A Novel Technique for the Quantification of Irinotecan HCl Injection

 

M.V. Kumudhavalli, C. Prakash, B.S. Venkateshwarlu, M. Kumar

Vinayaka mission’s College of Pharmacy, Yercaud main road, Kondappanaikenpatty,

Salem, Tamilnadu 636008, India

*Corresponding Author E-mail: prakashcv47@gmail.com

 

ABSTRACT:

This work aimed for the method development and validation of RP-HPLC method for quantitative determination of Irinotecan HCL in bulk and pharmaceutical formulation. More precise, accurate, sensitive, simple and economic HPLC method was developed and validated for quantitative determination of Irinotecan in bulk and dosage forms. Irinotecan was analyzed by using reverse phase Inertsil ODS 250 X 4.6 mm and 5µ particle size column. 0.02 M KH2PO4 buffer and acetonitrile were used as a mobile phase in the ratio of 40:60 v/v. The pH of the buffer was adjusted to 3.2 with o-phosphoric acid. The flow rate was 1.0 ml/min and the run time was 5 min. The analysis was carried out at 222 nm with UV detector at ambient temperature. The developed HPLC method was validated and stability studies were conducted under different conditions.  Irinotecan was eluted at retention time 2.1 min and the calibration curve was linear in the range 40-120 µg/ml concentration. Correlation coefficient was found to be 0.9999.  The Irinotecan test and standard stock solutions were stable over the period of 5 days.  The LOD and LOQ were found to be 0.8 ng/ml and 2.0 ng/ml. % RSD of Irinotecan content was < 0.5% and the assay was found to be 98.2-100%. The proposed method is observed to be rapid and selective, when compared to the methods reported in the literature. The retention time of Irinotecan was found to be very less (2.1 min). The method is most precise and economic with respect to solvent consumption.

 

KEYWORDS: Irinotecan, isocratic, HPLC analysis and Method validation.

 


INTRODUCTION:

Irinotecan ((S)-10 –[4-(-piperidino)piperidinocarbonyl oxoyl]-4,7-diethyl-4-hydroxy-1H-Pyrano[3,4,6,7] indolizino[1,2-b]diethyl-3,14[1H,12H]-dione mono  hydrochloride trihydrate) is an antineoplastic agent primarily used in the treatment of Metastatic colorectal cancer1-3. Irinotecan is a semi synthetic derivative of Comptothecin. Camptothecin is an alkaloid extracted from plant such as Camptotheca accuminata belongs to family nyssaceae. The empirical formula of entecavir is C33H38N4O6. It is marketed under the trade name Camptosar (Pfizer). According to literature review it was found that a very few methods are available for the estimation of Irinotecan4. Hence present study to develop a specific, precise, acoustic, linear, simple, rapid, validated and cost effective RP-HPLC, method for the estimation of the drug in dosage forms5-14. The proposed method has also been validated according to the ICH guidelines15,16

 

MATERIALS AND METHODS:

Experimental:

Chemicals and reagents:

Irinotecan reference standard - Reputed pharmaceutical company

Irinotecan injection 5 ml - Purchased commercial sample from the market

Acetronitrile - Spectrochem Pvt. Ltd., Mumbai

De ionized water - In house LABCONCO water purification system

KH2PO4 Buffer -Merck Pvt. Ltd., Mumbai

Ortho phosphoric acid - Merck Pvt. Ltd., Mumbai

H2O2 (3 % w/v) - Ranbaxy fine chemicals, Mumbai

NaOH - Loba chemie Pvt. Ltd., Mumbai

Zinc - Merck Pvt. Ltd., Mumbai

HCl - Merck Pvt. Ltd., Mumbai

 

Methods:

Instrumentation:

A Shimadzu HPLC system (10A VP) equipped with dual pump (LC 10AT) and SPD 10A VP UV-Vis detector was used for all the experiments. Data acquisition was performed by Class VP software. Analysis was carried out at 222 nm with a reverse phase Inertsil ODS column (250 X 4.6 mm, 5μm) at ambient temperature (250C). The mobile phase was a mixture of acetonitrile and phosphate buffer (0.02 M) in the ratio of 60:40 (v/v). The buffer pH was adjusted with O-phosphoric acid to 3.2. The flow rate was 1.0 ml/min and the runtime was 5.0 min. The mobile phase was degassed and filtered through 0.45 μm membrane filter before pumping into the HPLC system.

 

Preparation of KH2PO4 buffer solution:

Buffer solution with 0.02 M was prepared and the pH of the buffer was adjusted with O-phosphoric acid to pH 3.2.

 

Preparation of mobile phase:

KH2PO4 buffer solution and acetonitrile were mixed in the ratio of 40:60 v/v and was filtered through 0.45µm membrane filter and degassed for 10 min with nitrogen gas.

 

PROCEDURE:

CALIBRATION FOR HPLC METHOD:

Preparation of standard stock solution:

Weighed accurately 5 mg of Irinotecan standard and transferred to a 25 ml volumetric flask.  Dissolved with about 15 ml of mobile phase by sonication and finally filled up to the mark with mobile phase.  From the stock solution further dilutions were prepared with mobile phase to the required concentration

 

Preparation of test stock solution:

An accurately measured volume of injection, equivalent to 5 mg of irinotecan i.e., 0.25 ml was transferred into 25ml volumetric flask.  Dissolved it with 15ml of mobile phase by sonication and finally made up to the mark.  From this test stock solution further dilutions were prepared by diluting the appropriate volume of stock solution with mobile phase.

 

Figure 1: Chromatogram of standard Irinotecan HCl

 

Figure 2: Chromatogram of test Irinotecan HCl

 

Placebo interference:

0.25 ml of placebo was dissolved with 25 ml mobile phase and analysed. No peak observed at retention time of Irinotecan.

 

Figure 3: Chromatogram of Placebo

 

Initialization of the Instrument:

First, the column was placed on the instrument and switch on the instrument and washed with Double Distilled water for 30 min. Then run the mobile phase for 30 min for column saturation.

 

Chromatographic Conditions:

Based on the various trials, the following chromatographic conditions were finally optimized for the estimation of irinotecan.

Column- Inertsil ODS (250 x 4.6mm; 5µ)

Mobile phase-Buffer: Acetonitrile (40: 60)

Mode of separation-Isocratic

Buffer and strength-KH2PO4 buffer with 0.02M

pH of buffer-pH 3.2 with ortho phosphoric acid

Flow rate-1.0 ml/ min

Column temperature-Ambient

Run time-5.0 min

Injection Load-20 µl

Wave length of detection-222 nm with UV Detection

 

Method development:

Working standard of various concentrations was prepared by taking aliquots of standard solution which was injected

 

Method validation Linearity:

The ability (with in specified range) to obtain test results, which are directly proportional to the concentration of the analyte in the sample, is linearity test. To demonstrate the linearity minimum of 5 standard solution of irinotecan were prepared with concentration ranging from 50 to 150 % to the target assay concentration. Weighed accurately 5mg of Iirinotecan standard into 25ml of volumetric flask, then dissolved with 10 ml of mobile phase, sonicated the solution and finally made the volume up to the mark with mobile phase.

 

Figure 4: Chromatogram of Irinotecan HCl in optimized  chromatographic condition

 

Figure 5: Linearity Graph of Irinotecan HCl

 

 

Accuracy:

To validate the accuracy of test method recovery experiments were conducted at the concentration range of   80, 100, and 120 %. Test solutions were prepared from target test assay solution with a concentration of 80 µg/ml. Each test solution was prepared in triplicate and analysis was performed in triplicates. The assay content value at the beginning of validation was considered as true value (100 %) for recovery calculations. From analyzed data, % assay and % recovery were calculated and should be between 98 – 102 %.

 


Table 1: Linearity of Irinotecan HCl

Injection

Concentration (%)

Conc. of analyte (µg/ml)

Response

% Purity

1

50

40

4393513

98.8

2

75

60

6359898

98.89

3

100

80

8370847

99.6

4

125

100

10258390

99.8

5

150

120

12282459

99.8

Correlation coefficient 0.9999           Slope: 1.01636E±05. 

 

Table 2: Accuracy of Irinotecan HCl

Injection

Area

Quantity added (mg)

Quantity recovered (mg)

% Recovered

Mean% recovered

80 %prep.1

15021361

0.064

0.06374

99.5

 

     99.2

80% prep.2

15017844

0.064

0.06304

98.5

80% prep.3

15017644

0.064

0.06387

99.7

100%prep.1

16690402

 0.080

0.07864

98.3

 

     98.6

100%prep.2

16684430

 0.080

0.07888

98.6

100%prep.3

108460768

 0.080

0.07928

99.1

120%prep.1

183594422

0.096

0.09542

99.3

 

     99.5

120%prep.2

18359342

0.096

0.09513

99.5

120%prep.3

108460768

0.096

0.09427

99.7

                                                                                                                                            Mean

99.1

                                                                                                                                            S D

0.458

                                                                                                                                            % RSD

0.462


Method precision:

Measured accurately 0.25 ml of the formulation sample and transferred to 25ml of volumetric flasks.  Dissolved in mobile phase and finally made up to the mark.  The above procedure was repeated to make 6 different samples. Pipette out about 2ml of the above solutions and transferred to six different 5 ml volumetric flasks and filled up to the mark with mobile phase. 20 µl of each was injected in duplicate.  Calculate the % assay and % RSD. (table-3)

 

Robustness:

System suitability tests were performed after making the deliberate changes in the mobile phase to the developed HPLC conditions viz., flow rate (± 0.2 ml/min), composition (± 5 ml):  and buffer pH (± 0.2) as per method validation protocol. The results were shown in Table-4.

 

Table 3: Method precision of Irinotecan HCl

Injection

Peak area

% Assay

1

8345201

99.69

2

8342215

99.65

3

8343136

99.66

4

8343217

99.66

5

8343328

99.67

6

8344307

99.68

Mean

8343567

99.66

Standard deviation

1040.27

0.01472

%RSD

0.012

0.014


 

Table 4: Robustness of Irinotecan HCl

Parameter

% RSD of % purity

Tailing factor

Plate count

% Purity

Flow rate ± 0.2

        (ml)

1.2

0.92

1.3

2345

99.2

0.8

1.10

1.4

2549

99.5

Temperature± 5 şC

20

0.64

1.2

2285

99.1

30

0.70

1.3

2299

99.3

Mobile phase   composition ± 5 ml

45:55

1.10

1.3

2472

98.6

35:65

1.30

1.2

2283

98.7

pH of buffer ± 0.2

3.4

0.83

1.4

2457

99.5

3.0

1.01

1.2

2269

99.8

 


SUMMARY AND CONCLUSION:

More precise, accurate, sensitive, simple and economic HPLC method was developed and validated for quantitative determination of Irinotecan in bulk and dosage forms. Irinotecan was analyzed by using reverse phase Inertsil ODS 250 X 4.6 mm and 5µ particle size column. 0.02 M KH2PO4 buffer and acetonitrile were used as a mobile phase in the ratio of 40:60 v/v. The pH of the buffer was adjusted to 3.2 with o-phosphoric acid. The flow rate was 1.0 ml/min and the run time was 5 min. The analysis was carried out at 222 nm with UV detector at ambient temperature. The developed HPLC method was validated and stability studies were conducted under different conditions.  Irinotecan was eluted at retention time 2.1 min and the calibration curve was linear in the range 40-120 µg/ml concentration. Correlation coefficient was found to be 0.9999.  The Irinotecan test and standard stock solutions were stable over the period of 5 days.  The LOD and LOQ were found to be 0.8 ng/ml and 2.0 ng/ml. % RSD of Irinotecan content was < 0.5% and the assay was found to be 98.2-100%. The proposed method is observed to be rapid and selective, when compared to the methods reported in the literature. The retention time of Irinotecan was found to be very less (2.1 min). The method is also cost effective with respect to solvent consumption.

 

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15.    Validation of analytical methods, Definitions, and Terminology, ICH Harmonized Tripartite Guidelines ICH topic Q2A 1999.

16.    Validation of analytical procedures and methodology, Step – 4consenus, ICH Harmonized Tripartite Guidelines 1996.

 

 

 

 

Received on 13.06.2019           Modified on 17.07.2019

Accepted on 16.08.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(12): 6027-6030.

DOI: 10.5958/0974-360X.2019.01046.1