Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Ketorolac Tromethamine and Olopatadine Hydrochloride in Pure and Pharmaceutical Formulation

 

N. Sunitha, Y. Sujitha*, B. Thangabalan, S. Manohar Babu

Department of Pharmaceutical Analysis, SIMS College of Pharmacy, Mangaldas Nagar, Guntur-522 002 (A.P.)

*Corresponding Author E-mail: sujithayarlagadda@gmail.com

 

ABSTRACT:

A simple, fast, precise, selective and accurate RP-HPLC method was developed and validated for the simultaneous determination of  Ketorolac tromethamine and olopatadine HCl from bulk and formulations. Chromatographic separation was achieved isocratically on a Inertsil ODS C18 column (250×4.6 mm, 5 μ particle size) using a mobile phase 0.1 M Sodium di hydrogen orthophosphate: Acetonitrile in the ratio of 55:45. The flow rate was 1 ml/min and effluent was detected at 235nm. The retention time of Ketorolac and olopatadine  were 2.507 min and 4.933 min. respectively. Linearity was observed in the concentration range of 4-24μg/ml and 12-72μg/ml for Ketorolac and olopatadine respectively with correlation coefficient of 0.999 for both the drugs. Percent recoveries obtained for ketorolac and olopatadine were 100.03% and 100.04%, respectively. The method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. The method developed can be used for the routine analysis of Ketorolac and olopatadine from their combined dosage form.

     

KEYWORDS: RP-HPLC Method; UV-VIS detection; Keterolac and olopatadine ophthalmic formulation.

 

 

INTRODUCTION:

Ketorolac tromethamine is chemically 5-benzoyl-2,3 dihydro-1H-pyrrolizine-1-carboxylic acid,2-amino-2-(hydroxy methyl)-1,3-propanediol1 (Figure:1), is a non selective COX inhibitor. The primary mechanism of action responsible for ketorolac's anti-inflammatory2, antipyretic3 and analgesic4,5 effects is the inhibition of prostaglandin synthesis by competitive blocking of the enzyme cyclooxygenase6-8(COX).

 

Figure 1: Chemical structure of Ketorolac tromethamine

 

Figure 2: Chemical structure of Olopatadine HCl

 

Olopatadine HCl is chemically 11-[(Z)-3-(Dimethylamino)propylidene]-6-11-dihydrodibenz[b,e] oxepin-2-acetic acid hydrochloride9 (Figure:2), is a relatively selective histamine H1 antagonist10 that inhibits the in vivo and in vitro type 1 immediate hypersensitivity reaction11 including inhibition of histamine induced effects on human conjunctival epithelial cells13. It inhibits the release of histamine from mast cells 14,15.

The literature survey indicates that there is no method reported on simultaneous estimation of ketorolac tromethamine and olopatadine HCl by RP-HPLC12. So there is a need for development of suitable method for routine analysis and extend it for their determination in formulation.

 

The objective of this study was to develop simultaneous estimation of ketorolac tromethamine and olopatadine HCl in pure and tablet dosage form. This method was found to be linear, precise, accurate, sensitive, specific, and robust, and therefore suitable for routine analysis.

 

MATERIALS AND METHOD:

Chemicals and Reagents:

Ketorolac and olopatadine was obtained as a gift sample from Gold Fish Pharma, Hyderabad.  HPLC grade acetonitrile and analytical grade Sodium dihydrogen orthophosphate obtained from SD Fine Chemicals Ltd, Mumbai. HPLC grade water was used to prepare all solutions.

 

HPLC Instrumentation and Chromatographic conditions:

The analytical separations were carried out on a Shimadzu HPLC system equipped with Photo Diode Array detector. The output of signal was monitored and integrated using LC –solutions 2000 software. The analytical column was Inertsil ODS C18 (150 × 4.6mm, 5µ). Mobile phase consisted of 0.1M Sodium Dihydrogen orthophosphate and Acetonitrile in the ratio of 55:45. Mobile phase was mixed, filtered through 0.45µmembrane filter and degassed under ultrasonication. The mobile phase was used as diluent. The flow rate was 1 ml/min and runtime was 7 minute. The column was maintained at ambient temperature. UV detection was measured at 235 nm and the volume of sample injected was 20 μl.

 

Preparation of standard stock solution:

A stock solution was prepared by taking 20mg of ketorolac and 60mg of olopatadine in 50ml volumetric flask and make up the volume with mobile phase to get concentration of 400μg/ml and 1200μg/ml respectively. Resultant solution was filtered through whatman filter paper. The standard chromatogram for ketorolac and olopatadine was shown in figure 3.

 

Preparation of working standard solutions:

The working standard solutions were prepared by accurately transferring the   (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 ml) aliquots of the standard stock solution in a series of 10 ml volumetric flasks. The volume was made upto mark with mobile phase to obtain concentrations of 4 μg-24 μg/ml (for ketorolac) and 12 μg-72 μg/ml (for olopatadine).

 

Preparation of sample solution:

10 containers were accurately weighed from which weight of empty containers is subtracted. Volume equivalent to 20mgof ketorolac was taken in 150ml volumetric flask to which volume equivalent to 60 mg of olopatadine was added. Mobile phase (50 ml) was added and sonicated for 15min. The solution was filtered through whatman filter paper No.1 and 0.5ml of resulting solution was taken in 10ml volumetric flask and volume made upto mark with mobile phase to obtain 20 µg/ml of Ketorolac and 60 µg/ml of olopatadine.

 

RESULTS AND DISCUSSION:

HPLC method development and optimization16-18:

To optimize the chromatographic conditions, different columns, mobile phases, flow rates etc., were tested19.  0.1M Sodium Dihydrogen Ortho Phosphate and Acetonitrile in the ratio of 55:45 was preferred as mobile phase because it resulted in a greater response to Ketorolac and olopatadine after several preliminary investigatory runs compared with the different mobile phase combinations. The effect of the flow rate was studied in the range 0.9 to 1.1 ml/min and 1ml/min was preferred to be effective. Under these conditions, the analyte peak obtained was well-defined and free from tailing. The retention time (RT) was found to be 2.507min (for ketorolac) and 4.933min (for olopatadine). The optimized chromatographic parameters were listed in table 1.

 

Validation of the method20-24:

When method development and optimization are complete, it is necessary to accomplish method validation. The validation studies include linear range (correlation coefficient), method precision (RSD, %), method accuracy (% recovery and RSD, %), sensitivity studies (LOD and LOQ), and robustness.

 

 

Figure 3: Standard chromatogram for Ketorolac and Olopatadine

 

 

Table 1: Optimized chromatographic parameters

Optimized Chromatographic parameters

Elution

Isocratic

Mobile phase

0.1M NaH2PO4 and  Acetonitrile

(55:45)

Column

Inertsil ODSC18column

Flow rate

1ml/min

Detection

235nm

Injection volume

20μl

Temperature

Ambient

Retention time

2.507min (for ketorolac)

4.933min (for olopatadine)

Run time

7 min

 

System suitability studies: 

System-suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (RT), number of theoretical plates (N), tailing factor (T), and peak asymmetry (AS), resolution (RS) were evaluated. The system suitability test was performed using five replicate injections of standards before analysis of samples. The system suitability method acceptance criteria set in each validation run were: capacity factor > 2.0, tailing factor ≤ 2.0 and theoretical plates > 2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. System suitability parameters were shown in table 2.

 

Table 2: System suitability parameters

Parameters

Ketorolac olopatadine

Retention time

2.507min

4.933min

Theoretical plates

3618

5267

Peak asymmetric factor

1.294

1.195

 

Linearity:

The linearity of the method was evaluated by preparing six series of standard solutions of Ketorolac and olopatadine in the range of 4 μg-24 μg/ml (for ketorolac) and 12 μg-72 μg/ml (for olopatadine) in mobile phase and injecting the solutions into the HPLC system. Excellent correlation between peak area and concentration was observed for both drugs with R2 = 0.999 (Figure.3). The regression equation was found to be Y = 14.92x + 16.96 (for ketorolac) and Y = 22.74x + 58.97(for olopatadine). Statistical data are presented in table 3 and the calibration curves were shown in figures 4 and 5.

 

Precision:

System precision: (Repeatability)

To study precision, five replicate standard solutions of 20 µg/ml of Ketorolac and 60 µg/ml of Olopatadine were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated. Results of system precision studies were shown in table 4.

 

Figure 4: Calibration curve of Ketorolac

 

Figure 5: Calibration curve of Olopatadine

 

Method precision: (Reproducibility)

The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 5 times on the same day and on different days for concentration of sample solutions of 20µg/ml of ketorolac and 60 µg/ml of olopatadine.  The result was reported in terms of relative standard deviation (% RSD). Results of method precision studies were shown in table 5.

 

 

Table 3: Linearity results for Ketorolac and olopatadine

S.No

Concentration of

Ketorolac (μg/ml)

Ketorolac Peak Area (mV.sec)

Concentration of

Olopatadine (μg/ml)

Olopatadine Peak Area (mV.sec)

1

4

75.801

12

326.013

2

8

136.908

 24

594.218

 3

12

195.876

 36

889.563

4

16

256.068

 48

1155.966

5

20

317.625

 60

1450.482

6

24

373.232

 72

1669.688

 

Table 4: Results of system precision for ketorolac and olopatadine

S. No

Ketorolac

Olopatadine

Rt(Mins)

Area (Mv.S)

Rt(Mins)

Area (Mv.S)

1.

2.513

309.125

4.937

1488.372

2.

2.507

311.457

4.927

1461.328

3.

2.517

309.552

4.94

1461.625

4.

2.510

308.568

4.933

1467.432

5.

2.513

308.828

4.937

1462.664

Mean

2.512

309.506

4.929

1468.284

S.D

0.0037

1.1503

0.0072

11.495

%RSD

0.1490

0.3717

0.1480

0.782

 

Table 5: Results of Method precision for ketorolac and olopatadine

S.NO

Ketorolac Standard Area = 317.625(Mv.Sec)

Olopatadine Standard Area = 1450.482 (Mv.Sec)

Peak Area

% Labelled Claim

Peak Area

% Labelled Claim

1

293.805

92.49

1464.198

100.95

2

292.448

92.073

1472.554

101.521

3

291.031

91.628

1470.959

101.411

4

294.819

92.820

1475.77

101.743

5

291.031

91.627

1470.959

101.41

Mean

292.6268

92.1276

1470.888

101.407

S.D

1.6821

0.5277

4.2242

0.2892

%RSD

0.5749

1.793

0.2872

0.285

 

Table 6: Results of Intermediate precision for ketorolac and olopatadine

 Drugs

S.NO

Analyst – 1

Analyst – 2

AREA (Mv.sec)

% ASSAY

AREA (Mv.sec)

% ASSAY

KETOROLAC

Standard Area = 317.625(mV.sec)

1

309.125

97.323

311.958

98.215

2

310.093

97.629

312.749

98.464

3

310.629

97.780

310.593

97.786

4

310.647

97.803

312.285

98.318

5

311.295

98.007

311.693

98.132

MEAN

310.295

97.7084

311.8556

98.183

S.D

0.8101

0.2540

0.8083

0.2542

% RSD

0.2610

0.2599

0.2591

0.2589

OLOPATADINE

Standard Area = 1450.482 (mV.sec)

1

1450.372

99.999

1452.942

100.169

2

1449.324

99.920

1448.742

99.880

3

1448.657

99.874

1449.761

99.950

4

1447.946

99.825

1448.593

99.867

5

1449.693

99.945

1451.763

100.088

MEAN

1449.198

99.9126

1450.36

99.9908

S.D

0.9353

0.0666

1.9192

0.1327

% RSD

0.0645

0.0666

0.1323

0.1327

 

 

Intermediate precision

The intermediate precision of the proposed method was determined by performing the method by two analysts (Analyst 1 and Analyst 2) for concentration of sample solutions of 20µg/ml of ketorolac and 60 µg/ml of olopatadine. The percent relative standard deviation (% RSD) for peak responses was calculated. The results for intermediate precision were shown in table 6.

 

Accuracy:

Accuracy of the method was confirmed by the standard addition method, which was carried out by performing recovery studies at 3 different concentrations 80%, 100% and 120% of these expected, in accordance with ICH guidelines, by replicate analysis (n=3). Known amount of standard drug solution of ketorolac (2µg/ml) were added to pre analysed sample solutions (16µg, 20 µg, 24 µg /ml) of ketorolac and Known amounts of standard solutions of olopatadine (6µg/ml) were added to pre analysed sample solutions (48µg, 60µg, 72µg /ml) of olopatadine.  The closeness of obtained value to the true value indicates that the proposed method is accurate. Recovery studies were shown in table 7.Chromatogram for recovery study was shown in figure 6.

 

%Recovery = [(Ct –Cpa)/ Cs] × 100.

 

Where, Ct = Total concentration of analyte

Cpa = Concentration of pre-analysed sample

Cs = Concentration of standard added to pre-analysed sample

 

Figure 6: Recovery study chromatogram for Ketorolac and Olopatadine

 

Table 7: Results of recovery studies for ketorolac and olopatadine

Name

Amount of pre analysed sample

(µg/mL)

Amount of drug added  (µg/mL)

Area

(mV.sec)

Amount recovered

(µg)

% Recovery

Average

% Recovery

S.D

%

RSD

KETOROLAC

16

16

16

2

2

2

250.480

251.791

251.824

17.871

17.965

17.967

101.4

98.25

98.35

 

99.33

 

1.7904

 

 

1.802

20

20

20

2

2

2

305.376

308.002

307.234

21.867

22.055

22.000

103.0

102.75

100

 

101. 91

 

1.6645

 

 

1.633

24

24

24

2

2

2

385.74

386.171

385.948

25.951

25.98

25.965

97.55

99.00

98.25

 

98.26

 

0.7251

 

 

0.737

OLOPATADINE

48

48

48

6

6

6

1207.042

1211.186

1209.507

53.89

54.075

54.000

98.16

101.25

100

 

99.8

 

1.5543

 

 

1.557

60

60

60

6

6

6

1477.523

1476.181

1476.987

66.06

66.00

65.036

101.00

100.00

100.6

 

100.5

 

0.5033

 

 

0.5008

72

72

72

6

6

6

1716.980

1713.901

1714.780

78.07

77.93

77.97

101.16

98.83

99.5

 

99.83

 

1.1995

 

1.201

 

Figure 7: Assay chromatogram for Ketorolac and Olopatadine

 

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing parameters like flow rate of mobile phase and detection wavelength

·         Change in the detection wavelength by ± 2nm (233nm and 237nm)

·         Change in flow rate by ± 0.1 ml/minute (0.9 ml/min and 1.1 ml/minute)

 

After each change, sample solution was injected and % assay with system suitability parameters were checked.

Robustness values were given in tables 8, 9.

 

Table 8: Results of Robustness on ketorolac

Parameter

Rt (min)

Area(mV.sec) (n=3)

% RSD

Flow rate  (ml/min)

 

 

 

0.9ml/min

2.501

316

1.169

1.1ml/min

2.763

323.540

0.0363

Wavelength (nm)

 

 

 

233nm

2.499

303.633

1.504

237nm

2.510

313.062

1.452

 

Table 9: Results of Robustness on olopatadine

Parameter

Rt (min)

Area(mV.sec) (n=3)

% RSD

Flow rate (ml/min)

 

 

 

0.9ml/min

4.927

1467.45

0.1134

1.1ml/min

5.436

1670.64

0.1684

Wavelength (nm)

 

 

 

233nm

4.931

1487.609

1.1025

237nm

4.929

1464.133

0.8075

 

Specificity

Specificity of an analytical method is its ability to measure the analyte accurately and specifically in the presence of component that may be expected to be present in the sample matrix. Chromatograms of standard and sample solutions were compared in order to provide an indication of specificity of the method.

 

Limit of Detection and Quantitation:

Detection and Quantitation limit were calculated by the method based on the standard deviation (s) and slope of the calibration plot, using the formula  

Limit of Detection     =    s  × 3.3/S

 

Limit of Quantitation   =     s × 10/S

 

Where s = the standard deviation of the response.

S = the slope of the calibration curve (of the analyte).

Results of LOD and LOQ were shown in table 10.

 

Table 10: Results of LOD, LOQ for ketorolac and Olopatadine.

Sample

LOD (µg/ml)

LOQ (µg/ml)

Ketorolac 

(3.3×0.52771)/ 14.925

=0.1166

 (10× 0.52771) / 14.925=0.3535

Olopatidine 

(3.3×0.2892)/ 22.747

=0.0419

 (10× 0.2892)/

22.747=0.1271

 

Assay of pharmaceutical formulation:

10 containers were accurately weighed from which weight of empty containers is subtracted. Volume equivalent to 20mgof ketorolac was taken in 50ml volumetric flask to which volume equivalent to 60 mg of olopatadine was added. Mobile phase (30 ml) was added and sonicated for 15min. The solution was filtered through Whatman filter paper No.1 and 0.5ml of resulting solution was taken in 10ml volumetric flask and volume made upto mark with mobile phase to obtain 20 µg/ml of Ketorolac and 60 µg/ml of Olopatadine. And the % Assay results were shown in tables 11 and 12.Chromatogram for assay was shown in figure 7.

 

Table 11: Results of % assay for ketorolac

Drug

S. No

Amount found  (mg/5ml)

% Assay

Ketorolac

1

2

3

2.009

1.9855

2.0076

100.45

99.27

100.38

 

Table 12: Results of % assay for olopatadine

Drug

S.No

Amount found  (mg/5ml)

% Assay

Olopatadine

1

2

3

6.0029

6.0056

6.0022

100.04

100.09

100.03

 

CONCLUSION:

In the present investigation simple, sensitive and economical new analytical method was developed for the Ketorolac and Olopatadine by RP-HPLC technique. The developed and validated RP-HPLC method was found to be more economical due to the lower consumption of mobile phase (1mL/min), along with short analytical run time of 7.0 minutes.

 

The result of analysis of formulation and recovery studies obtained by HPLC method were statistically validated and high percentage of recovery studies suggest that the developed methods were free from interference of excipients used in formulation. The HPLC method was statistically validated in terms of accuracy, precision, linearity and reproducibility. Hence above methods can be employed in quality control laboratories to estimate the amount of Ketorolac and olopatadine in bulk and in commercial formulations. Summary of validated parameters were shown in table 13.

 

Table 13: Summary of validated parameters for proposed method

Parameters

Ketorolac

Olopatadine

Linearity

4-24µg/ml

12-72 (μg/mL)

Correlation coefficient

0.999

0.999

System precision  (% RSD)

0.3717

0.782

Method precision (% RSD)

1.793

0.285

% Recovery

100.03

100.04

LOD

0.1166

0.0419

LOQ

0.3535

0.1271

 

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Received on 12.09.2013          Modified on 27.09.2013

Accepted on 05.11.2013     ©A&V Publications All right reserved

Res. J. Pharm. Dosage Form. and Tech. 6(1): Jan.-Mar. 2014; Page 37-43