N H CH O H C NH O O H - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/50468/14/14_chapter6.pdfto porous silica gel for Chromatography R (5 µm) Mobile phase: dissolve 1.0 g

Chapter No. 6

PhD Thesis – Sardar Patel University Page 169

1. DRUG INFORMATION [1-6]

1.1 ATENOLOL:

PARAMETER DESCRIPTION

Name of Drug ATENOLOL

Chemical Structure

NH2

O

ONH

CH3

CH3

OH

Synonym Not Available

IUPAC names 2-(4-{2-hydroxy-3-[(1-methylethyl)amino]propoxy}phenyl)

acetamide

Chemical Formula C14H22N2O3

CAS Registry No. 29122-68-7

Molecular Weight 266.33 g/mol

PHYSICOCHEMICAL PROPERTIES OF DRUG

State & Colour White powder

Solubility Freely soluble in Methanol, Acetonitrile; soluble in Acetic acid,

DMSO,HCL and insoluble in Acetone, Ethyl acetate, CHCl3

Melting Range 152-156.5 0C

pKa 15.95

λmax 225 nm,275 nm(Acetonitrile),283 nm (Methanol)

PHARMACOLOGICAL DATA

Therapeutical Hypertension,

Chapter No. 6


Category Myocardial ischemia,

Catecholamine-induced tremor,

Chronic obstructive pulmonary disease(COPD)

Pharmacological

Uses

Atenolol is a cardio selective beta-adrenergic blocker possessing

properties and potency similar to propranolol, but without a

negative inotropic effect.

Mechanism of

Action

Like metoprolol, atenolol competes with sympathomimetic

neurotransmitters such as catecholamines for binding at β1-

adrenergic receptors in the heart and vascular smooth muscle,

inhibiting sympathetic stimulation. This results in a reduction in

resting heart rate, cardiac output, systolic and diastolic blood

pressure, and reflex orthostatic hypotension. Higher doses of

atenolol also competitively block beta(2)-adrenergic responses in

the bronchial and vascular smooth muscles.

Dose 12.5 – 100mg/day

STABILITY AND STORAGE DATA

Stability Drug is stable

Storage Store protected from light and moisture, at a temperature not

exceeding 300C.

1.2 NIFEDIPINE

PARAMETER DESCRIPTION

Name of Drug NIFEDIPINE

Chapter No. 6


Chemical Structure

Synonym Not Available

IUPAC names 3,5-dimethyl,2,6-dimethyl-4-(2-nitrophenyl)-1,4-

dihydropyridine-3,5-dicarboxylate

Chemical Formula C17H18N2O6

CAS Registry No. 21829-25-4

Molecular Weight 346.335 g/mol

PHYSICOCHEMICAL PROPERTIES OF DRUG

State & Colour Yellowish powder

Solubility Freely Soluble in Acetone, Acetonitrile, CHCL3, Soluble in

Methanol; Slightly Soluble in Isopropanol, Acetone and

Practically Insoluble in Water, HCL, NaOH.

Melting Range 173 – 176.50C

pKa Not available

λmax 341 nm,333 nm(ACN)

PHARMACOLOGICAL DATA

Therapeutical

Category

Vasodilator Agent

Calcium Channel Blocker

Tocolytic Agent

Pharmacological Nifedipine is a dihydropyridine calcium channel blocker. Its

main uses are as an antianginal (especially in Prinzmetal's

http://en.wikipedia.org/wiki/Dihydropyridine

http://en.wikipedia.org/wiki/Calcium_channel_blocker

http://en.wikipedia.org/wiki/Antianginal

http://en.wikipedia.org/wiki/Prinzmetal%27s_angina

Chapter No. 6


Uses angina) and antihypertensive, although a large number of other

indications have recently been found for this agent, such as

Raynaud's phenomenon, premature labor, and painful spasms of

the esophagus such as in cancer and tetanus patients. It is also

commonly used for the small subset of pulmonary hypertension

patients whose symptoms respond to calcium channel blockers

Mechanism of

Action

An Increased concentration of cytosolic Ca2+

causes increased

contraction in cardiac and vascular smooth muscle cells. The

entry of extracellular Ca2+

is more important in initiating the

contraction of cardiac myocytes (Ca2+

-induced Ca2+

release).

The release of Ca2+

from intracellular storage sites also

contributes to contraction of vascular smooth muscle,

particularly in some vascular beds. Cytosolic Ca2+

concentrations may be increased by various contractile stimuli.

Thus many hormones and neurohormones increase Ca2+

influx

through so-called receptor-operated channels, whereas high

external concentrations of K+ and depolarizing electrical stimuli

increase Ca2+

influx through voltage-sensitive, or "potential

operated," channels. The Ca2+

channel antagonists produce their

effects by binding to the a1 subunit of the L-type Ca2+

channels

and reducing Ca2+

flux through the channel.

Dose 60-90mg/day

STABILITY AND STORAGE DATA

Stability Drug light sensitive.

Storage Store protected from light and moisture, at a temperature not

exceeding 300C.

Marketed formulation of Atenolol and Nifedipine

Brand Name

(Formulation Type)

Contents Name of the manufacturing

Company

NILOL Atenolol 50mg/capsule Intas Pharmaceuticals Ltd

http://en.wikipedia.org/wiki/Antihypertensive

http://en.wikipedia.org/wiki/Raynaud%27s_phenomenon

http://en.wikipedia.org/wiki/Premature_birth

http://en.wikipedia.org/wiki/Esophagus

http://en.wikipedia.org/wiki/Cancer

http://en.wikipedia.org/wiki/Tetanus

http://en.wikipedia.org/wiki/Pulmonary_hypertension

http://en.wikipedia.org/wiki/Calcium_channel_blockers

Chapter No. 6


(Tablets) Nifedipine 20mg/capsule

2. LITERATURE SURVEY

2.1 Compendial methods of Atenolol:

Sr.

No.

Method Specifications Ref. No.

1 High Performance Liquid Chromatography

Column:15 cm x 4.6 mm, Octadecylsilane chemically bonded

to porous silica or ceramic microparticles (5 µm)

Mobile phase: dissolve 1.0 g of Sodium Octane-sulphonate

and 0.4 gm of Tetrabutyl ammonium hydrogen sulphate in

1000 ml of a mixture of 20 volumes of Tetrahydrofuran, 180

volumes of Methanol and 800 volumes of a 0.34 per cent w/v

solution of Potassium di-hydrogen phosphate and adjust the pH

to 3.0 with Phosphoric acid.

Flow rate: 1 ml/min

UV Detection: 226 nm

IP 2010

(7)


Column:15 cm x 4.6 mm, Octadecylsilane chemically bonded

to porous silica gel for Chromatography R (5 µm)

Mobile phase: dissolve 1.0 g of Sodium Octane sulphonate and

0.4 gm of Tetrabutyl ammonium hydrogen sulphate in 1000 ml

of a mixture of 20 volumes of Tetrahydrofuran, 180 volumes of

Methanol and 800 volumes of a 0.34 per cent w/v solution of

Potassium di-hydrogen phosphate and adjust the pH to 3.0 with

Phosphoric acid.

Flow rate: 1 ml/min


BP 2010

(8)


Column:30 cm x 3.9 mm, containing L1 packing

Mobile phase: dissolve 1.1 g of Sodium 1-heptanesulphonate

and 0.71 gm of anhydrous dibasic sodium phosphate in 700 ml

USP 2011

(9)

Chapter No. 6


of a mixture of water. Add 2 ml of Dibutylamine, and adjust

with 0.8 M phosphoric acid to pH 3.0. Add 300 ml of methanol,

mix, and pass through 0.5 µm or fine porosity filter and degas it

before use.

Flow rate: 0.6 ml/min

UV Detection: 226 nm.

2.1.1 Reported methods for estimation of Atenolol and its combinations:

Sr.

No.

Method Specification Matrix Ref. No.

1 High Performance Liquid Chromatography for

Atenolol

Column: ODS 25 mm x4.6 mm

Mobile phase: Phosphate buffer and Acetonitrile

(53:47 v/v)

UV Detection:230nm

Flow rate:1.2 ml/min

Bulk 10

2 UV and HPLC for Atenolol

Column: Purospher RP-18 (250 mm x 4.6 mm, 5

μm)

Mobile phase:10 mM Ammonium acetate buffer

(pH 7.0) and Acetonitrile (80:20 v/v)

UV Detection:226nm


UV Spectrophotometric method

UV Detection: 226nm

Solvent: Sodium acetate

Tablet 11

3 HPLC for Atenolol

Column: Lichrosorb, 10 µm RP 18-250 × 4.6 mm

Mobile phase: mixture of Acetonitrile: Methanol:

0.01 mol/L Phosphate buffer with pH adjusted to

6.0 with NaOH, containing 0.1% SDS (15:60:25

v/v/v)

Human

plasma

12

Chapter No. 6


Florescence Detection:

Excitation: 258 nm

Emission: 300nm


4 Stability indicating HPLC for Atenolol

Column: Waters µBondapak-C18 (3.9 × 300 mm,

5mm)

Mobile phase: Acetonitrile: Sodium phosphate

monobasic (0.08 M, pH 3.0) (10:90, v/v)

UV Detection:284nm


Tablet 13

5 Photo-degradation studies by HPLC for Atenolol

Column: RP-18 Alltima, Al-tech (1504.6 mm)

Mobile phase: Triethylamin (pH 4; 0.01 M) :

Acetonitrile (96:4 v/v)

UV Detection:220, 270, 335nm


Bulk 14

6

HPLC for Atenolol and Losartan

Column: C-18 Column (Microsorb-MV 100-

5,250 x 4.6 mm)

Mobile phase: Acetonitrile:Methanol:25mM

Phosphate buffer pH-3 (35:35:30v/v/v)

UV Detection:225nm


Tablet 15

7


Column: Thermo scientific C18

column(20cm×4.6 mm, 5µ)

Mobile phase: Acetonitrile : Phosphate buffer

(pH 3.6 adjusted with Anhydrous Disodium

hydrogen phosphate) (70:30 v/v)

UV Detection:229nm


Tablet 16

8 HPTLC for Atenolol, Losartan and Bulk and 17

Chapter No. 6


Hydrochlorothiazide

Plate: 10 cm× 20 cm Aluminium HPTLC plates

coated with 0.2-mm layers of silica gel 60

F254(Merck)

Mobile phase: Toluene: Methanol: Triethylamine

6.5:4:0.5 (v/v/v)


Tablet

9


Column: Supelcosil ODS analytical column

(25×0.46 cm, i.d., 5 µm)

Mobile phase: Acetonitrile : Phosphate buffer (pH

3 adjusted with Anhydrous Disodium hydrogen

phosphate) in the ratio of 45:55 v/v

UV Detection:227nm


Tablet 18

10

Simultaneous UV for Atenolol, Losartan and

Hydrochlorothiazide

Method 1:

UV Detection: 274.5-270.5nm, 226-222 nm and

252-248 nm for HCT, ATN, LOS

Solvent: Water

Method 2:

UV Detection: 272.5 , 224 and 250 nm for HCT,

ATN, LOS

Solvent: Water

Tablet 19

11

HPLCfor Atenolol, Losartan and

Hydrochlorothiazide

Column: C18 column (25×0.46 cm, i.d., 5 µm)

Mobile phase: 0.035M Potassium di-hydrogen

orthophosphate: Acetonitrile gradient

UV Detection:225nm

Tablet 20

12 UV Spectrophotometric for Atenolol and

Amlodipine

Bulk and

Tablet

21

Chapter No. 6


UV Detection: 238.4 nm and 273.4 nm for

Amlodipine and Atenolol respectively

Solvent: Water

13

HPLC for Atenolol and Amlodipine

Column: ODS (C18), 4.6 mm × 25 cm & (0.5 µm)

Mobile phase: Ammonium acetate buffer (pH was

adjusted to 4.5 ± 0.05 with glacial acetic acid),

Acetonitrile: Methanol (35:30:35 v/v/v)



Column temp.: 40 0C

Tablet 22

14

HPLC for Atenolol and Atorvastatin

Column: Phenomenex ODS C-18 column (250 mm

× 4.6 mm i.d. 5-µm particles)

Mobile phase: Acetonitrile, and Phosphate buffer

(pH 4.5±0.05 adjusted with Ortho phosphoric acid)

in the

ratio 72:28 (v/v)

UV Detection:238 nm


Tablet 23

15

HPLCfor Atenolol, Losartan and

Hydrochlorothiazide

Column: C18 Princeton SPHER ( 250 × 4.6 mm

and 0.5 µm)

Mobile phase: Acetonitrile: 50mM Potassium di-

hydrogen ortho phosphate (pH 3.5) ratio 50:50v/v.

UV Detection:270 nm


Bulk 24

16

HPTLC for Atenolol and Aspirin

Plate: Silica gel precoated with Aluminium plate 60

F254plates, [20 cm × 10 cm with 250 µm thickness]

Mobile phase: n-Butanol: Water: Acetic acid (8: 2:

0.2 v/v/v)

Bulk and

Tablet

25

Chapter No. 6



17 TLC for Atenolol and Indapamide

Bulk and

Tablet

26

18

HPTLC for Atenolol and Lercanidipine

Plate: Aluminum foil plates precoated with silica

gel 60F254

Mobilephase: Toluene: Methanol: Triethylamine

3.5:1.5:0.1 ( v/v/v)


Tablet 27

19 Stability indicating HPLC method for Atenolol

and Hydrochlorothiazide

Column: Hypersil-BDS C18column (250 × 4.6mm

i.d., 5 mm)

Mobile phase: 25mM Phosphate buffer (pH

3±0.05): Acetonitrile (85:15, v/v)

UV Detection:227 nm


Bulk and

Tablet

28

20

HPTLC for Atenolol and Chlorthalidone

Plate: TLC plates (20×10 cm) coated with silica gel

60 F254

Mobile phase: Chloroform: Methanol: Ethyl

acetate: Ammonia solution (75: 28: 2: 1.6v/v/v)


Bulk and

Tablet

29

21

Stability indicating HPLC for Atenolol, Aspirin,

Atorvastatin and Losartan

Column: Inertsil ODS C18

(150 × 4.6) mm with 5 µm particles

Mobile phase: Acetonitrile: Buffer consists of

0.1% Ortho-phosphoric acid (pH 2.9) Gradient

UV Detection:230 nm


Poly pill 30

22 Simultaneous UV for Atenolol and Nifedipine

Method 1 : Absorption correction method

Tablet

And

31

Chapter No. 6


UV detection: λmax at 276.5 nm and 328.5 nm for

Atenolol and Nifedipine respectively

Solvent: Methanol

Method 2: First order derivative

ZCP: Atenolol showed zero crossing point at 226.5

nm while Nifedipine showed zero crossing point at

235 nm

Solvent: Methanol

Capsule

2.2.1 Official methods of Nifedipine:

Sr.

No.

Method Specifications Reference

No.


Column: 150 mm x 4.6 mm

Stationary phase: Octadecysilane bonded to porous

silica(5µm).

Mobile phase: Water: Methanol: Acetonitrile

(55:36:9 v/v/v)

UV Detection: 235nm

Flow rate : 1.0 ml/min

I.P. -2010

(7)



Stationary phase: Octadecylsilyl silica gel for

chromatography (5 µm).

Mobile phase: Acetonitrile: Methanol: Water

(9:36:55v/v/v).

UV Detection: 235nm

Flow rate :1.0 ml/min

B.P.- 2010

(8)



Stationary phase: Octadecylsilyl silica gel for

chromatography (5 µm).

Mobile phase: Acetonitril: Methanol: Water

U.S.P. -2011

(9)

Chapter No. 6


(25:25:50v/v/v).

UV Detection:235nm

Flow rate :1.0 ml/min

2.2.2 Reported methods of Nifedipine and its combinations:

Sr.

No.

Method Specification Matrix Reference

No.

1 RP-HPLC for Nifedipine

Column: ODS 25 mm x 4.6 mm

Mobile phase: Methanol: Water(70:30

v/v) pH 3.0 by OPA

UV Detection:238nm

Tablet 32

2 HPLC method for Nifedipine in

human plasma by solid-phase

extraction

Column: Lichrocart Lichrospher 60 RP

select B

Mobile phase: 0.020 mol/L KH2PO4

(pH 4.8) and Acetonitrile (42:58v/v).

UV Detection:240nm

Plasma 33

3 HPLC of Nifedipine in Human

Plasma

Column: Supelcosil LC-18, 5 um, 15

cm 4.6 mm

Mobile phase: Acetonitrile: Methanol:

Water (35 : 17 : 48 v/v/v) adjusted to pH

4.0 with Phosphoric acid

Excitation wavelength: 330 nm

Emission wavelength: 440 nm


Human

plasma

34

4 HPLC-UV method for simultaneous

estimation of Nifedipine, Nateglinide

and Lovastatin

Column:C18 (25cm 4.6 mm)

Poly pill 35

Chapter No. 6


Mobile phase: Acetonitrile: Phosphate

buffer (0.01 M, pH 3.05) (60:40 v/v)

UV Detection: 236nm


5 HPLC for the determination of

Nifedipine in plasma and its use in

pharmacokinetic studies

Column: Nova Pak C–18 column

Mobile phase: Acetonitrile: Water (48:

52, v/v) pH 4.0 with Glacial acetic acid

UV Detection:240 nm

Flow rate:1-8 ml/min

Plasma 36

6 HPLC method for estimation of

Nifedipine and Atenolol

Mobile phase: Methanol:0.1 M

Disodium hydrogen phosphate

(75:25v/v)adjusted to pH 3.0 with acetic

acid

UV detection:238 nm


Tablet 36

7 HPLC method for estimation of

Nifedipine and Atenolol

Mobile phase: Methanol: Water: Acetic

acid (80:15:05 v/v)

UV detection:254 nm


Tablet 38

8 HPTLC method for estimation of

Atenolol and Nifedipine

Plate: TLC plates (20 ×10 cm) coated

with kieselguhr 60,GF 254.

Mobile phase: Cyclohexane: Methanol:

Ethylacetate: Ammonia solution

(5:1.5:3: 0.5 v/v/v)

Tablet 39

Chapter No. 6



3 DEVELOPMENT AND VALIDATION OF METHOD FOR

SIMULTANEOUS ESTIMATION OF ATENOLOL AND NEFEDIPINE BY

SECOND ORDER DERIVATIVE UV SPECTROSCOPY.

3.1 MATERIALS AND INSTRUMENTS

3.1.1 Materials and Reagents:

Standard drug sample Atenolol and Nifedipine were procured from West-

Coast Pharma Ltd., Ahmedabad, Gujarat, India.

Acetonitrile: Sisco Chem Pvt Ltd., Andheri, Mumbai, India.

Water: Distilled water collected from distillation assembly.

3.1.2 Instruments:

UV Spectrophotometer: Shimadzu-UV 1800 Spectrophotometer, Kyoto,

Japan

Weighing balance: Denver SI234, Germany

Digital pH meter: Systronic pH system 361, Ahmedabad, India

Sonicator: Electroquip Ultra sonicator, Ahmedabad, India

Ultra Centrifuge: REMI Research Centrifuge R24, Mumbai, India

Filters: Whatman filter paper

Pipette: Borosil pipettes of 1, 2, 5 and 10 ml capacity were used.

Volumetric flask: Borosil®-Volumetric flasks of 10, 25, 50, 100 and 200 ml

capacity were used. (All glassware were previously calibrated and made up

of low actinic glass)

Measuring cylinder: Measuring cylinder of 100 ml capacity was used.

3.1.3 Spectrophotometric Conditions

Mode: Spectrum

Scan speed: Fast

Wavelength range: 400-200 nm

Chapter No. 6


Absorbance scale: 0.00 – 2.00A

Initial base line correction: Water: Acetonitrile (60:40v/v)

3.2 PREPARATION OF SOLUTIONS

3.2.1 Preparation of Solvent mixture:

For the whole experimental work solvent was used in the ratio of 60:40 Water:

Acetonitrile.

3.2.2 Preparation of ATN standard stock solution

Accurately weighed 25 mg of ATN was transferred into 25 ml volumetric

flask and dissolved in 10.0 ml of ACN and diluted up to the mark with water

to get a stock solution having 1000 μg/ml concentration.

3.2.3 Preparation of ATN working standard solution

1ml of ATN standard stock solution was diluted to 10 ml with Solvent to get

working standard solution of 100 μg/ml concentration.

3.2.4 Preparation of solution for calibration curve of ATN

Appropriate aliquots (0.5, 0.75, 1.0, 1.25 and 1.50 ml) were withdrawn from

working standard stock solution of ATN and diluted up to 10 ml with solvent

to get standard solution having concentration 50, 75, 100, 125 and 150 µg/ml

of ATN.

3.2.5 Preparation of NIF standard stock solution

Accurately weighed 6.25 mg of NIF was transferred into 25 ml volumetric

flask and dissolved in 10.0 ml of ACN and diluted up to the mark with water

to get a stock solution having concentration of 400 μg/ml.

3.2.6 Preparation of NIF working standard solution

1ml of NIF standard stock solution was diluted to 10 ml with solvent to get

working standard solution containing 40 μg/ml of NIF.

3.2.7 Preparation of solution for calibration curve of NIF


working standard stock solution of NIF and diluted up to 10 ml with solvent to

get the standard solutions having concentration 20, 30, 40, 50 and 60 µg/ml of

NIF.

3.2.8 Preparation of standard solution containing mixture of ATN and NIF

Chapter No. 6


1 ml solution from Atenolol and Nifedipine standard stock solution were

mixed in volumetric flask and diluted up to 10 ml with solvent to get 100

μg/ml of ATN and 40 μg/ml of NIF.

3.2.9 Preparation of sample solution of marketed formulation

Accurately weighed 118.00 mg (equivalent to 50 mg of ATN and 20mg of

NIF) of tablet powder was transferred into 25 ml of volumetric flask, diluted

up to mark with solvent and sonicated for 30 minutes. The resulting solution

(1.0 ml) was transferred to 10 ml volumetric flask and diluted with solvent to

get a solution having concentration 100 μg/ml of ATN and 40 μg/ml of NIF.

3.3 OPTIMIZATION OF UV SPECTROSCOPIC METHOD

3.3.1 Selection of Wavelength for measurement:

The standard solutions of 10μg/ml of both the drugs ATN and NIF were

scanned over 200-400 nm. Data were recorded at an interval of 1nm. Overlay

of zero order spectra of ATN (10μg/ml) and NIF (10 μg/ml) is shown in

Figure 6.1.

Figure 6.1: Overlay of Zero order spectra of ATN(10μg/ml) and NIF (10 μg/ml)

The Second derivative spectrum of ATN exhibits a maximum absorbance at

245 nm while NIF reads zero and NIF exhibits a maximum absorbance at 218

nm while ATN reads zero which is shown in Figure 6.2 and 6.3 respectively

for ATN and NIF. Quantitative investigations using regression analysis have

established that the concentration of ATN and NIF correlates very well with

the measured second derivative peaks. Overlay of both the drugs ATN and

NIF is shown in Figure 6.4.

Chapter No. 6


Figure 6.2: Overlay of second order derivative of ATN

Figure 6.3: Overlay of second order derivative of NIF

Figure 6.4: Overlay of second order derivative of ATN and NIF

Chapter No. 6


3.3.2 Calibration Curves for ATN and NIF


working standard stock solution of ATN and of NIF and diluted up to 10 ml

with solvent to get the combined calibration standard solutions containing 50,

75, 100, 125 and 150 µg/ml of ATN and 20, 30, 40, 50 and 60 µg/ml of NIF.

The calibration standards were analysed by proposed method. The absorbance

of ATN and NIF were measured which was shown in Table 6.1and 6.2. The

calibration curves for ATN and NIF were constructed by plotting the

absorbance against respective drug concentrations which is shown in Figure

6.5 and 6.6.

Table 6.1: Calibration Curve data of ATN at 245 nm

Sr. No. Conc.

ATN (μg/ml)

Mean absorbance ± SD

(n=5)

%RSD

1 50 0.0062 ± 0.000100 1.61

2 75 0.0091 ± 0.000158 1.73

3 100 0.0118 ± 0.0002 1.69

4 125 0.0146 ± 0.000158 1.08

5 150 0.0173 ± 0.000100 0.57

Table 6.2: Calibration Curve data of NIF at 218 nm

Sr. No.

Conc. of NIF (μg/ml) Mean absorbance ± SD

(n=5)

%RSD

1 20 0.0025 ± 0.0001 0.48

2 30 0.0035 ± 0.0002 1.80

3 40 0.0045 ±0.0001 1.99

4 50 0.0054 ±0.0003 1.08

5 60 0.0066 ±0.0006 1.61

Chapter No. 6


Figure 6.5: Calibration Curve of ATN at 245 nm

Figure 6.6: Calibration Curve of NIF at 218 nm

3.4 METHOD VALIDATION

3.4.1 Linearity and range

Aliquots (0.5, 0.75, 1.0, 1.25 and 1.50 ml) were withdrawn from standard stock

solution of ATN and of NIF and diluted up to 10 ml with mobile phase to get the

combined standard solutions containing 50, 75, 100, 125 and 150 µg/ml of ATN

and 20, 30, 40, 50 and 60 µg/ml of NIF. The absorbance of ATN and NIF were

measured. The linear regression analysis data are shown in Table 6.3.

Table 6.3: Regression analysis data

Parameter ATN NIF

Linearity range (μg/ml) 50-150 μg/ml 20-60 μg/ml

Linearity equation y = 0.0001x + 0.0007 y = 0.0001x + 0.0005

Correlation co-efficient 0.9990 0.9981

Standard deviation of 0.000114000 0.000122000

y = 0.0001x + 0.0007

R² = 0.9990

0

0.005

0.01

0.015

0.02

0 50 100 150 200

Ab

sorb

an

ce

Concentration (μg/ml)

y = 0.0001x + 0.0005

R² = 0.9981

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0 10 20 30 40 50 60 70

Ab

sorb

an

ce

Concentration (μg/ml)

Chapter No. 6


intercept

Standard deviation of

slope

0.000114018 0.000114018

3.4.2 Precision

Method Repeatability: The precision of the method was checked by repeated

measuring the absorbance of ATN (50-150 μg/ml) and NIF (20-60 μg/ml)

solution (n=5) without changing the parameters of the proposed method

shown in Table 6.4 and 6.5 for ATN and NIF respectively.

Table 6.4: Repeatability data of ATN at 245nm

Conc.

(μg /ml) 50 75 100 125 150

Abs.

245nm

0.0062 0.0091 0.0118 0.0146 0.0173

0.0061 0.009 0.0116 0.0145 0.0174

0.0063 0.0092 0.0119 0.0145 0.0173

0.0062 0.0091 0.0117 0.0147 0.0175

0.0062 0.0091 0.0119 0.0147 0.0172

Mean 0.0062 0.0091 0.01178 0.0146 0.01734

S.D. 7.07E-05 7.07E-05 0.000130384 1E-04 0.000114

RSD 0.011405 0.00777 0.011068255 0.006849 0.006575

%RSD 1.14 0.77 1.10 0.68 0.65

Table 6.5: Repeatability data of NIF at 218nm

Conc.

(μg /ml) 20 30 40 50 60

Abs.

218nm

0.0025 0.0035 0.00451 0.0054 0.00665

Chapter No. 6


0.00256 0.00352 0.00452 0.00542 0.00667

0.00254 0.00357 0.00455 0.00543 0.00663

0.00253 0.00351 0.00456 0.00541 0.00664

0.00255 0.00355 0.00451 0.00545 0.00665

Mean 0.002536 0.00353 0.00453 0.005422 0.006648

S.D. 2.3E-05 2.91548E-05 2.35E-05 1.92E-05 1.48E-05

RSD 0.009078 0.008259139 0.005177 0.003548 0.002231

%RSD 0.90 0.82 0.51 0.35 0.22

Instrument Repeatability

The instrument precision was checked by repeated scanning and measuring of

the absorbance of ATN (100μg/ml) and NIF (40μg/ml) solutions (n = 6)

without changing the parameters of the proposed method. The %RSD values

for ATN and NIF were found to be 0.69% and 0.53%, respectively at 245 nm

and 218 nm (Table 6.6). Low relative standard deviation (<1) indicates that

the proposed method is repeatable.

Table 6.6: Instrument repeatability data for ATN and NIF

Sr.

No.

Concentration

of ATN

(µg/ml)

Abs. ATN

at 245 nm

Concentration

of NIF (µg/ml)

Abs. of NIF

at 218 nm

1 100 0.0117 40 0.00451

2 100 0.0118 40 0.00454

3 100 0.0117 40 0.00453

4 100 0.0119 40 0.00452

5 100 0.0118 40 0.00449

6 100 0.0117 40 0.00456

Mean 0.011766667 Mean 0.004525

SD 0.00008164 SD 0.0000243

RSD 0.006939064 RSD 0.005368

% RSD 0.69 % RSD 0.53

Chapter No. 6


Intra-day precision

Here, five different concentrations of ATN (50-150 μg/ml) and NIF (20-60

μg/ml) were prepared and absorbance were measured three times in a single

day and % RSD value was calculated to determine Intra-day variation which

was within limit (i.e. % RSD ≤2) as shown in Table 6.7 and 6.8.

Table 6.7: Intra-day precision data for ATN at 245 nm

Concentration(μg/ml) Mean absorbance± SD

(n=3)

%RSD

50 0.0061±0.0001 1.16

75 0.0090±0.0004 1.20

100 0.0117±0.0002 1.70

125 0.0145±0.0002 1.37

150 0.0172±0.0003 1.74

Table 6.8: Intra-day precision data for NIF at 218 nm

Concentration(μg/ml) Mean absorbance± SD

(n=3)

%RSD

20 0.0026±0.0001 1.89

30 0.0036±0.0002 1.50

40 0.0046±0.0001 1.98

50 0.0055±0.0004 1.70

60 0.0067±0.0006 1.85

Inter-day Precision

Five different concentrations of ATN and NIF were prepared, absorbance was

measured at three different days and % RSD value was calculated to determine

Inter-day variation which is within limit (i.e. % RSD ≤ 2) as shown in Table

6.9 and 6.10.

Table 6.9: Inter-day precision data for ATN at 245 nm

Conc. (μg/ml) Mean absorbance± SD

(n=3)

%RSD

50 0.0062±0.0001 1.61

Chapter No. 6


75 0.0091±0.0002 1.98

100 0.0118±0.0001 0.84

125 0.0146±0.0004 1.85

150 0.0173±0.0002 1.73

Table 6.10: Inter-day precision data for NIF at 218 nm

Conc. (μg/ml) Mean absorbance± SD

(n=3)

%RSD

20 0.0025±0.0001 0.48

30 0.0035±0.0003 1.80

40 0.0045±0.0001 1.99

50 0.0054±0.0004 1.08

60 0.0066±0.0006 1.61

3.4.3 Accuracy

Accuracy was determined by calculating the % recovery by standard addition

method. Known amount ATN and NIF of standard solutions (0, 4.0, 5.0 ml and

6.0 ml) added in pre-analysed sample solution of marketed formulation (1.0 ml)

which gave solutions having strength of 80%, 100% and 120% of middle

concentration from the range. Each solution was injected in triplicates and

recovery was calculated by measuring the absorbance.

The % recovery data obtained as shown in Table 6.11 , which suggests that the %

recovery ranges from 99.25- 101.15 % for ATN and 98.56-101.85 % for NIF.

Table 6.11: Recovery data of ATN and NIF

Level

Amt. of

sample

( μg/ml)

Amt. of std

drug

added(μg/ml)

Amt.

recovered

(μg/ml)

%Mean recovery ± SD

ATN NIF ATN NIF ATN NIF ATN NIF

80%

50

20 40 16

89.56 35.20

99.25±0.32 98.56±1.24 89.42 35.25

89.00 36.00

100% 50 20 50 20

100.95 41.17

100.75±0.80 101.85±1.41 99.87 40.95

101.45 40.10

120% 50 20 60 24 109.10 45.00

101.15±1.2 101.15±1.02 111.50 44.12

Chapter No. 6


110.17 44.40

3.4.4 Sensitivity

The sensitivity of method can be measured by Limit of Detection (LOD) and

limit of Quantitation (LOQ). The limit of Detection (LOD) and limit of

quantitation (LOQ) of the drug were calculated using following equations as

per ICH guideline.

The data of sensitivity obtained, is shown in Table 6.12 below,

Table 6.12: Results of sensitivity data for ATN and NIF

Parameter Results

ATN NIF

LOD (µg/ml) 5.21 2.0

LOQ (µg/ml) 15.81 7.25

3.5 ANALYSIS OF MARKET FORMULATION

For the analysis 50μg/ml solution of ATN and 20μg/ml solution of NIF from

marketed formulation (NILOL) were prepared, (n=5) and absorbance was

measured and %assay was calculated. The result of assay is shown in Table

6.13. Absorbance of both the drugs is shown in Figure 6.7 and 6.8.

Figure 6.7: Spectra of ATN (50μg/ml) and NIF (20μg/ml) (Marketed

Formulation)

Chapter No. 6


Figure 6.8: Second Derivative Spectra of ATN (50μg/ml) and NIF (20μg/ml)

(Marketed Formulation)

Table 6.13: Analysis of Marketed Formulation

Tablet Label claim (mg/tablet)

Assay ± SD (n=5)

(% of label claim)

NILOL ATN NIF ATN NIF

50 20 98.26±1.58 97.25±0.59

3.6 SUMMARY OF VALIDATION PARAMETER

The second order derivative method for simultaneous estimation of Atenolol

and Nifedipine was developed. The results obtained for each validation

parameter confirmed linearity, precision, accuracy of the developed analytical

method. The summary of developed analytical method is shown in Table 6.14.

Table 6.14: Summary of Validation Parameter

Parameter ATN NIF

Linearity Range (μg/ml) 50-100μg/ml 20-60μg/ml

LOD 5.21 μg/ml 0.52 μg/ml

LOQ 15.81 μg/ml 1.58 μg/ml

Recovery 99.25-101.15% 98.56-101.85%

Precision (%RSD)

Intra-day

0.85-1.07%

0.48-1.96%

Chapter No. 6


Inter-day 1.11-1.73% 1.56-1.99%

Correlation co-efficient(r2) 0.9990 0.9981

3.7 CONCLUSION

In this proposed method the linearity was observed in the concentration range of

50-100μg/ml and 20-60μg/ml with co-efficient of correlation, r2 = 0.9990 and r

2 =

0.9981 for ATN and NIF, respectively at 245nm and 218 nm. The additive present

in the tablet did not interfere with determination of ATN and NIF. So, the

developed Second order derivative UV spectroscopy method is simple, precise

and accurate and can be used for simultaneous determination of ATN and NIF

from pharmaceutical dosage forms. The method was validated as per International

Conference on Harmonization (ICH) guidelines.

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Chapter No. 6


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