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A validated HPTLC method for the quantification of podophyllotoxin in Podophyllum hexandrum and etoposide in marketed formulation
⁎Corresponding author. Address: Bioactive Natural Products Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India. Mobile: +91 9891374647; fax: +91 11 26059663. sahmad_jh@yahoo.co.in (Sayeed Ahmad)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A simple, sensitive, specific, rapid and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the quantification of podophyllotoxin and etoposide. Podophyllotoxin was quantified in the roots of Podophyllum hexandrum whereas etoposide in a marketed formulation. The method involved densitometric evaluation of both podophyllotoxin and etoposide after resolving it on silica gel plate using dichloromethane–methanol–formic acid (9.5:0.5:0.5 v/v/v) as the mobile phase. The method was validated for precision (inter-day, intra-day and inter-system), robustness, accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response (area) was linear within the concentration range of 150–2400 ng spot−1 for podophyllotoxin and 200–2000 ng spot−1 for etoposide. Instrumental precision was found to be 1.03–1.80 (% RSD) and 0.79–1.99 (% RSD) for podophyllotoxin and etoposide, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.71% for podophyllotoxin and 100.43% for etoposide, respectively. The HPTLC method for the quantification of podophyllotoxin and etoposide was found to be simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of P. hexandrum and several formulations containing these markers.
Keywords
Podophyllotoxin
Etoposide
HPTLC
Method development
Validation
1 Introduction
Podophyllum hexandrum Royle, belongs to family Berberidaceae, is a perennial herb which is distributed in restricted pockets throughout the alpine Himalayan region (Nayar and Sastry, 1990). P. hexandrum has been extensively used in Ayurvedic system of medicine for treatment of several ailments like constipation, cold, biliary fever, septic wounds, inflammation, burning sensation, mental disorder, monocytoid leukemia, Hodgkin’s and non Hodgkin’s lymphoma. The rhizomes of P. hexandrum yield cytotoxic lignan podophyllotoxin (Chartterjee, 1952) and several other lignans (Jackson and Dewick, 1984a,b; Sultan et al., 2010) due to which it possesses anti-tumor activity (Jackson and Dewick, 1986). The natural lignan, podophyllotoxin, is used for the semi-synthesis of anti-neoplastic drugs, etoposide and teniposide. Etoposide’s antineoplastic activity is achieved through DNA strand breakage, which likely results from the formation of a complex involving drug-DNA and the DNA unwinding enzyme topoisomerase II (Richter et al., 1987) while podophyllotoxin acts as an inhibitor of the microtubule assembly. Podophyllotoxin (Fig. 1a) is also the precursor for a new derivative CPH-82 (reumacon) that is being tested in Europe in phase III clinical trials for arthritis (Carlstrom et al., 2000). In addition, podophyllotoxin and podophyllin (Podophyllum resin) are considered as active constituents in dermatologic products for therapy of genital warts (Stoerh et al., 1978). Etoposide (Fig. 1b) is efficacious against small cell lung cancer, malignant lymphoma, leukemia and probably non-small cell lung cancer (Fleming et al., 1989; Radice et al., 1979). The methods so far reported for the analysis of podophyllotoxin and etoposide include their estimation using capillary electrophoresis (Ding et al., 2005), differential pulse voltammetry (Radi and Abd-Elghany Wahdan, 2007) electrochemistry (Radi and Abd-Elghany Wahdan, 2007) and OPLC (Erdelmeier et al., 1986) that showed low resolution owing to poor reproducibility. There are few HPLC (Cairnes et al., 1981; Bastos et al., 1995; Shirazi et al., 2001; Kato et al., 2003) methods available for the analysis of podophyllotoxin and etoposide but these methods required lengthy run times and complicated gradient elution systems using solvent mixtures. One HPTLC method (Mishra et al., 2005) for the determination of podophyllotoxin in plant has also been reported. Our HPTLC method is superior if we compare the limit of detection (LOD), limit of quantification (LOQ) and linearity range as compared to the reported HPTLC method. Further, in earlier report only podophyllotoxin in plant extract was quantified whereas in our method its semi-synthetic derivative etoposide was also quantified in a marketed formulation using the same mobile phase. With this background, we herein report a simple, specific and sensitive HPTLC method for the quantification of podophyllotoxin in the roots of P. hexandrum and etoposide in its marketed formulation. The proposed method has been validated as per the ICH guidelines (ICH, 2005) and similar to the methods reported by laboratory on HPTLC method development and validation for herbal drugs (Jha et al., 2010, Ahmad et al., 2011a,b).
2 Materials and methods
2.1 Chemicals and reagents
Standard Podophyllotoxin (98%) and Etoposide (98%) were procured from Sigma Aldrich, USA, respectively. Dried roots of P. hexandrum were procured from Yucca Enterprises, Mumbai, India, which were further authenticated by a Pharmacognosist and voucher specimens deposited in Bioactive Natural Product Laboratory, Department of Pharmacognosy, Jamia Hamdard. The marketed formulation (Etosid) containing etoposide (20 mg mL−1) was procured from local market, Delhi (Cipla India). All other chemicals used were of analytical reagent grade purchased from Merck India.
2.2 HPTLC instrumentation and experimental conditions
Sample solutions were applied with semiautomatic TLC sampler Linomat V (Camag, Switzerland) controlled by winCats software. The plates were developed in a 20 × 10 cm twin trough glass chamber (Camag, Switzerland). A TLC scanner III was used for scanning the TLC plates. Pre-coated silica gel aluminum plates 60F254 (E. Merck, Darmstadt, Germany) with a thickness of 0.2 mm were used for all determinations. The plates were pre-washed with methanol and activated at 60 °C for 5 min prior to chromatography. Five different volumes (0.3, 0.6, 1.2, 2.4, 4.8 μL) of standard solution of podophyllotoxin were applied on a 20 × 10 cm TLC plate for the preparation of the calibration curve of podophyllotoxin. Similarly, five different volumes (0.2, 0.4, 0.8, 1.0, 2.0 μL) of standard solution of etoposide were applied on a separate 20 × 10 cm TLC plate for the preparation of the calibration curve of etoposide. A constant application rate of 150 nL s−1 was employed for both podophyllotoxin and etoposide with a band width of 6.0 and 5.0 mm for podophyllotoxin and etoposide, respectively. The scanning speed was employed at 20 mm s−1 for both podophyllotoxin and etoposide and the slit dimension was kept at 5.0 × 0.45 mm and 4.0 × 0.45 mm for podophyllotoxin and etoposide, respectively. Twenty mL of mobile phase consisting of dichloromethane-methanol-formic acid (9.5:0.5:0.5, v/v/v) was used per plate. The optimized chamber saturation time for mobile phase was 15 min at room temperature (25 ± 2 °C) at relative humidity of 60 ± 5% RH. The plates were developed and scanned within 10 min using densitometric scanner III in the absorbance mode at 292 nm for both podophyllotoxin and etoposide. The source of radiation was deuterium lamp emitting a continuous radiation between 200 and 400 nm. The data obtained were analyzed by winCats software to get linear regression equation.
2.3 Preparation of standard solutions
A standard solution containing podophyllotoxin was prepared by dissolving 2.5 mg in 5.0 mL of methanol (500 μg mL−1) whereas a standard solution of etoposide was prepared by dissolving 5.0 mg in 5.0 mL of methanol (1000 μg mL−1). These stock solutions were used to make calibration curves of podophyllotoxin and etoposide.
2.4 Preparation of sample solutions
The dried and pulverized roots of Podophyllum hexandrum roots (60 g) were extracted with methanol (250 mL) in a soxhlet apparatus over water bath for 6 h. The extract was filtered and solvent was removed in Rota evaporator at 50 °C. The concentrated extract was dissolved in HPLC grade methanol and volume was adjusted to 100 mL. The sample solution for quantification of etoposide in formulation Etosid was prepared by diluting 0.2 mL (20 mg mL−1) in 2.0 mL of methanol.
2.5 Method validation
The developed method was validated as per the ICH guidelines (ICH, 2005). The linearity studied for both the constituents by applying different concentrations and LOD and LOQ was also determined. For the precision studies, repeatability and intermediate precision were determined. The robustness was carried out by deliberately changing solvent composition and detection wavelength whereas accuracy by recovery studies after spiking known concentration of standard to pre-analyzed samples. The specificity of the method was ascertained by comparing Rf values and U V spectra of peaks of both constituents in sample and in standard chromatogram.
3 Result and discussion
3.1 Optimization of solvent system
For the development of mobile phase, different trials were made using many solvents in different proportions. When mobile phase consisting of chloroform: methanol was used in the ratio of 8:2, v/v spot was observed at the almost same Rf value (0.66 and 0.67) for podophyllotoxin and etoposide. But it was found that the compactness of the peaks was poor. In order to improve the resolution between the peaks, a new mobile phase with the composition of dichloromethane: methanol and formic acid was used in the ratio of 9.5:0.5:0.5, v/v/v. This new mobile phase helped in achieving very compact spots at the same Rf value (Fig. 2a and b and Fig. 3a and b) for podophyllotoxin and etoposide in standard and in samples, respectively with good resolution.
3.2 Method validation
3.2.1 Linearity
A five point calibration curve was constructed by plotting peak area against concentrations. Linearity was evaluated by applying different concentrations (150–2400 ng spot−1) for podophyllotoxin and 200–2000 ng spot−1 for etoposide in triplicates. A good linearity relationship was found with r2 value of 0.9927 and 0.99786 for podophyllotoxin and etoposide, respectively (Table 1 and Fig. 4).
| Validation parameters | Podophyllotoxin | Etoposide |
|---|---|---|
| Linearity range (ng spot−1) | 150–2400 | 200–2000 |
| Correlation coefficient (r2) | 0.99271 ± 0.00013 | 0.99786 ± 0.00061 |
| Regression equation | Y = 1143.86 + 3.39∗X | Y = 6.213 + 1.23∗X |
| Limit of detection (ng spot−1) | 45.7 | 64.5 |
| Limit of quantification (ng spot−1) | 138.7 | 195.6 |
3.2.2 Precision
The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. System repeatability was determined in six replicates of a standard solution at three concentration levels of 400, 800 and 1600 ng spot−1 and 200, 400, 800 ng spot−1 for podophyllotoxin and etoposide, respectively. The results of repeatability were expressed in terms of relative standard deviation (% RSD). Intra-day precision was done by repeating the same assay six times on the same day. Intermediate precision was also assessed by the assay of three; six standard solutions were sets on different days (inter-day precision) and on different system (Inter-system precision). Precision data on the intra, inter-day and inter analyst variation for three different concentration levels are summarized in Table 2. The low % RSD indicated the method is precise for the analysis.
| Conc. (ng spot−1) | Podophyllotoxin | |||||
|---|---|---|---|---|---|---|
| Inter-day precision | Intra-day precision | Inter-system precision | ||||
| Mean peak area ± SD⁎ | % RSD⁎⁎ | Mean peak area ± SD | % RSD | Mean peak area ± SD | % RSD | |
| 400 | 2967.2 ± 53.63 | 1.80 | 3279.7 ± 42.0 | 1.28 | 3031.6 ± 53.82 | 1.77 |
| 800 | 5308.3 ± 82.43 | 1.55 | 5300.8 ± 57.8 | 1.09 | 5243.3 ± 93.84 | 1.79 |
| 1600 | 7016.0 ± 71.71 | 1.03 | 6968.6 ± 81.2 | 1.16 | 6889.6 ± 72.95 | 1.05 |
| Etoposide | ||||||
| 200 | 829.8 ± 11.39 | 1.37 | 869.3 ± 15.0 | 1.73 | 983.6 ± 11.58 | 1.17 |
| 400 | 1733.6 ± 24.85 | 1.43 | 1667.4 ± 32.24 | 1.93 | 1820.8 ± 14.55 | 0.79 |
| 800 | 2384.6 ± 47.61 | 1.99 | 2338.2 ± 41.36 | 1.76 | 2534.6 ± 39.81 | 1.57 |
3.2.3 Robustness of the method
By introducing small changes in the mobile phase composition and detection wavelength, the effects on the results were examined. Mobile phases having different compositions like dichloromethane: methanol: formic acid (9.7:0.3:0.5, v/v/v) and (9.3:0.7:0.5, v/v/v) were used and analysis was also done by changing detection wavelength (±2). Robustness of the method was determined at three different concentration levels of 400, 800 and 1600 ng spot−1 and 200, 400, 800 ng spot−1 for podophyllotoxin and etoposide, respectively. The effect of deliberate changes in the composition of mobile phase and detection wavelength was studied as % RSD and depicted in Table 3. Low % RSD indicated the method is robust.
| Mobile phase change (Dichloromethane: Methanol: Formic Acid) | Mean area ± SD⁎ | % RSD⁎⁎ | ||||
|---|---|---|---|---|---|---|
| Actual (v/v/v) | Used (v/v/v) | Level | Podophyllotoxin | Etoposide | Podophyllotoxin | Etoposide |
| 9.5:0.5:0.5 | 9.3:0.7:0.5 | −2 | 2809.9 ± 9.96 | 1525.7 ± 17.24 | 0.35 | 1.13 |
| 9.5:0.5:0.5 | 0 | 2936.2 ± 40.77 | 1670.8 ± 20.08 | 1.38 | 1.20 | |
| 9.7:0.3:0.5 | +2 | 3049.9 ± 37.65 | 1751.8 ± 31.94 | 1.23 | 1.82 | |
| Wavelength change | ||||||
| Actual (nm) | Used (nm) | Level | ||||
| 292 | 290 | −2 | 3058.6 ± 51.88 | 1725.7 ± 10.28 | 1.69 | 0.59 |
| 292 | 0 | 2950.51 ± 28.73 | 1665.8 ± 19.53 | 0.97 | 1.17 | |
| 294 | +2 | 3300.2 ± 60.51 | 1849.7 ± 33.19 | 1.83 | 1.79 | |
3.2.4 LOD and LOQ
In order to estimate the LOD and LOQ, blank solution (methanol) was spotted six times following the same method as explained above. The signal to noise ratio was determined. LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting known concentrations of reference solution until the average responses were approximately three or ten times the standard deviation of the responses for six replicate determinations. LOD and LOQ were calculated using the signal to noise ratio method and found to be 45.7, 138.7 ng spot−1 for podophyllotoxin and 64.5, 195.6 ng spot−1 for etoposide, respectively (Table 1).
3.2.5 Specificity
The specificity of the method was ascertained by analyzing standard drug and sample. The spots for podophyllotoxin and etoposide in sample were confirmed by comparing Rf and spectra of spot with that of standard. Purity of sample spot corresponding to podophyllotoxin and etoposide in samples was determined by taking the spectra and by comparing it with that of standard. The specificity of the newly proposed method was ascertained by superimposing the spectrum of both standard and sample peaks and confirmed for its purity (Fig. 5).
3.2.6 Recovery studies (Accuracy)
The pre-analyzed samples were spiked with 50%, 100% and 150% of the standard podophyllotoxin and etoposide, respectively and the mixtures were reanalyzed by using the proposed method. The experiment was conducted six times and the content of both constituents in respective samples was quantified and percentage recovery was calculated. This was done to check the recovery of the drug at different concentration levels in the samples. The results of the recovery study are depicted in Table 4.
| % of standard spiked to the sample | Theoretical content (μg spot−1) | Amount of drug recovered (μg ± SD⁎) | % of drug recovered | % RSD⁎⁎ |
|---|---|---|---|---|
| Podophyllotoxin | ||||
| 0 | 2.31 | 2.37 ± 0.04 | 102.59 | 1.85 |
| 50 | 3.46 | 3.42 ± 0.05 | 98.84 | 1.51 |
| 100 | 4.62 | 4.60 ± 0.03 | 99.56 | 0.86 |
| 150 | 5.77 | 5.78 ± 0.01 | 100.17 | 0.32 |
| Etoposide | ||||
| 0 | 2.78 | 2.85 ± 0.04 | 102.51 | 1.6 |
| 50 | 4.17 | 4.23 ± 0.08 | 101.43 | 1.9 |
| 100 | 5.56 | 5.43 ± 0.10 | 97.66 | 1.9 |
| 150 | 6.95 | 6.96 ± 0.12 | 100.14 | 1.8 |
3.2.7 Analysis of samples
The samples were spotted in triplicate on TLC plate and developed. The peak of podophyllotoxin comes at Rf of 0.66 in sample extract (Fig. 2b) whereas etoposide in formulation at Rf of 0.67 (Fig. 3b). No interference was observed in samples with immediate constituents and resolution between the peaks was also good.
4 Conclusion
The HPTLC method was developed and validated for the determination of podophyllotoxin in Podophyllum hexandrum roots and etoposide in a marketed formulation, which showed the presence of 1.15% w/w for podophyllotoxin in extract and 0.27% w/v for etoposide in formulation. The method was found simple, rapid, accurate, specific and robust for the analysis of podophyllotoxin in crude drug and etoposide in formulation using the same method. The proposed method can be adopted by any laboratory for the quality control of crude drugs and formulation that contains podophyllotoxin and etoposide as active markers or Podophyllum hexandrum roots as an ingredient.
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