DEVELOPMENT AND VALIDATION OF DROTAVERINE .... RPA1516215015.pdfHCl Molecular weight: 433.97 Brand name: DOTRA; DROTASPA Melting point: 208-211 C Pharmacokinetics: Drotaverine has

176 | P a g e International Standard Serial Number (ISSN): 2319-8141

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International Journal of Universal Pharmacy and Bio Sciences 4(3): May-June 2015

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093*** ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE……!!!

DEVELOPMENT AND VALIDATION OF DROTAVERINE INJECTION

BY RP HPLC K. PAVAN KUMAR *,ASIYA BEGUM

ANURAG GROUP OF INSTITUTIONS, VENKATPUR(v) , GHATKESAR,

HYDERABAD. .

KEYWORDS:

Drotaverine, HPLC, validation.

For Correspondence:

K. PAVAN KUMAR *

Address:

Anurag Group

of Institutions,

Venkatpur (V) ,

Ghatkesar, Hyderabad.

ABSTRACT

In the present investigation, we have developed a simple and sensitive

RP-HPLC method for the estimation of Drotaverine in injection dosage

form. The HPLC conditions were optimized to obtain an adequate

separation of eluted compound. Initially, various mobile phase

compositions were tried to elute the drug and the mobile phase and flow

rate selection was based on peak parameters such as peak height, peak

capacity, number of theoretical plates, tailing or symmetry factor, run

time and resolution. Finally the mobile phase containing the mixture of

acetonitrile: 25mM potassium dihydrogen ortho-phosphate & 1 %

triethylamine buffer pH 3.0 in the ratio 40:60 was selected/ optimized

for this combination. The optimum wavelength selected for detection

was 226 nm where better detector response was obtained with retention

time of ~5.0 minutes for drotaverine.



INTRODUCTION:

Drotaverine is an antispasmodic drug, structurally related to papaverine. Drotaverine is

a selective inhibitor of phosphodiesterase 4, and has no anticholinergic effects drotaverine has

been shown to possess dose-dependent analgesic effects in animal models.

Generic name: Drotaverine HCL

Chemicalname:1,2,3,4-tetrahydro-6,7-diethoxy-1-(3,4-diethoxyphenyl)methylene)- isoquinoline

hydrochloride.

Molecular formula:

Molecular formula: C24H31NO4.HCl

Molecular weight: 433.97

Brand name: DOTRA; DROTASPA

Melting point: 208-211 °C

Pharmacokinetics:

Drotaverine has antispasmodic effect mediated via inhibition of phosphodiesterase-IV, specific

for smooth muscle. It has a rapid and direct action on the smooth muscle. It acts to correct cyclic

AMP and Ca imbalance at the spastic site, thereby relieving smooth muscle spasm and pain.

Adverse Reactions / Side Effects: Vertigo, nausea, vomiting, dry mouth.

Special Precautions:

Exercise caution in patients with renal/hepatic/cardiac dysfunction. Pregnacy, lactation.

Other Drug Interactions:

May attenuate the action of levodopa. Concurrent use of analgesics, antimuscarinics or

benzodiazepines. Additive beneficial effect with concurrent use of analgesics, antimuscarinics or

benzodiazepines.



Dosage: tablet and injection

Use: Antispasmodic.

MATERIALS AND METHODS

1.1. Chemicals and Reagents

HPLC grade Acetonitrile (Molychem, Mumbai), methanol (Molychem, Mumbai),

triethyleamine (Molychem, mumbai), potassium dihydrogen ortho phosphate (Thermo

fisher scientific, Mumbai) and ortho phosphoric acid (Ranbaxy, SAS nagar). The standard

sample of drotaverine hydrochloride was provided by our guide and injections were

purchased from local pharmacy.

1.2. Instrumental conditions for drotaverine HCL

Column : Grace vydac C18 (250X4.6)mm 5µ

Flow rate : 1.0 mL/min

Wave length : 226 nm

Injection volume : 20 µL

Temperature : Ambient

Elution mode : Isocratic

Mobile phase :

40:60 v/v (Acetonitrile: 25mM potassium

dihydrogen ortho phosphate & 1% Triethylamine

pH 3.0)

Run time run : 8 mins

Retention time : ~5 mins



1.3. Selection of wavelength

To 10 mg of drotaverine hydrochloride in 10 mL volumetric flasks, added 5 mL of methanol,

dissolved by sonication and made up to the final volume with methanol. From this 1 mL was

pipetted out into a 10 mL volumetric flask, made up to the volume with methanol, from this 1

mL was pipetted out into a 10 mL volumetric flask and made up to the volume with methanol to

get the final concentration of 10 µg/mL of drotaverine hydrochloride . The wavelength was

selected by scanning the above standard drug between 200 to 400 nm. The scanned results

showed that reasonably maximum absorbance for standard drotaverine hydrochloride was

recorded at 226 nm; therefore 226 nm was selected as the detection wavelength for the HPLC

investigation.

1.4. Preparation of 20% ortho phosphoric acid

Take 2 mL of ortho phosphoric acid and transfer to 10 mL volumetric flask; the final volume

was made up with HPLC grade water and mix well.

1.5. Preparation of Mobile phase buffer pH 3.0

Take 3.4 g of potassium di hydrogen ortho phosphate and 10 mL of triethylamine in a beaker

containing 1000 mL of water. Dissolve the salt by sonication and adjust the pH of the solution to

pH 3.0 with 20 % ortho phosphoric acid.

1.6. Preparation of Mobile phase

Take 400 mL of acetonitrile and 600 mL mobile phase buffer pH 3.0 separately and mix well.

Degas the mobile phase by sonicating the solution for 10 mins and filter the solution with 0.45µ

particle size.

1.7. Preparation of stock solution. (1000 µg/mL)

Weighed 25.2 mg of drotaverine hydrochloride in 25 mL volumetric flask, 5 mL methanol was

added and dissolved by sonication and made up to the mark with methanol (1000µg/mL).

1.8. Preparation of intermediate standard solution. (20 µg/mL)

From the stock solution (1000 µg/mL), 2 mL was pipetted out into a 100 mL volumetric flask;

the final volume was made up with mobile phase (20 µg/mL).

1.9. Preparation of standard solution. (20 µg/mL)

From the intermediate standard solution (20µg/mL), 4mL was pipetted out into a 10mL

volumetric flask, final volume was made up with mobile phase (8µg/mL).



1.10. Preparation of sample solution. (8 µg/mL)

Take ten ampoules and pool them all and pipette out the solution equivalent to 100 mg of

drotaverine hydrochloride (5 mL) and transfer to a clear and dry 100mL volumetric flask. Make

up the solution with mobile phase and mix well (1000 µg/mL). From the above solution (1000

µg/mL), take 5mL and dilute to 25mL with the mobile phase (200 µg/mL). From the above

solution (200 µg/mL), take 4mL and dilute to 100mL with the mobile phase (8 µg/mL).

Table-1-Dilution chart of standard drotaverine hydrochloride and injections

drotaverine

hydrochloride

Weight Taken

25.2 mg

Dilution 25 2 100 4 10

Sample solution 5 mL

(100 mg)

Dilution 100 5 25 4 100

1.11. Calculation of amount present in the capsule.

The amount of drug present in each tablet can be found by using the following formula,

Sample peak area × Concentration of standard

Standard peak area × Concentration of sample

2.7. Validation Procedure

Accuracy

The sample solutions were prepared in different concentrations such as 50, 100 and 150 % from

the sample solution, each concentration was injected in triplicate and the assay was performed as

per the test method. From this % recovery and the amount present or recovered were calculated.

Preparation of 50 % solution

Pool 10 ampoules, and pipette out the solution equivalent to 50 mg of drotaverine hydrochloride

(2.5 mL) and transfer to a clear and dry 100 mL volumetric flask. Make up the solution with

mobile phase and mix well. From the above solution, take 5mL and dilute to 25mL with the

mobile phase. From the above solution, take 4mL and dilute to 100mL with the mobile phase.




Pool 10 ampoules, and pipette out the solution equivalent to 100 mg of drotaverine

hydrochloride (5 mL) and transfer to a clear and dry 100mL volumetric flask. Make up the

solution with mobile phase and mix well. From the above solution, take 5mL and dilute to 25mL

with the mobile phase. From the above solution, take 4mL and dilute to 100mL with the mobile

phase.


Pool 10 ampoules, and pipette out the solution equivalent to 50 mg of drotaverine hydrochloride

(7.5mL) and transfer to a clear and dry 100 mL volumetric flask. Make up the solution with

mobile phase and mix well. From the above solution, take 5 mL and dilute to 25mL with the

mobile phase. From the above solution, take 4 mL and dilute to 100 mL with the mobile phase.

Precision

Instrumental Precision

The standard solution of drotaverine hydrochloride containing 8.06 µg/mL was given in 6

replication injections, and the result of the injections was observed.

Assay precision

Assay precision was determined by performing interday assay and intraday assay.

Linearity

Linearity was determined in the range of 50-150 % (50, 75, 100, 125 and 150%) targeted

concentration of assay procedure. five series of standard solutions containing

4.03,6.05,8.06,10.08 and 12.10 µg/mL of drotaverine hydrochloride were injected in triplicate.

Preparation of 50 % Linearity standard. (4µg/mL)

2 mL of intermediate standard solution (20 µg/mL) of drotaverine hydrochloride is taken in a 10

mL volumetric flask and make up with mobile phase.

Preparation of 75 % Linearity standard. (6µg/mL)



Preparation of 100 % Linearity standard. (8 µg/mL)











Ruggedness

Analyst to Analyst Variation

Pool ten ampoules and pipette out the solution equivalent to 100 mg of drotaverine hydrochloride

(5 mL) and transfer to a clear and dry 100 mL volumetric flask. Make up the solution with

mobile phase and mix well (1000 µg/mL). From the above solution (1000 µg/mL), take 5 mL

and dilute to 25 mL with the mobile phase (200 µg/mL). From the above solution (200 µg/mL),

take 4 mL and dilute to 100 mL with the mobile phase (8µg/mL). From this 20 µL was injected

through column separately by two different analysts in the same HPLC system and same column.

The results were compared and calculated for two analysts as % of labeled amount substance,

RSD of assay results. It is said to be rugged if the assay result is within 97-103 % and the % RSD

is not more than (NMT) 2 %.

Robustness

Variation in Flow Rate

The above prepared standard solution was injected into HPLC with ±0.2 mL/min from the

standardized assay flow rate as per the method developed (1.0 mL/min.). The system suitability

parameters were evaluated as per the test method for flow rates (0.8, 1.0 and 1.2 mL/min.).

Variation in wave length

The above prepared standard solution, 20 µL was injected into HPLC with ± 2 nm wavelength

from the standardized assay wavelength as per the method developed (226 nm.) The system

suitability parameters were evaluated as per the test method for wavelengths (224 and 228 nm).

Statistical evaluation

The mean of six replicate analyses was calculated and the results are expressed in standard

deviation and percent relative standard deviation.

The standard deviation is calculated from the following formula

∑ (xi - x)2



SD =

N – 1

xi = Individual measurement in a set

x = Arithmetic mean of the set

N = Number of replicates taken in the set

The relative standard deviation is calculated from the formula

% RSD = SD × 100/ Mean

RESULT AND DISCUSSION :

In the present investigation, we have developed a simple and sensitive RP-HPLC method for the

estimation of drotaverine injection dosage form DROTIN injection In this method, mobile phase

containing Acetonitrile and 25mM potassium dihydrogen ortho phosphate & 1% Triethyle amine

(pH 3.0) buffer in the ratio 40:60 was selected/optimized after trials with different mobile phases

in different ratios, different column for better peak shape for drotaverine.


The optimum wavelength selected as 238 nm for detection where better detector response for the

drug was obtained whose UV spectra is presented in Figs.1

Fig.1-UV spectra of drotaverine

1.13. Optimization of mobile phase and flow rate using standards



The various composition of mobile phase under reverse phase mode was undertaken 3 trials

using the instrument (JASCO. HPLC) and UV/VIS JASCO UV-1570 detector whose results are

presented here:

In trial 1, mobile phase acetonitrile: 25mM KH2PO4 (35:65); flow rate 1.0 mL/min and column

Grace vydac C18 (250X4.6)mm 5µ was used for the standard drug drotaverine.

Fig 2.1 HPLC Chromatogram of trial-1for drotaverine

In trial 2, mobile phase acetonitrile: 25mM potassium dihydrogen ortho phosphate & 1 %

Triethylamine pH 3.0 (50:50); flow rate 1.0 mL/min was used for the standard drug drotaverine



Fig 2.2 HPLC Chromatogram of trial- 2 for drotaverine

In trial 3, mobile phase acetonitrile: 25mM potassium dihydrogen ortho phosphate & 1 %

Triethylamine pH 3.0 (40:60); flow rate 1.0 mL/min was used for the standard drug

drotaverine

Fig 2.3 HPLC Chromatogram of trial-3 for drotaverine



1.14. Assay of drotaverine injection samples

Fig 3.1 HPLC-Chromatogram of standard solution injection-1












Fig. 3.7-HPLC Chromatograms of sample tablet solution in injection-1





1.15. VALIDATION

Accuracy

The HPLC Chromatograms recorded for tablet sample solution 50 % recovery in triplicate

injections are presented in Figs.4.1-4.3

Fig. 4.1 HPLC Chromatograms for recovery 50 % in injection-1



Fig. 4.2 HPLC Chromatograms of recovery 50 % in injection-2

Fig. 4.3 HPLC Chromatograms of recovery 50 % in injection-3


injections are presented in Figs.4.4- 4.6.



Fig. 4.4-HPLC Chromatograms of recovery 100 % in injection-1






injections are presented in Figs.4.7-4.9





Fig.4.9-HPLC Chromatograms of recovery 150 % in injection-3



Precision

The precision of the analytical method was studied by analysis of multiple sampling of

homogeneous sample. The precision results were expressed as standard deviation or

relative standard deviation.

Fig.5.1-HPLC Chromatograms of precision in injection-1








Fig 5.5-HPLC Chromatograms of precision in injection-5




Interday precision

Fig.5.7-HPLC Chromatograms of interday precision standard injection-1












Fig.5.13-HPLC Chromatograms of interday precision sample injection-1





Linearity

The linearity chromatograms for 50% standard solution in triplicate injections are presented

in Figs. 6.1-6.3.

Fig. 6.1- HPLC Chromatograms for linearity -50 % in injection-1





The linearity chromatograms for 75% standard solution in triplicate injections are presented

in Figs. 6.4-6.6.



Fig.6.4-HPLC Chromatograms of linearity-75 % in injection-1





The linearity chromatograms for 100% standard solution in six injections are presented in

Figs. 6.7-6.12.













The linearity chromatograms for 125% standard solution in triplicate injections are

presented in Figs. 6.13-6.15.





Fig.6.15.-HPLC Chromatograms of linearity-125 % in injection-3



The linearity chromatograms for 150 % standard solution in triplicate injections are

presented in Figs. 6.16-6.18.





Fig.6.18-HPLC Chromatograms of linearity 150 % in injection-3

Ruggedness

Analyst to analyst variation

The chromatograms for the assay of drotaverine for the analyst to analyst variation

(Analyst-B) are presented in Figs.7.1-7.6 for standard and Figs.7.7-7.9 for sample tablet

solution.

Fig 7.1 HPLC-Chromatogram of analyst-B standard solution injection-1













Fig. 7.7-HPLC Chromatograms of analyst-B sample solution in injection-1







Robustness

The chromatograms for the standard containing drotaverine (8 ppm) for the flow rate 0.8

mL/min., 1.0 mL/min and 1.2 mL/min are presented in Figs.8.1-8.3.

Fig.8.1- HPLC Chromatogram for flow of 0.8 mL/min.





The chromatograms for the standard containing drotaverine (8 ppm) for the wavelength 226

nm, 224nm and 228 nm are presented in Figs.8.4-8.6.

Fig.8.4- HPLC Chromatogram for wavelength 224 nm.







Assay for Drotaverine Injection

Table 3- Assay of drotaverine in Injections

Injection.

No.

Standard

peak area

sample peak

area

Label claim

in mg/mL

1. 318211.00 314862.25

20

2. 319275.75 316753.75

3. 316879.50 320011.50

4. 317712.25

5. 318173.58

6. 317392.00

Mean 317940.680 317209.167

%RSD 0.26 0.82

Amount in mg/mL 20.11

% Assay 100.57

Table 4.1- Results of 50 % recovery studies of drotaverine in injections

Injection.

No.

Standard

peak area

sample peak

area

Label claim

in mg/mL

1. 318211.00 160761.85

20

2. 319275.75 163909.25

3. 316879.50 160258.50

4. 317712.25

5. 318173.58

6. 317392.00

Mean 317940.680 161643.200

%RSD 0.26 1.22

Recovered Quantity in mg 20.50

% Recovery 102.49




Injection.

No.

Standard

peak area

sample peak

area

Label claim

in mg/mL

1. 318211.00 314862.25

20

2. 319275.75 316753.75

3. 316879.50 320011.50

4. 317712.25

5. 318173.58

6. 317392.00

Mean 317940.680 317209.167

%RSD 0.26 0.82

Amount in mg/tablet 20.11

% Assay 100.57


Injection.

No.

Standard

peak area

sample peak

area

Label claim

in mg/mL

1. 318211.00 468322.00

20

2. 319275.75 467774.53

3. 316879.50 466559.27

4. 317712.25

5. 318173.58

6. 317392.00

Mean 317940.680 467551.933

%RSD 0.26 0.19

Recovered Quantity in mg 19.76

% Recovery 98.82



Table 4.4- Results of global % recovery studies

Different levels in

% % Recovery

50 102.49

100 100.57

150 98.82

Average 100.826

SD 1.552

% RSD 1.54

Table 5.1- Results of precision studies of standard drotaverine (Instrumental precision)

Injection No. Standard peak area Standard Retention time

in minutes

1. 318211.00 5.125

2. 319275.75 5.133

3. 316879.50 5.125

4. 317712.25 5.125

5. 318173.58 5.133

6. 317392.00 5.117

Mean 317940.680 5.126333

%RSD 0.26 0.12

Table 5.2- Results of interday and intraday precision studies of drotaverine (Assay precision)

Different levels in

% % Assay

Assay 100.57

Ruggedness 101.42

Interday 104.42

Average 102.135

% RSD 1.98



Table 6- Results of linearity studies of standard drotaverine

S.NO Concentration in µg/mL Standard peak area

1 4.03 166666.187

2 6.05 243434.210

3 8.06 317940.680

4 10.08 383875.017

5 12.10 464622.417

Graph 1- Linearity chart of drotaverine standard

Table 7.1-Ruggedness data of analyst –A

Injection.

No.

Standard

peak area

sample peak

area

Label claim

in mg/Ml

1. 318211.00 314862.25

20

2. 319275.75 316753.75

3. 316879.50 320011.50

4. 317712.25

5. 318173.58

6. 317392.00

Mean 317940.680 317209.167

%RSD 0.26 0.82


% Assay 100.57



Table 7.2-Ruggedness data of analyst –B

Injection.

No.

Standard

peak area

sample peak

area

Label claim

in mg/mL

1. 318211.00 318039.01

20

2. 319275.75 320674.72

3. 316879.50 320985.01

4. 317712.25

5. 318173.58

6. 317392.00

Mean 317940.680 319899.580

%RSD 0.26 0.51


% Assay 101.42



Table 8.1- Robustness data of flow rate.

Flow rate in

mL

Retentension Time in

mins Area

0.8 5.813 395236.21

1.0 5.125 318211.00

1.2 4.333 285341.56

Table 8.2-Robustness data for wavelength

Wave length

in nm

Retentension Time in

mins Area

224 5.133 311532.25

226 5.125 318211.00

228 5.133 295342.34

DISCUSSION

In the present investigation, we have developed a simple and sensitive RP-HPLC method for the

estimation of drotaverine in injection dosage form. The HPLC conditions were optimized to

obtain an adequate separation of eluted compound. Initially, various mobile phase compositions

were tried to elute the drug and the mobile phase and flow rate selection was based on peak

parameters such as peak height, peak capacity, number of theoretical plates, tailing or symmetry

factor, run time and resolution. Finally the mobile phase containing the mixture of acetonitrile:

25mM potassium dihydrogen ortho-phosphate & 1 % triethylamine buffer pH 3.0 in the ratio

40:60 was selected/ optimized for this combination. The optimum wavelength selected for

detection was 226 nm where better detector response was obtained with retention time of ~5.0

minutes for drotaverine.


The maximum absorption of drotaverine recorded at 226 nm, therefore the optimum wavelength

was selected as 226 nm for detection where better detector response for all the three drugs was

obtained. Fig (1)

1.17. Optimization of mobile phase

In trial 1(Fig.2.1), the Rt for the standard drug drotaverine was 13.108 mins for the mobile phase

acetonitrile: 25mM KH2PO4 (35:65); flow rate 1.0 mL/min and column Grace vydac C18

(250X4.6)mm 5µ. The peak shape was broad and retention time was very high. To get a good



peak shape and with optimum retention time peak modifier 1 % triethylamine was used in

mobile phase in trial-2.

In trial 2 (fig.2.2), the Rt for the standard drotaverine was 4.333 mins for the mobile phase

acetonitrile: 25mM potassium dihydrogen ortho phosphate & 1 % Triethylamine pH 3.0 (50:50);

flow rate 1.0 mL/min in the same column and conditions as above. The peak shape was good but

peak was eluting before 5 mins so the amount of organic solvent in mobile phase was reduced in

trial-3

In final trial (Fig.2.3), third trial was repeated by using mobile phase acetonitrile: 25mM

potassium dihydrogen ortho phosphate & 1 % Triethylamine pH 3.0 (40:60); flow rate 1.0

mL/min in the same column and conditions as above which gave satisfactory elution with far

retention times of 5.133 minutes than previous trials. So the above trial was optimized as final.

Therefore the above solvent composition of mobile phase containing acetonitrile: 25mM

potassium dihydrogen ortho phosphate & 1 % Triethylamine pH 3.0 in the ratio 40:60 was

selected as optimized mobile phase for this work.

1.18. Final assay method

In the final assay of injections (Tables 3),(Fig.3.1-3.9), the mean amount and % Assay present

are calculated to be 20.11 mg/mL and 100.57 % of drotaverine for the label claim of 20 mg/mL.

Complied with the Limit of IP2010

Limits 90.0-110.0% for injections and the RSD for standard

peak area and sample peak area of drotaverine are calculated to be 0.26 and 0.82 which complied

with ICH guidelines (RSD NMT 2.0).

Validation

Accuracy

It is the closeness of agreement between the values which is accepted either as conventional true

value or an accepted reference value found.

In 50 % recovery study for sample (tables 4.1) (Fig.4.1-4.3) in triplicate injections, % RSD of

standard and sample for drotaverine was found to be 0.26 and 1.22 which complied with ICH

guidelines (RSD NMT 2.0). The % recovery at 50 % of drotaverine was found to be 102.49 %.

In 100 % recovery study for sample (tables 4.2) (Fig.4.4-4.6) in triplicate injections, % RSD of





In 150 % recovery study for injection (tables 4.3) (Fig.4.7-4.9) in triplicate injections, % RSD of



The % global recovery of drotaverine at all three levels 50 %, 100 % and 150 % was found to be

100.826 and % RSD for drugs at these levels are 1.54, which complied with ICH guidelines

(Recovery % for all spiked levels should be in the range of 98.0-102.0 % and %RSD NMT 2.0).

Precision

In precision study for standards (tables 5), (Fig.5.1-5.6), the %RSD for the six replicate

injections of retention time for drotaverine was calculated to be 0.12, which complied with ICH

guidelines (RSD NMT 2.0).

In precision study for standards (table 5.1), (Fig.5.1-5.6), the %RSD for the six replicate

injections of Standard area for drotaverine was calculated to be 0.26, which complied with ICH


In precision study for assay (table 4.4) at three different levels, the %RSD for the three different

levels was calculated to 1.54, which complied with ICH guidelines (RSD NMT 2.0).

In precision study for assay (table 5.2) preformed Inter day and Intraday, the %RSD for the assay

preformed within day and between day was calculated to 1.98, which complied with ICH


Linearity

In linearity study in (Tables 6), (Fig.6.1-6.18), (Graph 1) for the standard of concentrations, 4.03,

6.05, 8.06, 10.08 and 12.10 µg/mL for the drotaverine. The correlation coefficient for

drotaverine was 0.999. It is complied with the Limit of ICH guidelines (NMT 0.999).

Ruggedness

In this, two different analysts (Tables 7.1-7.2) (fig.7.1-7.9) used the same HPLC system and

same column. The amount found to be 20.11 mg/mL for drotaverine for analyst A and 20.28

mg/mL for drotaverine for analyst B and the % recovery was found to be 100.57 and 101.42

% for analyst A and analyst B respectively.

RobustnessVariation in flow rate

Drotaverine standard preparations was prepared & injected into HPLC with ± 0.2 mL of flow.

The system suitability parameters were evaluated as per the test method for flow rates (±0.2



mL).as per my method the actual flow rate is 1 mL/min. So I established system suitability

parameters with 0.8 &1.2 mL flow rates.

The obtained area from chromatograms in (Fig.8.1-8.3) (tables 8.1) was found 395236.21,

318211.00 and 285341.56 for 0.8, 1.0 and 1.2 mL/min respectively and retention time was found

to be 5.813, 5.125 and 4.333 mins for 0.8, 1.00 and 1.2mL/mins respectively

Variation in wave length

Drotaverine standard preparation was prepared &injected into HPLC with ±2 nm of wave length

the system suitability parameters were evaluated as per the test method for wavelength (±2

nm).as per my method the actual wavelength is 226 nm. So I established system suitability

parameters with 224 nm and 228 nm wavelengths.

The obtained area from chromatograms in (Fig.8.4-8.6) (Tables 8.2) was found 311532.25,

318211.00 and 295342.34 for 224, 226 and 228nm respectively and retention time was found to

be 5.133, 5.125 and 5.133mins for 224, 226 and 228 nm respectively.



Table 16: Optimized chromatographic conditions for drotaverine

Parameters Drotaverine

Column : Grace vydac C18 (250X4.6)mm 5µ

Flow rate : 1.0 mL/min

Wave length : 226 nm

Injection volume : 20 µL

Temperature : Ambient

Elution mode : Isocratic

Mobile phase :

40:60 (Acetonitrile: 25mM potassium dihydrogen

ortho phosphate & 1 % Triethylamine pH 3.0)

Run time run : 8 mins

Retention time : 5 mins

Theoretical plates : 30575

Tailing factor : 1.00

Asymmetric factor : 1.00

Correlation

Coefficient(r2)

: 0.999



CONCLUSION

HPLC method for estimation of drugs drotaverine in liquid dosage form injection of DROTIN

Injection was developed by using Grace vydac C18 (250X4.6) mm 5µ column with mobile phase

composition of acetonitrile: 25mM potassium dihydrogen ortho phosphate & 1 % triethylamine

pH 3.0 in the ratio 40:60 by isocratic elution technique, the flow rate was 1.0 mL/min and UV

detection at 226 nm.

Peak parameters

The theoretical plate for drotaverine was 30575 which is above the acceptance limit.

The asymmetric factor for drotaverine was 1.00 which is within the acceptance limit of 1 ±

0.5.

The tailing factor for drotaverine was 1.00 which is within the acceptance limit of ≤ 2.

The HPLC method for estimation of drotaverine complied with all the peak parameters like

theoretical plates, asymmetric factor and tailing factor.

Accuracy

Accuracy was determined through recovery study of the drug at three different levels. For

drotaverine the mean recovery was found to be 100.826 % and the % Relative Standard

Deviation was found to be 1.54.

The acceptance criteria in ICH guidelines for accuracy in recovery should be in the range

of 98-102 %.The % Relative Standard Deviation should be ≤ 2.0.

Thus the concluded HPLC method complied with accuracy parameters of ICH guidelines,

so the method is said to be accurate.

Precision

Precision of the method and instrument was determined by six replicate injections of

standard solution.

The % Relative Standard Deviation for retention time and standard peak area was found to

be 0.12 and 0.26 respectively.

Precision of the assay was determined by repeating the assay at three different levels. The

% Relative Standard Deviation for assay was found to be 1.54 for drotaverine.

Precision of the assay was determined by repeating at within day and between day. The %

Relative Standard Deviation for assay pwas found to be 1.98 for drotaverine.



The ICH guidelines acceptance criterion for precision is that % Relative Standard

Deviation should be ≤ 2.0. The % relative standard deviation (RSD) of assay and recovery

studies was found to be ≤ 2 and hence the proposed method was found to provide high

degree of precision and reproducibility.

Thus the concluded HPLC method complied with precision parameters of ICH guidelines,

so the method is said to be precise.

Linearity

The Linearity for drug was proved in the range of 4.03-12.10 µg/mL for drotaverine HCl.

Linearity correlation Coefficient was found to be 0.999 for drotaverine. The ICH guidelines

acceptance criteria for linearity, correlation Coefficient (r2) should be ≥0.999.

Th us the concluded HPLC method complied with linearity parameter of ICH guidelines, so

the method is said to be linear in the specified range.

Ruggedness

The ruggedness of the method was determined by carrying out the assay by different

analyst and to check the reproducibility.

The result of different analyst was found satisfactory with % assay for set of analysis

preformed by different analyst was found to be 100.57 and 101.42 % for drotaverine HCl

respectively.

The ICH guidelines acceptance criteria for % recovery should be 98-102 %.

Thus the concluded HPLC method complied with ruggedness parameters for different

analyst of ICH guidelines, so the method is said to be rugged for different analyst.

Robustness

The robustness of the method was determined by carrying out the assay to different

experimental conditions like slight deliberate variation of the assay, instrumental variations

of wavelength and flow rate. After change in the experimental condition there was no

significant loss of sensitivity, selectivity and resolution of the drug peaks. So the method is

said to be robust for deliberate change of wavelength and flow rate.

Assay

The proposed HPLC method was showing the recovery of 100.57 % for drotaverine HCl

injection.



From the above results it can be concluded that this finalized method shall be used for

estimation of drotaverine in any marketed formulation. This method was fully validated

and shall produce a consistent and reproducible result.

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http://www.ingentaconnect.com/content/aoac/jaoac;jsessionid=66n1mphfde2i9.alice

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DEVELOPMENT AND VALIDATION OF DROTAVERINE .... RPA1516215015.pdfHCl Molecular weight: 433.97 Brand name: DOTRA; DROTASPA Melting point: 208-211 C Pharmacokinetics: Drotaverine has