A Robustustic Eco-Friendly Analytical Method to Quantify Topotecan in Pharmaceuticals by RP-HPLC

 

Gupta S. K.^1,2*, Sachan N.², Chandra P.², Sharma A. K.³

¹Sanskar College of Pharmacy and Research, Opp. Jindal Pipes Ltd. NH-24, Ahead Masuri Canal, Ghaziabad, Uttar Pradesh 201302 (India)

²School of Pharmaceutical Sciences, IFTM University, NH-24, Lodhipur, Moradabad, Uttar Pradesh, India

³Pharmacy Academy, IFTM University, NH-24, Lodhipur, Moradabad, Uttar Pradesh, India

 

ABSTRACT:

Topotecan is an anti-cancer drug used against ovarian cancer. It acts by inhibiting DNA-topoisomerase-I. A simple, robust, accurate and precise assay RP-HPLC method having rapid stability indication has been developed and subsequently validated for the quantitative estimation of Topotecan in API and in solid dosage form. The method so proposed is based on quantification of drug Topotecan in reversed phase using water’s C18 column which was maintained at an ambient temperature. The selected mobile phase consist of mixture of Acetonitrile: DMF: Water (60:10:30,v/v) at pH maintained to 2.5 with O-Phosphoric acid (1 : 100,v/v). The flow rate used was 1.0ml/min and injection volume was 20ml, detected at the wavelength of 297nm. The method was validated according to ICH guideline. The method was found to be accurate, robust, reproducible and linear. The drug was exposed to variable stressed conditions and was studied using the proposed method. The results reflects That there were no interfering peaks from exicipients, impurities or degradative products generated during stress conditions. Thus the proposed method for quantifying the Topotecan is specific and can be applied in the quality control and stability study of sample as API and solid dosage form.

 

 

INTRODUCTION:

Topotecan Hydrochloride chemically is (S)-10-[(dimethyl amino)methyl]-4-ethyl-4,9-dihydroxy-1H pyrano [3',4':6,7] indolizino [1,2-6] quinoline-3,14-(4H,12H)-dione mono hydrochloride. The drug is official in Martindale, the Extra Pharmacopoeia¹. Marketed Formulations of Topotecan Hydrochloride are as capsule and I.V. infusions. Structure of Topotecan is shown in Figure 1.

 

Figure 1 Structure of Topotecan

 

Topotecan is semi synthetically derived from campothecin, which is an alkaloid obtained form Campotheca acuminate; family Nyssacea., it is a anti-cancer drug with Topoisomerase I inhibitory activity. Topotecan destroy cancer cells by interfering with their growth.  Topotecan at neutral pH present in body as inactive carboxylate form and it is not a prodrug^2-6.

 

Few analytical methods for the determination of Topotecan by HPLC in plasma and in blood from pharmaceutical preparation where already exist as suggested by the literature^7-12. We describe a fast practical and precise method for the analysis of Topotecan in bulk drug. The aim of work is to develop a simple, accurate robust and cost effective HPLC method for bulk drug.

 

EXPERIMENTAL:

Chemicals and materials:

Topotecan bulk drug and Topotecan reference standard were supplied by Fresenius Kabi, Gurgaon. Capsule formulation containing 1mg of Topotecan was purchased from Parag Pharmacy, Delhi. Acetonitrile, Di Methyl Formamide, O-Phosphoric acid used were of HPLC grade. HPLC grade water was purified using Millipore Purification System (Millipore, Molsheim).

 

Instrument:

Analysis by HPLC was performed using an isocratic system consisting of a pump (Waters 515), UV/VIS detector (Waters 2489). The system was connected with the help of agilant software in a computer system for data connection and processing. The analytical column used was spherisorb RP C₁₈ 250 ´ 4.6mm, 5µm.

 

Chromatographic conditions:

The mobile phase used was the combination of Acetonitrile:Di Methyl Formamide:Water (60:10:30,v/v) and pH was maintained at 2.5 with O-Phosphoric acid (1:100,v/v) and was filtered through  4.5 mm membrane filler. The flow rate used was 1.0 ml/min and injection volume was 20ml and detected was done at the wavelength of 297nm at ambient temperature.

 

Standard and Sample preparation:

25 mg of Topotecan reference standard was weighed accurately and dissolved in 25ml volumetric flask with Di Methyl Formamide and make up the volume with Di Methyl Formamide. Take 5ml of this solution in 50ml volumetric flask and add 5ml of O-Phosphoric acid (1:100,v/v) to it and diluted it with water to volume to obtain a standard stock solution having a concentration of 100 mg/ml. The sample solution was also prepared in the same manner and with the same concentration. 20ml of each of the solution were injected.

 

Preparation of test Solution:

Twenty capsules were weighed accurately and powder was collected in a mortar. Powder equivalent to 10mg of Topotecan was taken from capsules powder in a 10mL calibrated volumetric flask. A small amount of mobile phase mixture was added to dissolve the solid content, and the solution sonicated for 10 min. volume of the solution was made up to the mark with the same solvent mixture. The solution was than filtered through 0.45mm membrane filter to remove undissolved impurities. This solution is appropriately diluted to produce 100mg/ml.

 

Forced degradation study :

The bulk drug and capsules of Topotecan were stressed under hermolytic, photolytic, hydrolytic and oxidative stress conditions in order to study the performance of the proposed method. The results indicate that the proposed method were optimized to obtain target degradation between 10-30% as per ICH guideline.

 

Result and Discussion:

Method development:

Different parameters were studied to perform the system suitability test over a freshly prepared standard stock solution of Topotecan.

 

System suitability results reflect in Figure No. 2:

 

Figure 2 General Chromatogram of Topotecan Hydrochloride

 

Retention Time                                    2.17

Tailing Factor                                      1.55

Theoretical Plates                                1400

Calibration Range                               25-200mg/ml

 

Method validation:

The describe method was validated as per ICH guideline for the assay of major components of bulk drug using following parameters^13-18.

 

Specificity and selectivity:

Specificity and selectivity parameters were studied for the determination of the presence of interfering impurities and endogenous components. A Topotecan reference solution was prepared and was compared with blank. Results indicate that the retention time of Topotecan is at 2.17 and none of the impurities have significant effect on the assay of drug. The results of assay were compiled in Table No.1

 

Table No. 1 Specificity and selectivity of the proposed Method

Accuracy:

Accuracy was determined by recovery studies of Topotecan, in this a known amount of Topotecan reference standard was added it to preanalyzed sample and subjected them to the proposed HPLC methods. Results of recovery study were shown in Table No. 2. The study was carried out at 3 different concentration levels. Each determination was performed in triplicate.

 

Table No. 2 Recovery result of the proposed Method

 

Precision:

Precision was studied to find out intra and interday variation in test methods of Topotecan in the concentration range of 20mg to 200 mg/ml.  For three time on the same day and inter day. Precision was determined by analyzing corresponding standard daily for a period of three days. The % RSD in case intraday and interday was found to be 0.37 and 0.56 respectively.

 

Linearity:

Linearity of a solution is done by preparing standard solution sets of predetemined concentration level. The linearity range for Topotecan was found to be 25-200 mg /ml. Calibration curves containing the standard of 25 mg/ml to 200mg/ml were used for determination of the linearity of the Topotecan. Result of Linearity is compiled in Table No. 3 and a Lineratiy curve is shown in Fig No. 3

 

Table No. 3 Data indicating linearity of the proposed method

 

Stability:

Stability of reagents, mobile phase, standard and sample solutions were studied for 48 hours and compared with the freshly prepared solutions and was found to be stable.  

 

Assay of the Solid dosage form:

The proposed validated method was successfully applied to determine Topotecan Solid dosage form. The result obtained for Topotecan Capsules was comparable with corresponding labeled amounts as shown in Table 4.

 

Figure 3 Linearity Curve

 

Table 4 Assay of Solid dosage form

 

CONCLUSION:

An HPLC method with UV detection was developed and was validated for the determination of Topotecan drug substance. The proposed method has advantage of simplicity and convenience for the quantitation and cab be used for the assay of their dosage form. The method was found to be specific precise, accurate and sensitive for the estimation of Topotecan in Capsule dosage form. Hence it can be conveniently adopted for routine analysis.

 

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Accepted on 29.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4387-4390.