A Simple and Sophisticated RP-HPLC Approach for Quantifying of Irinotecan in API and in Pharmaceutical Dosage Form
Shiv Kumar Gupta1*, Neetu Sachan2, Phool Chandra3, Arun Kumar Sharma4
1Saraswathi College of Pharmacy, NH-09/24, Anwarpur, Pilkhuwa, Hapur, Uttar Pradesh, India.
2Department of Pharmaceutical Sciences, Maharana Pratap Group of Institutions Mandhana Campus,
3 Kms from IIT Kanpur - 209217, Uttar Pradesh, India.
3College of Pharmacy Teerthankar University, N.H.-9, Delhi Road, Moradabad - 244001, Uttar Pradesh, India.
4Pharmacy Academy, IFTM University, NH-9, Lodhipur, Moradabad, 244001, Uttar Pradesh, India.
*Corresponding Author E-mail: shivki4u@gmail.com
ABSTRACT:
Irinotecan is an anti-cancer drug used for the treatment of ovarian cancer. It works by inhibiting DNA-topoisomerase, type I. A simple, accurate and precise assay and rapid stability indicating RP-HPLC method has been developed and subsequently validated for the quantitative estimation of Irinotecan in API and its parental preparation. Proposed method is based on identification of drug Irinotecan in reversed phase mode using water’s C18 column maintained at an ambient temperature. The optimum mobile phase consist of mixture of Acetonitrile: Mixed phosphate buffer (potassium dihydrogen phosphate (0.01mm) with 1.0ml triethylamine) (30:70v/v) and pH was adjusted to 2.5 with O-Phosphoric acid (1:100,v/v). The flow rate was maintained at 0.8 ml/min. The injection volume was 20ml and detected at the wavelength of 221nm. The method was validated according to ICH guideline. The method was found to be accurate, reproducible and linear. The drug was exposed to variable stressed conditions and was analyzed by proposed method. There were no interfering peaks from exicipients, impurities or degradative products due to variable stress conditions. Thus the proposed method is specific for the quantitative estimation of Irinotecan and can be successfully applied in the quality control and stability sample of API and its parental preparation.
KEYWORDS: RP-HPLC, Irinotecan, Stressed degradative condition.
INTRODUCTION:
Irinotecan Hydrochloride chemically is 4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl [1,4'-bipiperidine]-1'-carboxylate, mono hydrochloride, trihydrate]. Its chemical structurte is shown in Figure 1. The drug is official in Merk Index1. It is available as I.V. infusions.
Figure 1: Irinotecan
Irinotecan is a semi synthetic derivative of campothecin (an alkaloid obtained fromCampotheca acuminate, belonging to family Nyssaceae), is an anti-tumor drug with Topoisomerase I inhibitory activity. Irinotecan interferes with the growth of cancer cells, which are eventually destroyed. Since the growth of normal cells may also be affected by the medicines, other effects may also occur. Irinotecan is found to be predominantly in the inactive carboxylate form at neutral pH and it is not a prodrug2-5.
Literature survey reveals few analytical methods for the determination of Irinotecan by HPLC in plasma and in blood from pharmaceutical preparation6,7.We describe a fast practical and precise method for the analysis of Irinotecan in bulk drug. The aim of the work is to develop a simple, accurate and cost effective HPLC method for bulk drug.
Irinotecan bulk drug and Irinotecan reference standard were supplied by Fresenius Kabi, Gurgaon. Irinotecan vial(2ml) containing 20mg/ml of Irinotecan was purchased from Parag Pharmacy, Delhi. Acetonitrile, Potassium dihydrogen phosphate, Triethylamine, 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 ´ 4.6 mm, 5µm.
The mobile phase was a mixture of Acetonitrile: Mixed phosphate buffer (potassium dihydrogen phosphate (0.01mm) with 1.0ml triethylamine) (30:70,v/v) and pH was adjusted to 2.5 with O-Phosphoric acid (1: 100,v/v). The flow rate was maintained at 0.8 ml/min. The injection volume was 20ml and detected at the wavelength of 221 nm at 25°C temperature.
50 mg of Irinotecan reference standard was accurately weighed and dissolved in 50 ml volumetric flask with 20ml of mobile phase and sonicate to dissolve and make up the volume up to the mark with mobile phase.
Take 5ml of this solution in 50ml volumetric flask and add 5 ml 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:
A 2ml vial of Irinotecan Hydrochloride was taken and transferred it to a 10ml calibrated volumetric flask. Around 5 mL of mobile phase mixture was added, and the solution sonicate for 10 min. Volume was made up to the mark with the same solvent mixture. The solution was filtered through 0.45 mm membrane filter. This solution is appropriately diluted to produce 100 mg/ml.
Forced degradation study of drug substance and drug product8-19
In order to establish whether the analytical method and the assay were stability indicating, bulk drug of Irinotecanand its formulation were stressed under hermolytic, photolytic, hydrolytic and oxidative stress conditions. The degradation conditions were optimized as per ICH guideline to maintain it in between 10 to 30%. For this study weighed amount of the Irinotecan Hydrochloride was treated with various stress conditions and analyzed by HPLC
Acidic degradation:
10.02 mg of Irinotecan Hydrochloride was weighed accurately and was kept with 1 ml of 0.1N HCl into a 10 ml volumetric flask. It was kept at room temperature for 2 h, and volume was made up with diluent.
Alkaline degradation:
10.11 mg of Irinotecan Hydrochloride was weighed accurately and was mixed with 1 ml of 0.01N NaOH into 10 ml volumetric flask. It was kept at room temperature for 2 h, and volume was made up with diluent.
Oxidative degradation:
10.16 mg of Irinotecan Hydrochloride was mlixed with 1 ml of 3%v/v H2O2 into 10 ml volumetric flask and was heated at 50°C for 45min, cooled and diluted to volume with diluent.
Water degradation:
In a 10 ml volumetric flask, 10.08 mg of Irinotecan Hydrochloride was added to 1 ml of HPLC grade water, and placed at room temperature for 1 h, and diluted to volume with diluent.
Thermal degradation:
Weigh accurately about 10.26 mg of thermally degraded Irinotecan Hydrochloride into 10 ml volumetric flask, 2ml of diluents was added, sonicate to dissolve, and diluted to volume with diluents.
RESULT AND DISCUSSION:
Method Development:
The proposed chromatographic method was first optimized on various columns like C-18 and C-8 with various mobile phase as single solvent or mixture of solvents with and without buffers (Acetate, phosphate at different pH) to add on. Finally, the best chromatographic resolution was obtained with Agilant software, spherisorb RP C18, (250 ´ 4.6 mm, 5µm) column. System suitability test was carried out on freshly prepared standard stock solution of Irinotecan Hydrochloride to check various parameters.
System suitability results were subject in Table 1
Table 1: System Suitability parameters (n=5)
|
S. No. |
Parameters |
Found |
Acceptable limits |
|
1 |
Theoretical Plates |
9796 |
Should be ≥1,000 |
|
2 |
Retention Time |
2.171 |
|
|
3 |
Calibration Range |
20-140 mg/ml |
|
|
4 |
Tailing Factor |
1.554 |
USP Tailing should be ≤ 3 |
Method validation:
The describe method has been validated for the assay of major components of bulk drug using following parameters20-26.
Specificity and selectivity were studied for examination of the presence of interfering endogenous components, a reference solution containing IrinotecanHydrochloride was prepared and was compared with blank Fig 2. Results indicate that the retention time of IrinotecanHydrochloride at 2.171 and none of the impurities were interfering in its assay. The results of assay were compiled in Table 2
|
S. No. |
Bulk drug |
||
|
Actual Amount Claim (in mg) |
Found (in mg) |
% Claim |
|
|
1. |
20.0 |
19.23 |
96.15 |
|
2. |
20.0 |
19.97 |
99.85 |
|
3. |
20.0 |
19.86 |
99.30 |
|
|
|
Mean |
98.43 |
Linearity was studied by preparing standard solution sets of different concentration level. The linearity range was found to be 20-140mg /ml. Calibration curves containing the standard of 20mg/ml to 140mg/ml were used for determination of the linearity of the Irinotecan Hydrochloride. Linearity range was found to be in between 20mg/ml to 140 mg/ml with Regression Equation y=0.0072x +0.0015 and with Correlation (R2) as 0.9997. Result of Linearity is expressed in Fig 3
Figure 2: General Chromatogram of Irinotecan Hydrochloride
Figure 3: Linearity Curve of Irinotecan Hydrochloride
Precision was studied to find out intra and interday variation in test methods of Irinotecan Hydrochloride 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.
Accuracy was determined by recovery studies of Irinotecan Hydrochloride, a fixed known concentration of IrinotecanHydrochloride reference standard was taken and subjected them to the proposed HPLC methods. Results of recovery study were shown in Table 3. The study was carried out at 3 different concentration levels. Each determination was performed in triplicate.
|
Sample |
Labeled amount (mg) |
Amount added (mg) |
Amount recovered (mg)* |
% Recovery |
Mean Recovery |
SD |
RSD |
|
1 |
24.89 |
4.68 |
29.43 |
99.52 |
99.553 |
0.05 |
0.050 |
|
2 |
4.98 |
29.75 |
99.61 |
||||
|
3 |
4.981 |
29.73 |
99.53 |
||||
|
4 |
25.1 |
9.98 |
34.69 |
98.88 |
98.933 |
0.18 |
0.178 |
|
5 |
10.14 |
34.93 |
99.13 |
||||
|
6 |
10.06 |
34.73 |
98.79 |
||||
|
7 |
24.94 |
14.89 |
39.68 |
99.62 |
99.670 |
0.16 |
0.161 |
|
8 |
14.61 |
39.49 |
99.85 |
||||
|
9 |
14.81 |
39.57 |
99.54 |
||||
|
Mean Recovery in presence of Placebo |
99.386 |
||||||
|
Standard Deviation |
0.396 |
||||||
|
Relative Standard Deviation |
0.398 |
||||||
Determination of limits of quantification and detection:
The limit of detection (LOD) and limit of quantitation (LOQ) for Irinotecan Hydrochloride was determined at a signal-to-noise ratio of 3:1 and 10:1, respectively. For the proposed method LOD & LOQ was found to be 10.70µg/ml and 32.42µg/ml repectively.
Robustness:
The robustness of an analytical method is the conditions when various parameters were intentionally variated by a small amount do not alter the ability of the method to remain unaffected by small changes in parameters. To calculate the robustness of the method, various experimental conditions like flow rate, column temperature, pH and mobile phase composition were purposely altered, and chromatographic changes were evaluated. At all conditions it was found that no significant change in the retention time of Irinotecan Hydrochloride was observed.
Stability:
Stability of reagents, mobile phase, standard and sample solutions were studied first for 24 hours and then for 48 hours and compared with the freshly prepared solutions and was found to be stable. The study was based on comparing results of area under curve, peak purity and its resolution. The value of area under curve was within 0.5% after 48 hours. The results indicate that the solutions were stable for 48 hours at ambient temperature as there was no formation of any unknown peak and solution remains stable.
Stress degradation studies of Irinotecan Hydrochloride in the powdered state:
The stress degradation studies were carried out as per the guidelines of the regulatory bodies. When Irinotecan Hydrochloride was subjected for degradation under stressed condition than results reflects that the drug was found to be not much affected under photolytic degradation, thermal degradation and is slightly got degraded in aqueous hydrolytic degradation but when it comes with the oxidative degradation, than drug got much degraded with 17.83% by using 3% of hydrogen peroxide and it gets degraded by 11.60% in acidic conditions while about 9.16% in alkaline condition.
Assay of the Parental dosage form:
The proposed validated method was successfully applied to determine Irinotecan Hydrochloride in partental dosage form. The result obtained for Irinotecan Hydrochloride vail was comparable with corresponding labeled amounts as shown in Table 5.
Table 5: Assay of Solid dosage form.
|
No. of Determinants |
Labelled Amount (mg/ml) |
Average Amount Determined |
% Assay Value |
% RSD |
|
6 |
20 (mg) |
99.85 |
99.85 |
0.075 |
An HPLC method with UV detection was developed and was validated for the determination of Irinotecan Hydrochloride 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 IrinotecanHydrochloride in parental dosage form. Hence it can be conveniently adopted for routine analysis.
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Received on 17.05.2023 Modified on 13.01.2024
Accepted on 30.06.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(10):4722-4726.