Development and validation of Spectrophotometric method and TLC Densitometric Determination of Irinotecan HCl in pharmaceutical dosage forms

1 Introduction

Irinotecan HCl (Fig. 1) (7-ethyl-10-[4-(l-piperidino)-1-piperidino]carbonyloxy camptothecin; CPT-11), a semisynthetic water-soluble derivative of camptothecin, an alkaloid isolated from Camptotheca acuminata, has unique antitumor activity, preventing DNA synthesis by inhibiting topoisomerase I. Irinotecan-HCl is a new drug which has antitumor activity in a wide range of malignancies, such as metastatic colorectal cancer, upper gastrointestinal, pancreatic, lung and breast cancer, and gynaecological malignancies (Chollet et al., 1998; Hwang et al., 2003). Camptothecin is an alkaloid extracted from a plant known as C. acuminata belonging to the family Nyssaceae (Sawada et al., 1991; Kawato et al., 1991). Irinotecan (Budavari, 2001; Kono and Hara, 1991; Poujol et al., 2003; De et al., 2003) inhibits the action of enzyme topoisomerase I which produces reversible single strand breaks in DNA during DNA replication in tumor growth. Irinotecan hydrochloride is a pale yellow to yellow crystalline powder, with the empirical formula C₃₃H₃₈N₄O₆·HCl·3H₂O and molecular weight of 677.19. It is slightly soluble in water and organic solvents. Literature survey reveals many Chromatographic methods for the determination of Irinotecan in biological fluids like plasma, blood and urine (Shende and Gaud, 2009; Murali et al., 2007; Yang et al., 2005; Owens et al., 2003; Hang et al., 2009; Baylatry et al., 2010; Rivory and Robert,1994; Edmond et al., 2005). In the present research work an accurate and economical UV Spectrophotometric and TLC Densitometric method has been developed for the estimation of Irinotecan in formulation and bulk drug substances.

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Figure 1

Structure of Irinotecan HCl.

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2 Materials and method

2.1

2.1 UV spectrophotometric method

UV–Visible double beam spectrophotometer, Shimadzu model-1700 having spectral bandwidth 3 nm and of wavelength accuracy ±1 nm, with 1 cm quartz cells was used. All other chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals Corporation Ltd., Mumbai, India. Deionized and ultrapure water used in all experiments was obtained from Milli-Q system (Millipore). Silica gel 60F₂₅₄ TLC plates (20 × 10 cm & 10 × 10 cm, layer thickness 0.2 mm, Merck, Germany) were used as stationary phase. The injection formulation available in Indian market under the trade name of “Irinocam” of 5 ml (100 mg) per vial, manufactured by Dr. Reddy's Laboratories, Hyderabad, India. Solubility of the drug was checked in solvents like water, methanol, DMSO.

The spectra of drugs in this solution were recorded. Absorbance of drug exhibited distinct λ_max in water. Hence water was selected as a solvent for further studies. However, the drug is freely soluble in water hence water was chosen as a solvent for developing the method and the cost of water is low as compared to the other solvents.

2.1.1

2.1.1 Preparation of stock solution

Weigh and transfer accurately, equivalent to 100 mg of Irinotecan as working standard into 100 ml volumetric flask, dissolve and dilute up to mark with distilled water. Transfer 10 ml solution from the stock to 100 ml volumetric flask with distilled water to produce a concentration of 100 μg/ml, use this as standard stock solution.

2.1.2

2.1.2 Preparation of working standard solution

Weigh accurately 100 mg of Irinotecan, transfer to a 100 ml volumetric flask, and make up to volume with water and filter. Take 10 ml from that solution to 100 ml volumetric flask and make up the volume to 100 ml with distilled water. From the stock solution different concentrations of Irinotecan, were prepared and scanned in UV region at 200–400 nm. The wavelength corresponding to maximum absorbance (λ_max) was found at 268 nm (Fig. 2).

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Figure 2

UV spectra Irinotecan HCl.

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2.1.3

2.1.3 Preparations of sample solution

Ten injection vials were weighed and mixed properly. A quantity equivalent to 100 mg of Irinotecan was weighed into 100 ml volumetric flask. To this flask, 20 ml distilled water was added and sonicated for 5 min with continuous shaking, the solution was cooled to ambient temperature and diluted up to mark with the same solvent. The solution was then filtered through Whattman filter paper No. 41. From the filtrate, appropriate dilutions were made in distilled water to obtain the desired concentration of 10 μg/ml. Measure the absorbance at 341 nm of standard and sample solution of same concentration and calculate the percent purity of sample. From the stock solution (100 μg/ml), working standard solutions of drugs were prepared by appropriate dilution and were scanned in entire UV range to determine the λ_max. Standard solutions were prepared having concentration 2–10 μg/ml for Irinotecan. The absorbance of these standard solutions was measured at 341 nm.

3 Densitometry chromatographic conditions

The instrument used in the present study was Camag HPTLC system comprising Camag Linomat V automatic sample applicator, Hamilton syringe (100 μl), and Camag TLC scanner III with Wincats software. The HPTLC system consisted of Linomat V auto sprayer connected to a nitrogen cylinder, a twin trough glass chamber (10 × 10 cm), saturated with filter paper for 30 min. Separation was performed as stationary phase and using a mobile phase comprising of toluene/ethyl acetate/methanol/carbon tetrachloride, in the volume ratio of 9.2:5:0.9:0.8 (v/v/v/v) respectively. After development, plates were observed under UV light. Scanning wavelength for Irinotecan was 317 nm. High performance thin layer chromatography was performed on 10 × 10 cm precoated silica gel 60F₂₅₄ precoated plates from E. Merck. The adsorbent has a very large surface area; it may absorb air and other impurities from atmosphere, particularly volatile impurities, after the pack has been opened. The non-volatile impurities adsorbed by layer can lead to irregular baseline in scanning densitometry. To avoid possible interference from such impurities in quantitative analysis, plates were prewashed with methanol, dried, and activated for 30 min at 135 °C with the plates being placed between two sheets of glass to prevent deformation of aluminium during heating.

4 Validation of method

The method was validated as per the ICH (ICH, 2002) in terms of linearity, accuracy and specificity, intra-day and inter-day precision, repeatability of measurement of peak area as well as repeatability of sample application. To spectrophotometric study the accuracy and precision of the above proposed methods, recovery studies were carried out by the addition of known amount of standard drug solutions of Irinotecan preanalysed solution. The resulting solution was then analyzed by proposed methods. Results of recovery studies were found to be satisfactory and are reported in Tables 2 and 3. TLC Densitometry linearity was studied in the concentration range, aliquots of standard stock solution of Irinotecan (100 ng/μl were applied on the TLC plate under nitrogen stream. TLC plates were developed under above established conditions. Area under peak was recorded and plotted against concentration. The specificity of the method was ascertained by analyzing standard drug and sample. The spot for drug was confirmed by comparing the R_f and spectra of the sample spots with those of standard drug. To check the accuracy of the method, recovery studies were carried out by the addition of standard drug solution to pre-analyzed sample solution at three different levels 80%, 100% and 120%. Chromatogram was obtained and the peak areas were noted. At each level of the amount, three determinations were carried out (Table 2). The intra-day precision was determined by analyzing standard solutions of Irinotecan in range of 200–1200 ng/band for three times on the same day while inter-day precision was determined by analyzing corresponding standards on three different days over a period of one week (Table 3). Robustness studies were carried out by examining the effect of small, deliberate variation of the analytical conditions on the peak areas of the drug. Factors varied were volume of mobile phase (±0.5%), and time from application to development (0, 10, 20, and 30 min) and from development to scanning (0, 30, 60, and 90 min). One factor at a time was changed to study the effect. The robustness of the method was checked at amount of 800 ng/band. The limit of detection (LOD) and limit of quantitation (LOQ) were determined on the basis of signal to noise ratio. LOD was the amount of the applied sample producing a peak area that is equal to the sum of the mean blank area and three times the standard deviation. LOQ was the amount of the applied sample producing a peak area that is equal to the sum of the mean blank area and ten times its standard deviation. Stock solution of Irinotecan was prepared and different volumes of stock solution in the range of 200–1200 ng/band were spotted in triplicate. The amount Irinotecan by spot versus average response (peak area) was graphed and the equation for this was determined. The standard deviations (S.D.) of responses were calculated. The average of standard deviations was calculated (A.S.D.). Detection limit was calculated by (3.3 × A.S.D.)/b and quantification limit was calculated by (10 × A.S.D.)/b, where “b” corresponds to the slope obtained in the linearity study of method (Table 3).

5 Results and discussion

UV Spectrophotometric and HPTLC methods were found to be accurate, economic and rapid for routine simultaneous estimation of Irinotecan, in bulk and formulation dosage forms. For UV Spectrophotometric method, linearity was obtained in concentration range of 10–50 μg/ml, for both the drugs; with regression 0.9998 intercept −0.01876 and −0.02843 and slope 0.09661 Irinotecan, respectively. Standard stock solution was suitably diluted with distilled water to obtain concentration ranging from 2–10 μg/ml. Absorbance of these solutions was measured at 268 nm. Analysis of formulation is described in Table 1, the regression equation and correlation coefficient were found to be Y = 1.2653 × −0.327 and 0.9994 respectively. The relative standard deviation values are below 2% indicating the precision of the method. The validations of the proposed methods were further confirmed by recovery studies. The % recovery vary from 99.14 ± 0.043 to 101.31 ± 0.05, indicating good accuracy of methods. The high % recovery value indicates non interference from excipients used in formulations.

The specific range derived from the linearity studies: The range was calculated from the linearity graph. From the lower to higher concentration between which the response is linear, accurate and precise. Acceptance criteria: R.S.D. < 2.0. The range for Irinotecan was found to be 200–1200 ng/ml. Use of pre-coated silica gel HPTLC plates with toluene/ethyl acetate/methanol/carbon tetrachloride, in the volume ratio of 9.2:5:0.9:0.8 (v/v/v) resulted in good separation of the drug. Six different concentrations of mixture of Irinotecan were prepared from stock solution of Irinotecan (100 μg/ml). The drug peak-area was calculated for each concentration level and a graph was plotted of drug concentration against the peak area. The plot was linear for Irinotecan in the concentration range of 10–100 μg/spot. The limit of detection (LOD) was found to be 36 ng/spot for Irinotecan. Limit of quantitation (LOQ) for Irinotecan was determined experimentally by spotting six replicates of each drug at LOQ concentration. The LOQ of was found to be 57 ng/spot. A representative calibration curve was obtained by plotting peak area of compound against the concentration over the range of 200–1200 ng/spot. The drug was satisfactorily resolved with R_f value 0.34 ± 0.08. The drug content was found to be 101.09 with a R.S.D.% of 0.38. The low values of R.S.D.% indicated the suitability of this method for routine analysis of Irinotecan in pharmaceutical dosage forms. The intra-day and inter-day relative standard deviations were found in the range 0.41–1.09% and 0.45–1.24% respectively. Repeatability of sample application and measurement of peak area were carried out using six replicates of same spot (800 ng/spot of Irinotecan). The intra-day and inter-day variation for the Irinotecan was carried out at three different concentration levels of about 200, 800 and 1200 ng/spot. The smaller values of intraday and inter-day variation in the analysis indicate that the method is precise. R.S.D. for repeatability of measurement of peak area and repeatability of sample application was found to be 0.674% and 1.876%, respectively. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions.

6 Conclusion

The UV and HPTLC methods developed in this study have the advantage of simplicity, precision, accuracy, and convenience. It is of potential value for the analysis of Irinotecan as the bulk drug and in commercial formulations. The developed and validated RP- TLC technique is precise, specific and accurate indicating for the determination of drug. Statistical analysis proves that the method is reproducible and selective for the analysis of Irinotecan as bulk drug and formulations. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase –unlike HPLC – thus reducing the analysis time and cost per analysis.