DEVELOPMENT AND VALIDATION OF ZOLEDRONIC ACID BY USING RP-HPLC Method. Shanmugasundaram.P*.,...

January-March 2013 Volume 1 Issue 1 Page 62

INTERNATIONAL JOURNAL OF FRONTIERS IN SCIENCE AND TECHNOLOGY

Research Article

ISSN 2321 – 0494

www.ijfstonline.org Received on : 25.02.2013,Revised and Accepted on : 20.03.2013

DEVELOPMENT AND VALIDATION OF ZOLEDRONIC ACID BY USING RP-HPLC

METHOD

Shanmugasundaram.P*., Rasagna.,Viswanath.V., Ravichandiran V,M.Sumithra.

School of Pharmaceutical Sciences, VISTAS, Vels University, Pallavaram, Chennai –

600117.

Abstract

The present work deals with the development of a precise, accurate, simple, specific,

reliable and less time consuming RP-HPLC method for the estimation of zoledronicacid.A

newer RP-HPLC method was developed for bulk drug and formulations. The proposed method

gives reliable assay results with short analysis time using mobile phase of triethylamine (pH3) :

acetonitrile. The proposed method was developed by HPLC Shimadzu Separation Module LC-

20AT Prominence Liquid Chromatograph using Hypersil silica, C18 250× 4.6mm, 5µ column

column with an injection volume of 20 l was injected and eluted with a mobile phase

composition of Triethylamine buffer PH

3 : Acetonitrile (50:50), which is pumped at a flow rate

of 1.1ml/min and detected by uv detector at 215nm. The retention time was found to be 3.649

min thus the method is not time consuming and can be used in laboratories for the routine

analysis of formulation. The method was validated in accordance with ICH parameters. The

method was also specific and robust.

KEYWORDS: ICH parameters, RP-HPLC, zoledronicacid

Corresponding author: Dr.P.Shanmugasundaram, E.Mail:[email protected]

INTRODUCTION

Zoledronic acid is an Antihypocalcemic, bone density conservation agent chemically

known as (1-hydroxy-2 (1h-imidazol- 1-yl)-1-phasphonoethyl)phosphoric acid. It is almost

white powder and freely soluble in water. It is mainly used to treat high blood calcium levels

namely hypercalcaemia and also used in cancer chemotherapy to treat bone cancer problems

that may occur with multiple myeloma. Figure 1 gives the structure of zoledronicacid

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MATERIALS & METHODS:

Equipment and Apparatus Used:

Single Pan balance (Metler Toledo)

Control Dynamics pH meter (Metler Toledo)

HPLC Shimadzu Separation ModuleLC-20AT Prominence Liquid Chromatograph

UV Visible Detector (SPD 20A Prominence)

Chromatographic data Software: Spinchrom

Hypersil silica, C18 250× 4.6mm., 5µ column.

Vacuum filter pump,(NUPORE filtrartion systems, 0.45 µ, 47mm)

Mobile phase reservoir

Ultrasonicator

Reagents used:

Acetonitrile HPLC grade

triethylamine,laboratory reagent

Water (HCL)

Orthophosphoric acid

METHOD DEVELOPMENT

Chromatographic parameters:

Instrument : HPLC Shimadzu Separation Module LC-20AT Prominence

Liquid Chromatograph

Detector : U.V – Visible detector

Column : Hypersil silica, C18 250× 4.6mm, 5µ column

Temperature : 30oC

Flow rate : 1.1ml/min

Wave length : 215 nm

Runtime : 10 min

Injection volume : 20 l

Mobile Phase: : Triethylamine buffer PH

3Acetonitrile(50:50)

Diluent : Mobile phase

Retention time : 3.649 min




Selection of Mobile phase:

A number of trials were made to find out the ideal solvent system (mobile phase) for

eluting the drug. The mobile phase containing Methanol: Water (50:50), Methanol: Phosphate

buffer PH

3 (50:50) , Acetonitrile : Triethylamine buffer PH

3 (50:50) was tried. Better peak

resolution and adequate retention time were obtained with the ratio of Triethylamine buffer PH

3: Acetonitrile (HPLC grade) (50:50)

Preparation of Mobile phase:

The mobile phase was prepared by mixing Triethylamine : Acetonitrile: (pH adjusted 3.0

with orthophosphroic acid) in the ratio of 50:50. The mobile phase is then sonicated using UV-

Sonicator to remove the impurities and dissolved gases, as they may lead to unwanted peaks in

the chromatogram.

Preparation of Triethylamine buffer:

Dissolve 4.0 ml of Triethylamine in 100oml of water to produce 0.02% buffer pH is

adjusted to 3.0 with 10% v/v Orthophosphoric acid.

Preparation of standard stock solution:

Stock solution of zolidronicacid(1mg/ml) was prepared by dissolving 25mg of

Zoledronicacid in 25 ml of volumetric flask containing mobile phase. The solution was

sonicated for about 10 min and then made up to volume with mobile phase. Daily working

standard solutions of Zolidronicacid was prepared by suitable dilution of the stock solution with

appropriate mobile phase.

Preparation of sample solution:

5 vials of zoledronicacid injection 25ml was transferred into volumetric flask, 70 ml of diluent

was added to it and was shaken by mechanical stirrer and sonicated for about 10 minutes by

shaking at intervals of five minutes each and was diluted up to the mark with diluent and the

solution was filtered through 0.45 m filter before injecting into the HPLC system.

Procedure:

Separately inject the blank, triplicate injections of standard and duplicate injections of

sample preparations to the HPLC system and record the chromatograms. The results for sample

were shown in table.

METHOD VALIDATION

1 LINEARITY:

The linearity of the method was demonstrated over the concentration range of 60-140µg

/ ml. Aliquots of 80-120 µg / ml were prepared from stock solution and labeled as solution 1, 2,



3, 4,5 respectively. The solutions were injected in to HPLC system as per test procedure. A

calibration curve was plotted for concentration Vs peak area and was given in the Figure1.


Acceptance criteria:

Correlation Coefficient (r2) should be not less than 0.9990.

2 .PRECISION

Method precision:

Five samples of 100 µg/ml solutions were prepared from stock solution and injected into

the HPLC system as per test procedure.


% Relative standard deviation of results should not be more than 2.

System precision:




% Relative standard deviation of results should not be more than 2.

Intraday Precision:

Intraday precision of test method is demonstrated by 7 injections of the same batch(same conc)

of samples at 0, 1, 2, 3, 4,5 and 6 hrs on same day.

Interday Precision:

Interday precision of test method is demonstrated by 5 injections of the same batch(same conc)

of samples on 5 successive days.

3 . ACCURACY

The accuracy of the method was evaluated by determination of recovery of

zoledronicacid at three levels of concentrations. The sample solutions were spiked with

zoledronicacid standard solutions corresponding to 80, 100, and 120% of nominal analytical

concentrations. (80µg/ml, 100µg/ml and 120µg/ml). The results showed good recovery within

limits (98% – 102%).

4 SPECIFICITY

A) zoledronicacid Identification

Solutions of Standard and Sample were prepared as per test procedure and injected into the

HPLC system.

B) Stress degradation studies by treating with alkali and acid:

Zoledronicacid was treated with 0.1M NaOH and 0.1M HCl. Solutions of sample was

prepared as per test procedure and injected into the HPLC system.




5 ROBUSTNESS:

The robustness of the proposed method was determined by analysis of aliquots from

homogenous lots by differing physical parameters like flow rate and mobile phase composition

which may differ but the responses were still within the specified limits of the assay.

Effect of variation of Mobile Phase Composition

A study was conducted to determine the effect of variation in mobile phase ratio by Changing

the ratio of mobile phase i.e Triethylamine: Acetonitrile: (pH adjusted 3.0withorthophosphroic

acid)in the ratio of 50:50. Standard solution was prepared and injected into the HPLC system

and recorded the chromatograms.

Acceptance criteria

Tailing Factor of zoledronicacid standard should not be more than 2 for Variation in

composition of mobile phase.

The % RSD of asymmetry of zoledronicacid standard should be not more than 2 for

Variation in composition of mobile phase.

Effect of variation in Buffer PH

A study was conducted to determine the effect of variation in Buffer PH. Standard solution

was prepared and injected into the HPLC system by maintaining PH 3.5and 4.5. The effect of

variation in Buffer PHwas evaluated. The results were discussed in the Table: 5

Acceptance criteria

The tailing factor of standard should be not more than 2.0 for Variation in flow.

The % RSD of Asymmetry of zoledronicacid standard should be not more than 2 for variation

in flow.

Effect of variation of Flow Rate

A study was conducted to determine the effect of variation in flow rate. Standard solution

was prepared and injected into the HPLC system by keeping flow rates 0.8 ml / min and 1.2 ml /

min. The results were discussed in the Table: 6

Acceptance criteria

The tailing factor of standard should be not more than 2 for Variation in flow.

The % RSD of ofzoledronicacid standard should be not more than 2 for variation in

flow.


January-March 2013 Volume 1 Issue 1 7

Effect of variation in Temperature

A study was conducted to determine the effect of variation in temperature. Standard

solution was prepared and injected into the HPLC system by keeping temperature 30˚c, 27˚c

and 25˚c. The effect of variation of temperature was evaluated. The results were discussed in the

Table: 7


Acceptance criteria

The tailing factor of standard should be not more than 2.0 for Variation in flow.

The % RSD of asymmetry of zoledronicacid standard should be not more than 2.0 % for

variation in flow.

6 RUGGEDNESS:

Ruggedness is the degree of reproducibility of the results obtained under a variety of

conditions. It is checked that the results are reproducible under differences in conditions,

analysts and instruments. And hence the proposed method was found to be rugged.

The Chromatograms were shown in Fig:8

7 LIMIT OF DETECTION (LOD)

The parameter LOD was determined on the basis of response and slope of the regression

equation. The LOD for this method was found to be 0.989.

8 LIMIT OF QUANTIFICATION (LOQ)

The parameter LOQ was determined on the basis of response and slope of the regression

equation. The LOQ for this method was found to be 2.1872.

9. SYSTEM SUITABILITY

A Standard solution of zoledronicacid working standard was prepared as per procedure

and was injected six times into the HPLC system. The system suitability parameters were

evaluated from standard Chromatograms obtained by calculating the % RSD of retention times,

tailing factor, theoretical plates and peak areas from six replicate injections.

Acceptance criteria: The % RSD for the retention times of principle peak from 6

replicate injections of each Standard solution should be not more than 2

The number of theoretical plates (N) for the zoledronicacid peaks should be not less than

2000. The Tailing factor (T) for the zoledronicacid peaks should be not more than 2

From the system suitability studies it was observed that all the parameters were within limit.

Hence it was concluded that the Instrument, Reagents and Column were suitable to perform the

assay.



The results were discussed in Table 3

ASSAY

Assay of different formulations available in the market was carried by injecting sample

corresponding to equivalent weight into HPLC system. And percentage purity was found out

by following formulae. Recovery studies were carried out.]


Calculated the percentage purity of zoledronicacid present in tablet using the formula:

Splarea x Stddilx Avgwt x P

Percentage purity = ----------------------------------------

Stdarea x Spldil x L.C x 100

Where,

P = (%) potency of zoledronicacid working standard use

L.C = Label claim

Avgwt = Average weight of tablets

RESULTS AND DISCUSSIONS

LINEARITY: In the new RP-HPLC method for zoledronicacid by using Methanol:

Acetonitrile: Acetate Buffer (40:40:20) as a mobile phase we obtained a linearity curve by

taking the sample at concentrations ranging from 80-120 µg/ml. Retention time – 3.42. From

the peak response obtained correlation coefficient was found to be 0.999.

ACCURACY

The accuracy of the method was evaluated by determination of recovery of

zoledronicacid at three levels of concentrations. The sample solutions were spiked with

zoledronicacid standard solutions corresponding to 80, 100, and 120% of nominal analytical

concentrations. (80µg/ml, 100µg/ml and 120µg/ml). The results showed good recovery within

limits (98% – 102%).

PRECISION

Method precision:



The % Relative standard deviation of results should be 0.09.

System precision:





The % Relative standard deviation of results should be 0.10.


SYSTEM SUITABILITY

A Standard solution of zoledronicacid working standard was prepared as per procedure

and was injected six times into the HPLC system. The system suitability parameters were

evaluated from standard Chromatograms obtained by calculating the % RSD of retention times,

tailing factor, theoretical plates and peak areas from six replicate injections.

The % RSD for the peak areas of principle peak from 6 replicate injections of each Standard

solution should be 0.07 (limit should be less than-2)

ROBUSTNESS: The studies were carried out by using different chromatographic condition

like follows:

a) Change in flow rate

b) Change in organic phase composition in the mobile phase

c) Change in pH of mobile phase

The % RSD was found to be below 2% i.e. within the limit. Thus the results show that the

method is precise under different a chromatographic condition which is shown in table below.

LIMIT OF DETECTION (LOD)

The LOD for this method was found to be 0.989

LIMIT OF QUANTIFICATION (LOQ)

The LOQ for this method was found to be 2.1872

All the ICH validation parameters were found to be within the limits

CONCLUSION:

The praposed analytical method is simple, economical

,rapid,sensitive,reproduciable,and accurate for the estimation of zolidronic acid.A newer RP-



HPLC method was developed for bulk drug and formulations. The praposed method gives

reliable assay results with short analysis time using mobile phase of triethylamine (pH3) :

acetonitrlie

The above method does not suffer from any interferance due to common

excipients. Thus it was show that praposed methods could be successfully appiled toestimate

commercial pharmaceutical products contining zolidronic acid .Thus the above findings will

enable the quantification of the drug for future investigation in the field of analytical chemistry.


Figure 1: Zoledronicacid structure

Figure 2: linearity

Figure 3: chromatogram of of zoledronicacid.by RP-HPLC Method




Figure 4: System Suitability of zoledronicacid.

Table 1: Standard Concentration To Peak Response For zoledronicacid

Parameters

Zoledronicacid

215nm

Concentration (µg/ml) 80-120 µg/ml



Correlation 0.999

Slope 22481.8

Y- intercept 17879

Acecptance criteria: The Correlation coefficient (r2) should be more than 0.99


Table 2: specificity of zoledronicacid.

Parameters Zoledronicacid

Area under curve

2106934

20699684

2107438

2341854

2345450

Mean 2676908

SD 25079

%RSD 0.80

Retention time (min) 3. 42



Theoretical plates 5919

Tailing factor 1. 24


Table 3: System suitability for Zoledronicacid by RP-HPLC

Parameter Observed Value

USP tailing 1.24

Retention time 3.43




REFERENCES

[1] Michael , schartz , krull. Analytical method development and validation 2004; 24-46

[2] Sharma bk. Instrument methods of chemical analysis ,Introdiuction analysis chemistry ,goel

publishing house meerut, 23th edition; 2004, p. 223-38

[3] Basic edition in analytical chemistry,17th

edition; 2001, p. 323-42

S.No Area response of Zoledronic acid

1.0 2086027

2.0 2084140

3.0 2087209

4.0 2088028

5.0 2088003

6.0 2087320

Average 2086788

SD 2145

%RSD 0.07



[4] Willard hh, Merritt ll. Instrumental method of analysis, cbs publishers and distributors ,

New delhi, 7th

edition; 1986, p. 256-64

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Simultaneous Estimation Of Ranitidine Hydrochloride And Domperidone In Combined

Tablet Dosage Form. Ijpsr, 2010; 1(8).

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