Statistical comparison of the results

with the reference method shows excellent agreement and indicates no significant

difference in accuracy and precision.

Ceccato A60 et al., (2002) have developed a sensitive method for the separation

and determination of amlodipine enantiomers in plasma based on solid-phase extraction

(SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid

chromatography (LC). The SPE technique is used to isolate the drug from the biological

matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to

mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned

with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A

1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed

with ammonium acetate buffer and secondly with a mixture of water and methanol

(65:35, v/v), while the final elution step is obtained by dispensing methanol containing

2.5% of ammonia. The eluate is then collected and evaporated to dryness before being

dissolved in the LC mobile phase and injected into the LC system. The stereo selective

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analysis of amlodipine is achieved on a Chiral AGP column containing α1-acid

glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate

buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass

spectrometry with an APCI interface in the positive-ion mode. The chromatographed

analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion

transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(−)-propranolol

used as internal standard (IS). The method was validated considering different

parameters, such as linearity, precision and accuracy. The limit of quantitation was found

to be 0.1 ng/ml for each amlodipine enantiomer.

Basavaiah K61 et al., (2003) have developed a simple, accurate, sensitive and

economical procedure for the estimation of amlodipine besylate and felodipine, both in

pure form and in formulations. The method is based on the reduction of iron(III) by the

studied drugs in acid medium and subsequent interaction of iron(II) with ferricyanide to

form Prussian blue. The product exhibits absorption maximum at 760 nm in both cases.

Beer's law is obeyed in the concentration ranges 5.0–15.0 and 1.5–5.0 μg/ml, for

amlodipine and felodipine, respectively. The molar absorptivities are 1.76×104 and

4.24×104 l/mol cm. The corresponding Sandell sensitivities are 23.18 and 9.06 ng/cm2.

The limits of detection as well as quantification are reported. Seven replicate analyses of

solutions containing three different concentrations of each drug were carried out and the

percent error and the RSD values have been reported. The results demonstrate that the

method is equally accurate and precise as the official methods as found from the t- and F-

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values. The reliability of the method was established by recovery studies using standard-

addition technique.

Ceccato A62 et al., (2003) have developed and validated a high performance

liquid chromatographic method for the determination of amlodipine residues in swab

samples in order to control a cleaning procedure. The swabbing procedure was optimized

in order to obtain a suitable recovery of amlodipine from stainless steel. A mean recovery

close to 90% was obtained when two swabs moistened with methanol were used. The

residual amlodipine was chromatographed at 25°C in the isocratic mode on a RP-18

stationary phase using a mobile phase consisting of acetonitrile, methanol and pH 3.0

triethylamine solution (15:35:50 v/v/v). UV detection was performed at 237 nm. The

method was shown to be selective and linear into the concentration range varying from

0.39 to 1.56 mg/ml. Accuracy and precision of the method were also studied. The limits

of detection and quantitation were evaluated to be 0.02 and 0.08 mg/ml, respectively. The

stability of amlodipine at different steps of the sampling procedure and the precision of

the swabbing procedure were also investigated.

Bahrami Gh63 et al., (2004) have described a fast, sensitive and specific high

performance liquid chromatographic method using fluorescence detection for analysis of

amlodipine in human serum. Amlodipine is extracted from serum by ethyl acetate and

involves precolumn derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and

reverse-phase chromatography on C18 column. The mobile phase was sodium phosphate

buffer (pH 2.5) containing 1 ml/l triethylamine and methanol at flow rate of 2.8 ml/min.

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Propranolol was used as internal standard. The standard curve was linear over the range

0.25–16 ng/ml of amlodipine in human serum. The within-day and between-day precision

studies showed good reproducibility with coefficients of variation less than 12% for all

the analytes. The limit of quantification was 0.25 ng/ml of serum. The method has been

applied to a bioequivalence study after administration of 10 mg amlodipine in 12 normal

subjects.

Zarghi A64 et al., (2005) have developed a rapid, simple and sensitive high-

performance liquid chromatography (HPLC) method for quantification of amlodipine in

plasma. The assay enables the measurement of amlodipine for therapeutic drug

monitoring with a minimum detectable limit of 0.2 ng ml−1. The method involves simple,

one-step extraction procedure and analytical recovery was about 97%. The separation

was performed on an analytical 125 × 4.6 mm i.d. Nucleosil C8 column. The wavelength

was set at 239 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen

phosphate buffer and acetonitrile (63:37, v/v) adjusted to pH 3.5 at a flow rate of

1.5 ml min–1. The calibration curve was linear over the concentration range 0.5–

16 ng ml−1. The coefficients of variation for inter-day and intra-day assay were found to

be less than 10%.

Murlidhar S S65 et al., (2005) have developed and validated a stability

indicating reversed-phase HPLC method for simultaneous estimation of amlodipine

(AM) present as amlodipine besylate (AB), and benazepril hydrochloride (BH) from their

combination product. The proposed RP-HPLC method utilizes a Zorbax SB C18, 5 μm,

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250mm×4.6mm i.d. column, mobile phase consisting of phosphate buffer and acetonitrile

in the proportion of 65:35 (v/v) with apparent pH adjusted to 7.0, and UV detection at

240 nm using a photodiode array detector. AB, BH, and their combination drug product

were exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the

stressed samples were analysed by the proposed method. Peak homogeneity data of AM

and BH peaks obtained using photodiode array detector, in the stressed sample

chromatograms, demonstrated the specificity of the method for their estimation in

presence of degradants. The described method was linear over a range of 6–14 μg/ml for

AM and 12–28 μg/ml for BH. The mean recoveries were 99.91 and 100.53% for AM and

BH, respectively. F-test and t-test at 95% confidence level were used to check the

intermediate precision data obtained under different experimental setups; the calculated

value was found to be less than critical value.

Mohammadi A66 et al., (2007) have developed and validated a simple, rapid,

precise and accurate isocratic reversed-phase stability-indicating HPLC method for the

simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial

tablets. The method has shown adequate separation for AM, AT from their associated

main impurities and their degradation products. Separation was achieved on a Perfectsil®

Target ODS-3, 5 μm, 250 mm × 4.6 mm i.d. column using a mobile phase consisting of

acetonitrile–0.025 M NaH2PO4 buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and

UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis

and heat to apply stress conditions. The linearity of the proposed method was investigated

in the range of 2–30 μg/ml (r = 0.9994) for AT and 1–20 μg/ml (r = 0.9993) for AM. The

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limits of detection were 0.65 μg/ml and 0.35 μg/ml for AT and AM, respectively. The

limits of quantitation were 2 μg/ml and 1 μg/ml for AT and AM, respectively.

Degradation products produced as a result of stress studies did not interfere with the

detection of AT and AM and the assay can thus be considered stability-indicating.

Famei Li67 et al., (2007) have developed a novel, specific and sensitive ultra

performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method

for the determination and pharmacokinetic study of amlodipine in human plasma. The

analysis was carried out on an ACQUITY UPLCTM BEH C18 column (50mm×2.1 mm,

i.d., 1.7μm) with gradient elution at a flow-rate of 0.35 ml/min. The mobile phase was

water and acetonitrile under gradient conditions (both containing 0.3% formic acid) and

nimodipine was used as the internal standard. Detection was performed on a triple-

quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via

Turbo ion spray ionization (ESI). Linear calibration curves were obtained over the

concentration range 0.15–16.0 ng/ml, with a lower limit of quantification of 0.15 ng/ml.

The intra- and inter-day precision (R.S.D.) values were below 15% and the accuracy

(R.E.) was −2.3% to 6.9% at all three QC levels.

Tapan Kumar Pal68 et al., (2008) have developed and validated a simple,

sensitive and specific liquid chromatography–tandem mass spectrometry method for

quantification of metoprolol succinate (MPS) and amlodipine besylate (AM) using

hydrochlorothiazide (HCTZ) as IS in human plasma. Both the drugs were extracted by

simple liquid–liquid extraction with chloroform. The chromatographic separation was

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performed on a reversed-phase peerless basic C18 column with a mobile phase of

methanol–water containing 0.5% formic acid (8:2, v/v). The protonated analyte was

quantitated in positive ionization by multiple reaction monitoring with a mass

spectrometer. The method was validated over the concentration range of 1–100 ng/ml for

MPS and 1–15 ng/ml AM in human plasma. The MRM transition of m/z 268.10–103.10,

m/z 409.10–334.20 and m/z 296.00–205.10 were used to measure MPS, AM and HCTZ

(IS), respectively.

Ramakrishna K69 et al., (2008) have describes a simple isocratic reverse phase

HPLC method for the determination of four genotoxic alkyl benzenesulfonates (ABSs)

viz. methyl, ethyl, n-propyl and isopropyl benzenesulfonates (MBS, EBS, NPBS and

IPBS) in amlodipine besylate (ADB). Good resolution between benzene sulfonic acid

(BSA), MBS, EBS, NPBS, IPBS and ADB was achieved with Inertsil ODS 3V

(150mm×4.6mm, 5μm) column using a 65:35 (v/v) mixture of 1% triethyl amine, pH

adjusted to 3.0 with ortho phosphoric acid and acetonitrile as mobile phase. The flow rate

was 1.0 ml/min and the elution was monitored at 220 nm. This method was validated as

per International Conference on Harmonization (ICH) guidelines and is able to quantitate

MBS, EBS, NPBS and IPBS at 21, 32, 35 and 28ppm levels, respectively with respect to

5 mg/ml of ADB. The method is linear in range of 75–180ppm of ABSs, which matches

the range of 50–120% of estimated permitted level (150 ppm) of ABSs. ABSs were not

present in the three studied pure and tablet batches of ADB.

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Vaijanath G. Dongre70 et al., (2008) have developed a simple, precise, specific

and accurate reverse phase HPLC method for the simultaneous determination of

metoprolol succinate (MS) and amlodipine besylate (AB) in tablet dosage form. The

chromatographic separation was achieved on Hypersil BDS cyano (250mm×4.6 mm,

5μm) column using PDA detector. The mobile phase consisting of buffer (aqueous

triethylamine pH 3) and acetonitrile in the ratio of 85:15 (v/v) at a flow rate of 1.0

mL/min was used. The method was validated according to the ICH guidelines with

respect to specificity, linearity, accuracy, precision and robustness.

Gengliang Yang71 et al., (2009) have developed an on-line solid-phase extraction

(SPE)-HPLC method for simultaneous screening of nicardipine and amlodipine in human

plasma. A short monolithic poly (glycidyl methacrylate-co-ethylene glycol

dimethacrylate) [p(GMA-EDMA)]-based weak cation-exchange (WCX) column was

prepared and employed as the selective extraction sorbent, which exhibited good

permeability and biocompatibility. During the on-line SPE protocol, high-abundance

proteins (human serum albumin, immunoglobulin G, immunoglobulin A and transferrin)

and most matrixes in plasma were fast removed while nicardipine and amlodipine were

effectively trapped on this monolithic column. Furthermore, the monolithic WCX sorbent

could be continuously reused more than 300 times without obvious changes in analytes

extraction and proteins cleanup. The proposed method was linear over a range of 0.5–

50.0 ng mL−1 for both analytes with a linear regression coefficient greater than 0.998, and

the limit of detection (LOD) for each analyte was 0.2 ng mL−1. Validation assays also

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demonstrated acceptable precision and adequate recovery for simultaneous quantitative

screening of nicardipine and amlodipine in human plasma.

Deepak Sharma72 et al., (2011) have developed an isocratic reversed phase high-

performance liquid chromatographic (HPLC) method with ultraviolet detection at 268 nm

for the determination of amlodipine besylate (ADB) and nebivolol hydrochloride in

dosage formulation. Good chromatographic separation was achieved by using a stainless

steel analytical column, the Lichrospher ODS RP-18 column (250 × 4 mm), particle size

5 μm. The system was operated at ambient temperature (25 ± 2 °C) using a mobile phase

consisting of acetonitrile (ACN) and a phosphate buffer (pH 3.0), mixed in a ratio of 40 :

60 at a flow rate of 0.8 ml/minute. The slope, intercept, and correlation coefficient were

found to be 8818.2, - 18159, and 0.9993 for amlodipine and 9048.7, 108595, and 0.9998

for nebivolol, respectively. The proposed method was validated for its specificity,

linearity, accuracy, and precision.

Hany W. Darwish73 et al., (2011) have developed a three simple, specific,

accurate and precise spectrophotometric methods manipulating ratio spectra for the

simultaneous determination of Amlodipine besylate (AM) and Atorvastatin calcium (AT)

in tablet dosage forms. The first method is first derivative of the ratio spectra (1DD), the

second is ratio subtraction and the third is the method of mean centering of ratio spectra.

The calibration curve is linear over the concentration range of 3–40 and 8–32 μg/ml for

AM and AT, respectively. These methods are tested by analyzing synthetic mixtures of

the above drugs and they are applied to commercial pharmaceutical preparation of the

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subjected drugs. Standard deviation is <1.5 in the assay of raw materials and tablets.

Methods are validated as per ICH guidelines and accuracy, precision, repeatability and

robustness are found to be within the acceptable limit.

Sushant K Shrivastava74 et al., (2012) have developed and validate a simple and

rapid isocratic reversed-phase high performance liquid chromatographic method (RP-

HPLC) for the simultaneous estimation of amlodipine and telmisartan in combined

dosage form. The chromatographic separation was achieved by using mobile phase

acetonitrile and 0.05M sodium dihydrogen phosphate buffer (60:40) adjusted to pH 6.0, a

C-18 column, perfectsil target ODS3 (150 mm × 4.6 mm i.d., 5 μm). The mobile phase

was pumped at a flow rate of 0.8 mL/min and the eluents were monitered at 254 nm.

Retention times were 4.0 min and 8.2 min for amlodipine and telmisartan respectively.

The method was validated in terms of accuracy, precision, linearity, range, specificity,

limit of detection and limit of quantitation. Linearity for amlodipine besylate and

telmisartan was established in the range of 5-30 and 10-60 μg/mL, respectively. The

recoveries for the two compounds were above 96%. This method was found to be

efficient, accurate, precise, specific and economic and is suitable for routine quality

control analyses.

Shah S.K75 et al., (2012) have developed a rapid, simple and sensitive high-

performance liquid chromatography (HPLC) method for quantification of olmesartan

medoxomil (OLM) and amlodipine besylate (AM) in plasma. The assay enables the

measurement of OLM and AM for therapeutic drug monitoring with a minimum

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detectable limit of 2 ng mL. The method involves a simple, one-step extraction procedure

and analytical recovery was above 50%. The Separation was performed on an analytical

250 X 4.6 mm Eurospher 100- 5 C18 column. The wavelength was set at239 nm. The

mobile phasewas a mixture of acetonitrile:0.05 M ammoniumacetate buffer: 0.1

mLtriethylamine at pH 6.8 was selected at a flow rate of 1.0 mL/min. The calibration

curve for the determination of OLM and AM in plasma was linear overthe ränge 2–2500

and 8–10,000 ng/mLAM and OLM. The coefficients of Variation for interday and

intraday assay were found to be < 15%.

Niveen A. Mohamed76 et al., (2012) have developed and validated a simple and

sensitive kinetic spectrophotometric method for determination of amlodipine besylate

(AML). The method was based on the condensation reaction of AML with 7-chloro-4-

nitro-2,1,3-benzoxadiazole in an alkaline buffer (pH 8.6) producing a highly colored

product. The color development was monitored spectro- phometrically at the maximum

absorption λmax 470 nm. The factors affecting the reaction were studied and the

conditions were optimized. The stoichiometry of there action was determined, and the

reaction pathway was postulated. Moreover, both the activation energy and the

specificrate constant (at70°C) of there action were found to be 6.74kcalmole/l and

3.58s/1, respectively. The initial rate and fixed time methods were utilized fo

rconstructing the calibrationgraphs for the determination of AML concentration. Under

the optimum reaction conditions, the limits of detection andq uantification were 0.35 and

1.05 mg/mL, respectively .The precision of the method was satisfactory; the relative

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standard deviations were 0.85–1.76%. The recovery percentages ranged from

99.5571.69% to 100.6571.48%.

Chang H77 et al., (2012) have described a sensitive, specific liquid

chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantitative

determination of amlodipine and bisoprolol, using clenbuterol as the internal standard

(IS). The analytes and IS were isolated from 100 μL plasma samples by a simple liquid–

liquid extraction (LLE). Reverse-phase high performance liquid chromatography

(RP-HPLC) separation was accomplished on a Diamonsil C18 column (50 mm ×4.6 mm,

5 μm) with a mobile phase composed of methanol–water–formic acid (75:25:0.01, v/v/v)

at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 3 min.

Multiple reacting monitoring (MRM) transitions of m/z [M+H]+409.1→237.9

(amlodipine), m/z [M+H]+ 326.2→116.0 (bisoprolol) and m/z [M+H]+ 277.0→203.0

(clenbuterol, IS) were used to quantify amlodipine, bisoprolol and IS, respectively. The

method was sensitivewith a lower limit of quantitation (LLOQ) of 0.2 ng/mL for both

amlodipine and bisoprolol, and the linear range was 0.2–50 ng/mL for both amlodipine

and bisoprolol (r2 > 0.9961). All the validation data, such asaccuracy, precision and inter-

day repeatability, were within the required limits.

Venkateswarlu P78 et al., (2012) have developed and validated a rapid and

sensitive liquid chromatography – tandem mass spectrometric (LC–MS/MS) assay

method for the simultaneous quantification of telmisartan and amlodipine in human

plasma. Carbamazepine was used as a ninternal standard. Analytes and the internal

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standard were extracted from human plasma by solid – phase extraction technique using

Waters Oasiss HLB 1cm3 (30 mg) extraction cartridge. There constituted samples were

chromatographed on a Hypurity advance C18 column (50mmX4.6 mm, 5 μm) using a

mixture ofacetonitrile–5mM ammonium acetate buffer (pH-4.0)(50:50,v/v)as the mobile

phase at a flow rate of 0.8mL/min. The calibration curve obtained was linear (r2 = 0.99)

over the concentration range of 2.01–400.06 ng/mL for telmisartan and 0.05–10.01ng/mL

for amlodipine. Method validation was performed as per FDA guidelines and the results

met the acceptance criteria. A run time of 2.5min for each sample made it possible to

analyze more than 400 human plasma samples per day.

Jianzhong Shentu79 et al., (2012) have reported an automated method (XLC–

MS/MS) that uses online solid-phase extraction coupled with HPLC–tandem mass

spectrometry here for the first time to quantify amlodipine in human plasma. Automated

pre-purification of plasma was performed using 10 mm × 2 mm HySphere C8 EC-SE

online solid-phase extraction cartridges. After being eluted from the cartridge, the analyte

and the internal standard were separated by HPLC and detected by tandem mass

spectrometry. Mass spectrometric detection was achieved in the multiple reaction

monitoring mode using a quadrupole tandem mass spectrometer in the positive

electrospray ionization mode. The XLC–MS/MS method was validated and yielded

excellent specificity. The calibration curve ranged from 0.10 to 10.22 ng/mL, and both

the intra- and inter-day precision and accuracy values were within 8%.

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Shankar Ganesh G80 et al., (2013) have developed and validated a novel and

accurate liquid chromatography tandem mass spectrometry method using electrospray

ionization mode for the simultaneous determination of amlodipine (AMD), valsartan

(VAL) using losartan (LOS) as an internal standard (IS), and hydrochlorothiazide (HCT)

using furosemide (FSD) as an IS. The separation was carried on Aquasil C18

(50mmX2.1mm5mm) reversed phase column using acetonitrile and water containing

0.1% formicacid (50:50,v/v) as the mobile phase. The method was validated in terms of

linearity, accuracy and precision over the concentration range of 1–1000 ng/mL. The

intra and inter-day precision and accuracy, stability and extraction recoveries of all the

analytes were in the acceptable range.

Anandkumar R. T81 et al., (2013) have developed a simple, sensitive and

specific liquid chromatographic method with UV detection (230 nm) for the simultaneous

estimation of hydrochlorothiazide, amlodipine and losartan in tablet dosage form and

telmisartan as an internal standard. Separation was achieved with a phenomenex luna 5μ

CN 100R, 250 × 4.60 mm 5 micron size column, ambient temperature with a low

pressure gradient mode with mobile phase containing acetonitrile, water and 0.4% of

potassium dihydrogen phosphate buffer pH 2.7 adjusted with orthophosphoric acid

(45:35:20). The flow rate was 1 mL min−1 and eluent was monitored at 230 nm. The

selected chromatographic conditions were found to effectively separate

hydrochlorothiazide, amlodipine and losartan with retention time of 3.9, 4.9 and 5.8 min

respectively. The linearity range of hydrochlorothiazide, amlodipine and losartan found

in the range of 12.5–62.5 μg ml−1, 2.5–12.5 μg ml−1 and 50–250 μg ml−1 respectively.

133

Weiyong Li82 et al., (2013) have developed a sensitive, simple and rapid high-

performance liquid chromatography coupled with positive ion electrospray ionization-

tandem mass spectrometry (HPLC–ESI-MS/MS) method for the simultaneous

determination of amlodipine, atorvastatin and its metabolites ortho-hydroxy atorvastatin

and para-hydroxy atorvastatin in human plasma. The analytes were extracted from human

plasma through liquid–liquid extraction method. A mixture of methyl tert-butyl ether and

ethyl acetate (50:50, v/v) was used as the extractant. The chromatographic separation was

achieved on a CAPCELLPAK CR 1:4 (5 μm, 150 mm × 2.0 mm i.d.) column within

6.0 min with the mobile phase consisted of acetonitrile and ammonium acetate buffer

(20 mM) containing 0.3% formic acid (50:50, v/v). Data acquisition was carried out in

multiple reaction monitoring (MRM) mode. The method was validated successfully.

Mahmoud Alawi83 et al., (2013) have quantified a simple liquid

chromatography/ion trap mass spectrometry method for amlodipine and atorvastatin with

its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma

was developed. Analytes with internal standard were extracted by protein direct

precipitation with acetonitrile. Adequate chromatographic separation was achieved using

Phenomenex Synergi 4u polar-RP 80A (150 mm × 4.6 mm, 4 μm) column in the isocratic

elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by

trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of

0.50 mL/min. Standard solutions for the analytes were prepared using amlodipine

besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt,

para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard.

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The method validation intends to investigate specificity, sensitivity, linearity, precision,

accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard

calibration levels were prepared by pooled human plasma to attain final dynamic range of

0.2–20.0 ng/mL for amlodipine, 1.5–150 ng/mL for atorvastatin, 1.0–100.0 ng/mL for

ortho-hydroxy atorvastatin and 0.2–20.0 ng/mL for para-hydroxy atorvastatin. Clinical

bioequivalence study was successfully investigated by the application of this validated

bioanalytical method in order to evaluate bioequivalence of two commercial products

10 mg amlodipine/80 mg atorvastatin in a single dose.

Pei Hu84 et al., (2013) have developed a sensitive and rapid ultra performance

liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method to determine

olmesartan and amlodipine levels in human plasma and urine simultaneously.

Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and

mass spectrometric analysis was performed using a QTrap5500 mass spectrometer

coupled with an electro-spray ionization (ESI) source in the positive ion mode. The

MRM transitions of m/z 447 → 207 and 409 → 238 were used to quantify olmesartan and

amlodipine, respectively. This assay method has been fully validated in terms of

selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability,

matrix effect and recovery. The linearity of this method was found to be within the

concentration range of 0.2–500 ng/mL and 4–5000 ng/mL for olmesartan in human

plasma and urine and 0.1–50 ng/mL and 2–1000 ng/mL for amlodipine in human plasma

and urine. Only 2 min were needed for an analytical run.

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Rasha A. Shaalan85 et al., (2013) have developed a simple and selective HPLC-

DAD stability indicating method for the simultaneous determination of the three

antihypertensive drugs amlodipine besylate (AML), valsartan (VAL) and

hydrochlorothiazide (HCT) in their combined formulation. Effective chromatographic

separation was achieved using Zorbax SB-C8 column (4.6 X 250 mm, 5 μm ps) with

gradient elution of the mobile phase composed of 0.025M phosphoric acid and

acetonitrile at a flow rate of 1 mL/min. The multiple wavelength detector was set at 238

nm for measurement of AML and 225 nm for both VAL and HCT. Quantification was

based on measuring the peak areas. The three compounds were resolved with retention

times of 4.9, 6.4 and 8.3 min for HCT, AML and VAL respectively. Analytical

performance of the proposed HPLC procedure was statistically validated with respect to

system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection

and quantification limits. The linearity ranges were 5–200, 5–200 and 10–200 μg/mL for

AML, VAL and HCT respectively with correlation coefficients >0.9993. The three drugs

were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation,

photolysis and thermal degradation.

Manju Misra86 et al., (2013) were developed and validated a high-performance

liquid chromatographic (HPLC) and UV spectrophotometric methods for the quantitative

determination of amlodipine besylate (AM) and benazepril hydrochloride (BZ). Different

analytical performance parameters such as linearity, precision, accuracy, specificity, limit

of detection (LOD) and limit of quantification (LOQ) were determined according to

International Conference on Harmonization ICH Q2B guidelines. The RP-HPLC method

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was developed by the isocratic technique on a reversed-phase Shodex C- 18 5e column.

The retention time for AM and BZ was 4.43 min and 5.70 min respectively. The UV

spectrophotometric determinations were performed at 237 nm and 366 nm for AM and at

237 nm for BZ. Correlation between absorbance of AM at 237 nm and 366 nm was

established and based on developed correlation equation estimation of BZ at 237 nm was

carried out. The linearity of the calibration curves for each analyte in the desired

concentration range was good (r2 >0.999) by both the HPLC and UV methods. The

method showed good reproducibility and recovery with percent relative standard

deviation less than 5%. Moreover, the accuracy and precision obtained with HPLC co-

related well with the UV method.

Chun-Mei Fu87 et al., (2013) have developed and validated an RP-HPLC method

for the simultaneous determination of Ramipril (RP) and Amlodipine (AL) in tablets. The

linearity of the proposed method was investigated in the range of 0.01–0.25

mg/mL(r2¼0.9998) for RP and 0.014–0.36 mg/mL (r2¼0.9997) for AL .The limits of

detection (LOD) were 0.06 μg/mL and 0.02 μg/mL for RP and AL, and the limits of

quantitation (LOQ) were 0.2 μg/mL and0.07 μg/mL, respectively .Some major impurities

and degradation products did not disturb the detection of RP and AL and the assay can

thus be considered stability-indicating.

Chul Soon Yong88 et al., (2014) have been developed a simple, rapid, accurate,

precise and robust HPLC method for the simultaneous determination of fimasartan and

amlodipine in tablet dosage form. Furthermore, stability of active ingredients was

137

evaluated under normal and stress conditions. The isocratic elution was accomplished by

Nucleosil C18 column (250 mm x 4.6 mm, 5 mm) at 40 °C. The mobile phase consisted

of acetonitrile and 0.02 M monopotassium phosphate buffer (pH 2.2) in the ratio of 50:50

(v/v) was eluted at 1.0 ml/min. The eluent was monitored by the UV detector for

fimasartan and amlodipine at 237 nm for 8 min, detection time. The validation of HPLC

method was carried out in accordance with the ICH guidelines.

Nina Alizadeh89 et al., (2014) have established two simple, rapid and sensitive

spectrophotometric methods for amlodipine besylate (ADB) in pure form and in

pharmaceutical preparations have been developed. The methods are based on the charge

transfer reactions between the drug as electron donor with 7,7,8,8-

tetracyanoquinodimethane (TCNQ) and tetracyanoethylene (TCNE) as p-acceptors in

order to the formation of charge transfer (CT) complexes. These reactions give colored

products which have maximum absorption bands at 745 and 396 nm for TCNQ and

TCNE, respectively. Beer’s law is obeyed in the concentration ranges 20–110 μg mL/1

and 5–35 μg mL/1 for ADB using TCNQ and TCNE reagents. The molar absorptivities

are 2.73 · 103 and 6.43 · 103 L mol/1 cm and the Sandell (S) sensitivities are 0.14 μg

cm-2 and 0.063 μg cm-2 using TCNQ and TCNE reagents, respectively, which indicate

the high sensitivity of the proposed methods. The relative standard deviations (R.S.D.:

0.94 and 0.73) obtained using TCNQ and TCNE reagents, respectively, refer to the high

accuracy and precision of the proposed method.

138

Sherine S. Diab90 et al., (2014) have developed a Four, accurate, precise, and

sensitive spectrophotometric methods for the simultaneous determination of a ternary

mixture containing amlodipine besylate (AM), olmesartan medoxomil (OL) and

hydrochlorothiazide (HZ), where AM is determined at its λmax 364.6 nm (0D), while (OL)

and (HZ) are determined by different methods. Method (A) depends on determining OL

and HZ by measuring the second derivative of the ratio spectra (2DD) at 254.4 and

338.6 nm, respectively. Method (B) is first derivative of the double divisor ratio spectra

(D-1DD) at 260.4 and 273.0 nm for OL and HZ, respectively. Method (C) based on

successive spectrophotometric resolution technique (SSRT). The technique starts with the

ratio subtraction method then measuring OL and HZ at their isoabsorptive point at

260.0 nm, while HZ is measured using the amplitude of first derivative at 335.2 nm.

Method (D) is mean centering of the ratio spectra (MCR) at 252.0 nm and 220.0 nm for

OL and HZ, respectively. The specificity of the developed methods is investigated by

analyzing laboratory prepared mixtures containing different ratios of the three drugs and

their combined dosage form. The obtained results are statistically compared with those

obtained by the official or reported methods, showing no significant difference with

respect to accuracy and precision at p = 0.05.

139

RP-HPLC METHOD DEVELOPEMNT AND VALIDATION FOR THE

SIMULTANEOUS ESTIMATION OF LISINOPRIL AND AMLODIPINE

BESYLATE

3.5. EXPERIMENTAL PROTOCOL

3.5.1 MATERIALS AND METHODS

3.5.1.1 Chemicals and Reagents

The reference sample of lisinopril and amlodipine besylate was supplied by Cipla

Pharmaceutical Ltd., Mumbai, India. HPLC grade water, acetonitrile and methanol were

purchased from E. Merck (India) Ltd., Mumbai, India. Potassium dihydrogen phosphate

and orthophosphoric acid of AR Grade were obtained from S.D. Fine Chemicals

Ltd.,Mumbai, India. HPLC grade water was obtained following distillation in glass and

passage through a Milli-Q system (Millipore, Bangalore, India) and was used to prepare

all the solutions. Tablet Formulation of Lisinipril and Amlodipine besylate in combined

dosage form, AMLOPRESS-L, with 5mg LSNP and 5mg AMD lable claim

manufactured by Cipla Pharmaceutical Ltd.

3.5.2 Selection of Initial Conditions for Method Development

3.5.2.1. Determination of absorption maxima by UV-Visible Spectroscopy:

Lisinopril:

Weigh accurately equivalent to about 10mg of Lisinopril and transfer into a 10ml

volumetric flask. Add about 7mL of Milli-Q water and sonicate for 15 minutes with

intermediate shaking. Dilute to volume and mix well. Transfer 1mL above centrifuged

solution into separate 10 mL volumetric flask. Dilute to volume and mix well. Filter

140

through 0.45μ PVDF membrane filter .The wavelength was selected by scanning the

above standard drug between 200 to 400 nm.

Figure 3.5.1: UV Spectrum of Lisinopril

Amlodipine besylate:

Weigh accurately equivalent to about 10mg of Amlodipine besylate and transfer

into a 10ml volumetric flask. Add about 5mL of Milli-Q water and sonicate for 15

minutes with intermediate shaking. Dilute to volume and mix well. Transfer 1mL of

above centrifuged solution into separate 10 mL volumetric flask. Dilute to volume and

mix well. Filter through 0.45μ PVDF membrane filter .The wavelength was selected by

scanning the above standard drug between 200 to 400 nm.

141

Figure 3.5.2: UV Spectrum of Amlodipine besylate

Isobestic Point:

It is the point at which both the drugs in a particular combination will have same

absorbance at a single wavelength. The Isobestic point of Lisinopril & Amlodipine

besylate is found to be 212nm.

Figure 3.5.3: Isobestic point of Lisinopril and Amlodipine besylate

142

3.5.2.2 HPLC INSTRUMENTATIONS AND CONDITIONS

An isocratic HPLC system (Analytical Technologies Limited) consisted of P2230

plus HPLC pump, variable wavelength programmable UV 2230 plus detector system,

Rhenodyne valve with 20μL fixed loop and Analchrom 2006 as operating software. The

chromatographic column used was Hypersil ODS-BP C18 column (250 mm × 4.6 mm;

5.0 μm). Analytical balance (wensar) was used for weighing purpose. Before the analysis,

the mobile phase was filtered through a 0.2 µm filter (Gelman science, India) and

degassed using Branson sonicator (Branson Ultrasonics Corporation, USA) at the flow

rate of 1.0ml per minutes. Sample solutions were also filtered through a 0.2 µm filter and

aliquots of 20 µL were injected into the chromatographic system.

143

3.6 METHOD DEVELOPMENT OF LISINOPRIL AND AMLODIPINE

BESYLATE:

3.6.1 Trial I:

Chromatographic conditions:

Flow rate : 1.0 ml/min

Column : Hypersil BDS C18, 250X4.6mm,5 m

Wave length : 212nm

Column temperature : ambient

Injection volume : 20l

Run time : 8mins

Mobile Phase : methanol: water (75:25 %v/v)

Figure 3.6.1: Chromatogram Lisinopril and Amlodipine besylate - Trail I

Conclusion: Lisinopril and Amlodipine peaks do not pass USP tailing factor. The next

trail is performed by changing the mobile phase.

144

3.6.2. Trial: II

Mobile phase: Acetonitrile: water (75:25 v/v)

Chromatographic conditions:

Flow rate : 1.0 ml/min

Column : C18, 250X4.6mm, 5 m, hypersil BDS

Detector wave length : 212nm

Column temperature : ambient

Injection volume : 20l

Run time : 8 mins

Diluent : Acetonitrile: water (75:25)

Figure 3.6.2: Chromatogram Lisinopril and Amlodipine besylate - Trail II

Conclusion: Lisinopril and Amlodipine peaks do not pass USP tailing factor. The next

trail is performed by changing the mobile phase.

145

3.6.3. Trial: III

Preparation of Phosphate buffer

Weigh 13.6 grams of Potassium dihydrogen phosphate into a 1000ml beaker,

dissolve and diluted to 1000ml with HPLC water. Adjusted the pH to 6.0 with ortho

phosphoric acid.

Chromatographic conditions:

Mobile phase: Acetonitrile: phosphate buffer (60:40 %v/v)

Figure 3.6.3: Chromatogram Lisinopril and Amlodipine besylate - Trail III

Conclusion: Lisinopril and Amlodipine peaks do not pass USP tailing factor. The next

trail is performed by changing the mobile phase.

146

3.6.4. Trial: IV (OPTIMIZED CONDITIONS)

Preparation of Phosphate buffer:

Weigh 13.6 grams of Potassium dihydrogen phosphate into a 1000ml beaker,

dissolve and diluted to 1000ml with HPLC water. Adjusted the pH to 6.0 with ortho

phosphoric acid.

Preparation of mobile phase:

Mix a mixture of above buffer 700mL (70%) and 300 mL of methanol HPLC

(30%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45 µ filter under

vaccum filtration

Mobile phase: Buffer: methanol (70:30 % v/v)

Chromatographic conditions:

Detector wave length : 212nm

Injection volume : 20l

Run time : 7min

Figure 3.6.4: Chromatogram Lisinopril and Amlodipine besylate - Trail IV

147

Conclusion: The above method is finalized for Lisinopril and Amlodipine besylate

peaks. From the above tests Methanol: Phosphate Buffer (30:70%v/v) was chosen as the

mobile phase, as

Drug was providing less retention time

More peak area and good peak as well in this mobile phase

Drug is not forming any precipitate

Stable throughout the experiment

3.7. METHOD DEVELOPMENT OF LISINOPRIL AND AMLODIPINE

BESYLATE (HPLC)

Preparation of Phosphate buffer

Weigh 13.6 grams of Potassium dihydrogen phosphate into a 1000ml beaker,

dissolve and diluted to 1000ml with HPLC water. Adjusted the pH to 6.0 with ortho

phosphoric acid.

Preparation of mobile phase

Mix a mixture of above buffer 700mL (70%) and 300 mL of methanol HPLC

(30%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45 µ filter under

vaccum filtration

Preparation of Standard Solution

Accurately weigh and transfer 10mg of Lisinopril and Amlodipine besylate

working standard into two 10 mL volumetric flask add about 7 mL of Diluent and

148

sonicate to dissolve it completely and make volume up to the mark with the same solvent.

(Stock solution).

From the above stock solution take 1ml in 10 ml volumetric flask and make up the

volume with diluent to give 100ppm. From the above solution pipette 4.0 ml into a 10ml

volumetric flask and dilute up to the mark to give 40ppm.

Preparation of Sample Solution

Weigh lisinopril and amlodipine besylate Tablets (five) and calculate the average

weight. Accurately weigh and transfer the sample equivalent to 10 mg of Lisinopril &

Amlodipine besylate into a 10 mL volumetric flask. Add about 7 mL of diluent and

sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well

and filter through 0.45µm filter.Pipette out 1ml of stock solution in 10ml volumetric flask

and make up the volume with diluents to give 100ppm. Further pipette 4.0 ml of the

above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

Mix well and filter through 0.45µm filter with diluen to give 40ppm. Mix well and filter

through 0.45µm filter.

Chromatographic Condition:

A mixture of Methanol: Phosphate Buffer at pH 6 adjusted with orthophosphoric

acid (30:70v/v) was used as mobile phase and was filtered through 0.45μ membrane filter

prior to use. The flow rate of mobile phase was maintained at 1mL/min. Detection was

carried out at 212nm (Figure 3.5.3) at the ambient temperature. Total run time 7.0 min

149

was used with injection volume of 20 μL. The retention time of the drugs LSNP and

AMD were 3.883 and 2.716 minutes respectively (Figure 3.7.2).

Figure 3.7.1: Chromatogram of Blank

Figure 3.7.2: Chromatogram of Standard Solution of Lisinopril and Amlodipine

Figure 3.7.3: Chromatogram of Sample Formulation of Lisinopril and Amlodipine

150

3.8. RESULT AND DISCUSSION:

METHOD VALIDATION:

The proposed method has been extensively validated in terms of specificity,

linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ),

robustness and system suitability. The accuracy was expressed in terms of percent

recovery of the known amount of the standard drugs added to the known amount of the

pharmaceutical dosage forms. The precision (% RSD) was expressed with respect to the

repeatability, intraday, and interday variation in the expected drug concentrations. The

optimized HPLC method for the analysis of fresh quality control and samples were

validated in accordance with the ICH guidelines as described in chapter 1.

3.8.1. Specificity:

Commonly used excipients (starch, microcrystalline cellulose and magnesium

stearate) were spiked into a pre-weighed quantity of drugs. The chromatogram was taken

by appropriate dilutions and the quantities of drugs were determined. Blank are injected

at the retention time of main peaks. On the basis of the chromatogram (Figure 3.7.1) we

can say that there is no interference of blank with retention time of active component.

151

3.8.2. Precision:

Figure 3.8.1: Chromatograms of Precision studies of Lisinopril and Amlodipine

besylate

152

Table 3.1: Results of Lisinopril for precision studies

S.NoRetention

TimePeak Area % Assay % Mean %RSD

1 2.6500 4393.1825 101.65

101.58 0.078

2 2.7167 4390.0012 101.57

3 2.7167 4382.4421 101.39

4 2.6333 4395.3385 101.70

5 2.7000 4389.2901 101.70

6 2.6667 4387.3311 101.51

Table 3.2: Results of Amlodipine besylate for precision studies

S.NoRetention

TimePeak Area % Assay % Mean %RSD

1 3.8000 184.1203 101.29

101.23 0.158

2 3.8833 183.2286 100.78

3 3.8833 183.0512 100.67

4 3.7833 183.9186 101.82

5 3.8333 184.0822 101.27

6 3.8167 184.5423 101.54

Acceptance Criteria:

The % relative standard deviation of assay of Lisinopril & Amlodipine besylate

peak area for the six replicate injections should be not more than 2.0%

153

Observation:

The % RSD of assay of six replicate injections of Lisinopril & Amlodipine

besylate was found to be within the limits.

3.8.3. Accuracy:

The accuracy of the method was evaluated in triplicates by recovery studies by

recovery studies at three different concentration levels of 50%, 100% and 150% for

LSNP and AMD. Known amounts of standard drug concentrations were added to the

sample and peak area was determined and analysing the resulting mixture as described in

table 3.3 and 3.4.

Figure 3.8.2.A: Chromatograms of Accuracy of Lisinopril and Amlodipine besylate

(50%)

154

Figure 3.8.2.B: Chromatograms of Accuracy Lisinopril and Amlodipine besylate(100%)

Figure 3.8.2.C: Chromatograms of Accuracy of Lisinopril and Amlodipine besylate(150%)

155

Table 3.3: Results for Accuracy of lisinopril

Table 3.4: Results for Accuracy of Amlodipine besylate

Level (%)

Retention time

Peak area % RecoveryMean

recovery%RSD

50 2.7167 94.4391 98.96

98.78 0.18650 2.6833 94.2132 98.53

50 2.7333 94.1324 98.87

100 2.6500 184.1203 100.21

99.56 0.06100 2.7167 184.3122 100.28

100 2.7167 184.2964 98.21

150 2.7500 264.9989 100.76

100.77 0.07150 2.7500 264.7123 100.91

150 2.7667 264.6129 100.66

Level (%)

Retention time

Peak area % Recovery%Mean recovery

%RSD

50 3.8667 2311.2591 98.53

98.33 0.2950 3.8500 2302.3133 98.27

50 3.9167 2309.2199 98.28

100 3.8000 4393.1825 101.29

101.37 1.17100 3.8833 4396.2132 101.41

100 3.8333 4311.3122 101.40

150 3.9000 6483.2932 98.81

98.72 0.12150 3.9333 6489.0132 98.70

150 3.9667 6477.3126 98.66

156

Acceptance Criteria:

Recovery of drug at each level should be between 98.0% to 102%

The %RSD for recovery of triplicate preparations at each level should not be more than

2.0%

Observation:

The recovery results indicating that the test method has an acceptable level of

accuracy.

3.8.4. Linearity:

Weigh accurately about 10 mg of LSNP and AMD sample into a 10ml clean dry

conical flask add about 7ml of diluent and sonicate to dissolve it completely and make

volume up to the mark with the same solvent. Further dilute the above stock solutions

into a series of dilutions were made with diluents to get 20 to 80μg/ml concentrations

respectively. The chromatograms were recorded at 212nm. The calibration curve was

obtained by plotting the ratio of peak area of drug versus analyte concentration Using the

data, regression equation was established, linearity plot was constructed by using least

square method and correlation coefficient was calculated.

157

Linearity 50% Linearity 75%

Linearity 100% Linearity 125%

Linearity 150% Linearity 175%

158

Linearity 200%

Figure 3.8.3: Chromatograms of Linearity studies of lisinopril and amlodipine

besylate

Figure 3.8.4: Linearity Plot of Lisinopril

159

Figure 3.8.5: Linearity Plot of Amlodipine besylate

Table 3.5 Results of Linearity Studies

S.No Lisinopril Amlodipine besylate

Conc(µg/ml )

Peak AreaConc

(µg/ml )Peak Area

1 20 2317.7917 20 94.4391

2 30 3224.4565 30 138.4136

3 40 4393.189 40 184.4734

4 50 5479.0435 50 227.371

5 60 6493.9932 60 264.9989

6 70 7390.1958 70 309.8992

7 80 8486.251 80 355.4852

Slope 102.6 4.309

Intercept 280.4 9.521

Correlation Coefficient

0.9999 0.999

160

Acceptance Criteria:

Correlation Coefficient should be not less than 1.

Observation:

The correlation coefficient for lisinopril & Amlodipine besylate met the

acceptance criteria of NLT 0.999.

3.8.5. LOD and LOQ

LOD and LOQ for LSNP and AMD by this method were evaluated on the basis of

signal-to-noise ratio method described in ICH guidelines. A signal-to-noise ratio between

3 or 2:1 is generally considered acceptable for estimating the detection limit. A typical

signal-to-noise ratio required for LOQ is 10:1. According to a formula given by Miller,

the limit of detection (LOD) and limit of quantification (LOQ) were calculated.

Table 3.6 Results for LOD and LOQ

Drug LOD (μg/ml) LOQ (μg/ml)

LSNP 0.012 0.037

AMD 0.027 0.083

161

Figure 3.8.6: Chromatograms of LOD and LOQ of Lisinopril and Amlodipine

besylate

Observation

The LOD and LOQ values from the above demonstrate that the method is

sensitive for the determination of Lisinopril & Amlodipine besylate.

3.8.6. Ruggedness

Ruggedness is a measure of reproducibility of test results under normal variation

of the operating conditions, e.g. analyst-to-analyst, system-to-system, column-to-column

and day-to-day.

162

System to system /Analyst to Analyst/column to Column variability study was

conducted on different HPLC systems, different columns and different analysts under

similar conditions at different times.

Figure 3.8.7: Chromatogram of Ruggedness studies of lisinopril and amlodipine

besylate by using System 1

163

Table 3.7: Results of Ruggedness studies by using System 1 for lisinopril

S.No Peak area %recovery%Mean recovery

Standard deviation

%RSD

1 4393.1825 100.21

100.10 0.988 0.0987

2 4390.2132 100.14

3 4382.3126 99.94

4 4386.2162 100.04

5 4392.3121 100.19

6 4388.6891 100.10

Table 3.8: Results of Ruggedness studies by using System 1 for Amlodipine besylate

S. No Peak area %recovery%Mean recovery

Standard deviation

%RSD

1 184.1263 101.30

101.13 0.220 0.217

2 184.1342 101.30

3 183.6823 101.04

4 184.2364 101.36

5 183.3591 100.85

6 183.4681 100.92

164

Figure 3.8.8: Chromatogram of Ruggedness studies of lisinopril and amlodipine

besylate by using System 2

165

Table 3.9: Results of Ruggedness studies by using System 2 for lisinopril

S.No Peak area %recovery% Mean recovery

Standard deviation

% RSD

1 4393.2132 100.21

100.10 0.1214 0.1213

2 4390.2162 100.14

3 4381.3192 99.92

4 4382.2632 99.94

5 4391.4162 100.17

6 4389.6781 100.12

Table 3.10: Results of Ruggedness studies by using System 2 for Amlodipine

besylate

S. No Peak area %recovery%Mean recovery

Standard deviation

% RSD

1 184.1231 101.30

101.13 0.2195 0.2171

2 184.1426 101.31

3 183.6831 101.04

4 184.2341 101.36

5 183.3682 100.86

6 183.4631 100.91

166

Figure 3.8.9: Chromatogram of Ruggedness studies of lisinopril and amlodipine

besylate by using Column1

167

Table 3.11: Results of Ruggedness studies by using column 1 for lisinopril

S. No Peak area %recovery%Mean recovery

Stand Deviation

%RSD

1 4393.6843 100.22

100.19 0.0448 0.044

2 4392.7642 100.20

3 4390.8632 100.15

4 4391.7162 100.17

5 4395.3268 100.26

6 4390.4491 100.14

Table 3.12: Results of Ruggedness studies by using column 1 for Amlodipine

besylate

S. No Peak area %recovery%Mean recovery

Stand deviation

%RSD

1 185.2369 101.94

101.60 0.428 0.421

2 184.4123 101.46

3 183.3642 100.86

4 184.4162 101.47

5 185.2231 101.93

6 185.1843 101.91

168

Figure 3.8.10: Chromatogram of Ruggedness studies of lisinopril and amlodipine

besylate by using column 2

169

Table 3.13: Results of Ruggedness studies by using column 2 for lisinopril

S. No Peak area %recovery%Mean recovery

Stand deviation

%RSD

1 4393.6833 100.22

100.20 0.0447 0.046

2 4390.7182 100.15

3 4393.8136 100.22

4 4395.7211 100.27

5 4391.3246 100.16

6 4392.4133 100.19

Table 3.14: Results of Ruggedness studies by using column 2 for Amlodipine

besylate

S. No Peak area %recovery% Mean recovery

Stand deviation

%RSD

1 184.3264 101.41

101.46 0.1678 0.165

2 184.4321 101.48

3 183.9348 101.19

4 184.3642 101.44

5 184.6841 101.62

6 184.7386 101.65

Acceptance Criteria:

The % relative standard deviation of Lisinopril and amlodipine besylate for the

six replicate injections should be not more than 2.0% obtained from standard solution.

Observation:

The % RSD of six replicate injections of lisinopril and Amlodipine besylate was

found within the limits.

170

3.8.7. Robustness

The robustness was studied by the standard solutions with slight variations in the

optimized conditions. They are ± 0.2 ml of the flow rate and ± 5° in the temperature.

Figure 3.8.11: Chromatogram of Robustness studies of lisinopril and amlodipine

besylate with changes in flow rate (0.8ml)

171

Figure 3.8.12: Chromatogram of Robustness studies of lisinopril and amlodipine

besylate with changes in flow rate (1.0ml)

172

Figure 3.8.13: Chromatogram of Robustness studies of lisinopril and amlodipine

besylate with changes in flow rate (1.2ml)

173

Figure 3.8.14: Chromatogram of Robustness studies of lisinopril and amlodipine

besylate with a changes in temperature (20°C)

174

Figure 3.8.15: Chromatogram of Robustness studies of lisinopril and amlodipine

besylate with a changes in temperature (25°C)

175

Figure 3.8.16: Chromatogram of Robustness studies of lisinopril and amlodipine

besylate with a changes in temperature (30°C)

176

Table 3.15 Robustness study for LSNP and AMD

Condition varied

Changed condition

Area (n=6) % Assay

LSNP±S.D AMD±S.D LSNP AMD

Flow rate (ml/min)

0.8 4393.0335±0.007 184.79±0.035 100.19 101.68

1.0 4393.1357±0.013 184.60±0.041 100.21 101.57

1.2 4392.9927±0.018 184.18±0.037 100.20 101.38

Temperature (OC)

20 4393.3025±0.004 184.25±0.167 100.21 101.37

25 4393.1032±0.008 184.39±0.105 100.21 101.37

30 4393.3396±0.005 184.19±0.096 100.21 101.33

3.8.8. System Suitability:

System suitability was performed by calculating chromatographic parameter

namely, theoretical plate, tailing factor, and resolution and retention time. The results are

given in table 3.16.

Table 3.16 System Suitability data for LSNP and AMD

Parameters LSNP AMD

Retention time (Rt) (min) 3.88 2.71

Tailing factor 1.06 1.1

Plate count 6800 7458

%RSD 1.18 0.56

177

Table 3.17. Optical Characteristic of the proposed method

Parameters Lisinopril Amlodipine besylate

λ max (nm) 212

Calibration curve range (μg/ml) 20 to 80

Regression equation Y = 102.6x+280.4 Y = 4.309x+9.521

Slope 102.6 4.309

Intercept 280.4 9.521

Correlation coefficient (r2) 0.9999 0.999

%RSD 1.18 0.58

LOD 0.012 0.037

LOQ 0.027 0.083

Plate count 6800 7458

Tailing factor 1.06 1.10

Retention Time (min) 3.883 2.716

% Recovery 98.33 – 101.37 98.90 – 100.70

3.9. SUMMARY

In this study an isocratic RP-HPLC method for the simultaneous determination of

Lisinopril and Amlodipine besylate in formulation was developed and optimized. The

results of the study demonstrate the benefit of applying this approach in selecting

optimum conditions for the determination of drugs in formulation. The validation study

supported the selection of the assay conditions by confirming that the assay was specific,

accurate, linear, precise and robust. Therefore the proposed HPLC method can be used as

a routine quality control analysis in a pharmaceutical environment.

178

Validation Parameters

Acceptance Criteria HPLC Results

Precision

The %RSD of peaksobtained from the 6replicate injections

should be NMT 2.0%

Lisinopril Amlodipine besylate

0.078 0.158

Accuracy

The % recovery at eachlevel shall be NLT

98.0% and NMT 102.0%of the added amount.

Lisinopril Amlodipine besylate

98.33 – 101.37 98.90 – 100.70

LinearityThe Correlation

coefficient shall be NLT0.999

Lisinopril Amlodipine besylate

0.999 0.999

LODDetection limit near to

3.00.012 0.027

LOQQuantification limit near

to 100.037 0.083

Robustness

The %RSD of peaksobtained from the 6replicate injections

should be NMT 2.0%.

Lisinopril Amlodipine besylate

0.044 – 0.121 0.165 – 0.421

RuggednessAll the system suitabilityparameters should passfor all the conditions.

The system suitability parameters passed for all the Conditions.

System Suitability

For 6 replicate injections Lisinopril Amlodipine besylate

The %RSD NMT 2.0% 1.18 0.56

Tailing factor NMT 2.0% 1.06 1.1

Plate Count NLT 3000 6800 7458