A high performance thin layer chromatographic method for the estimation of colchicine in different formulations.

BACKGROUND: Colchicine is a main alkaloid present in bitter and sweet variety of colchicum (Colchicum luteum Baker), which have been reported to possess anti-rheumatic, anti-gout, and anticancer potential. Colchicum is an important ingredient of several Unani and Herbal formulations. Quantification of colchicine will play a great role in quality control of these formulations. Hence, a high-performance thin layer chromatographic (TLC) method has been developed for the analysis of colchicine in Unani formulations of various dosage forms such as hubb (tablet) and capsules.
MATERIALS AND METHODS: The samples were applied on aluminum TLC plates precoated with silica gel 60-F254 and developed using mobile phase toluene-dichloromethane-methanol in equal proportions. Quantification was done by densitometric scanning at 350 nm, which showed a linear response in the range of 50-500 ng/spot. The developed method was validated as per the International Conference on Harmonization guidelines for linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantification. RESULTS AND
CONCLUSION: The developed method was applied for quantitative estimation of colchicine in different Unani and Herbal formulations. The method was found simple, selective, accurate with a wide range of linearity, hence suitable for the quality control of different formulations and varieties of colchicum with respect to colchicine content.

Colchicine is a toxic natural product and alkaloid, obtained from Colchicum luteum Baker and Colchicum autumnale. It was used to treat rheumatic complaints, especially gout, and still finds huge consumption,[12] despite dosing issues concerning its toxicity. However, because of certain limitations such as dosage, time of introduction of therapy, no standardized treatment recommendation has been established till date. Lethal outcome after ingestion of as little as 7.0 mg of colchicine, as well as survival after ingestion of 60 mg colchicine has been reported. Toxic effects occur in association with serum colchicine levels >5.0 ng/mL.[3] Considering the acute toxicity caused by colchicine ingestion, which has caused fatal accidents including death in some cases,[4] the estimation of colchicine in the various formulation is of prime importance.[5] Keeping in view of the above aspects, the present experimental work was carried out for quantification of colchicine in different marketed formulations to confirm, whether these formulations contain toxic or nontoxic amount.

Materials and Methods

Development of high-performance thin layer chromatographic method

The effort was made to develop a mobile phase, which can be used for the routine analysis of colchicine by hit and trial method. After selection of the suitable solvent system, ultraviolet (UV) spectra of selected peak of colchicine in chromatograms of standard and samples were taken to ascertain the detection wavelength for the analysis.

Preparation of standard calibration plot

Different volumes (0.1, 0.2, 0.4, 0.5, 1.0, 2.0, and 4.0 µL) from stock solution (250 µg/mL) of standard colchicine were spotted in duplicate on thin layer chromatographic (TLC) plate to obtain concentrations of 50–1000 ng/spot. The data of peak height and peak area versus drug concentration were treated by linear least square regression by Wincats 3.00 software (CAMAG, Switzerland), which was installed with the CAMAG high-performance TLC (HPTLC) system.

Preparation of samples

Ten tablets were weighed, powdered and transferred to 100 mL conical flask containing 75 mL of distilled water. The solution was sonicated at 70°C for 45 min followed by filtration through Whatman filter paper no. 41. The process was repeated thrice for complete extraction and filtrates were pooled. It was concentrated to 50 mL on a water bath and extracted using chloroform in triplicate (each 50 mL). The chloroform extract was evaporated to dryness on a water bath and the residue obtained was re-dissolved in 5.0 mL of methanol, which was filtered through 0.45 µ filter and used for analysis. Similarly, samples were prepared for capsules and crude drugs.

High-performance thin layer chromatographic instrumentation

The samples were spotted in the form of width 4.0 mm with a CAMAG microlitre syringe on precoated silica gel aluminium plate 60F-254 (20 cm × 10 cm with 0.2 mm thickness, E. Merck, Germany) using a Linovat V (CAMAG, Switzerland). A constant application rate of 120 nl/s was employed, and space between two bands was 10 mm. Linear ascending development was carried out in twin trough glass chamber saturated with the mobile phase. The optimized chamber saturation time for the mobile phase was 30 min at room temperature. The length of chromatogram run was 80 cm subsequent to the development. TLC plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed on CAMAG TLC scanner III in the absorbance mode at 350 nm. The source of radiation utilized was deuterium and tungsten lamp. The slit dimension was kept 4.0 mm × 0.45 mm, and 20 mm/s scanning speed was employed.

Method validation

The developed method was validated as per the International Conference on Harmonization (ICH) guidelines[6] for linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, robustness, and specificity.

Results and Discussion

Optimization of high-performance thin layer chromatographic method

Different solvent systems were tried for the separation of colchicine in standard and in different samples. The solvent system toluene:dichloromethane:methanol, (10:10:10, v/v/v) was found suitable for the separation with a sharp and compact peak of colchicine. The λmax (350) was ascertained by taking UV spectra of selecting peaks of colchicine in standard and samples chromatogram.

Validation parameters

The standard chromatogram showed peaks of colchicine at Rf 0.68 ± 0.02 [Figure 1a]. The peak area thus obtained with different concentration of standards were treated with linear least square regression to get the regression equation Y = 441.0 + 12.79x (r2 = 0.994), which was used for quantification of colchicine in different samples. The pre-analyzed samples were spiked with standard colchicine at four different concentration levels, that is, 0, 50, 100, and 150% and the mixtures were re-analyzed by the proposed method. The recovery experiment was conducted in triplicate, which demonstrated a good recovery of 96.64–102.14% [Table 1]. Robustness of the method was carried out by introducing small changes in the composition of mobile phase and detection wavelength. The difference in the area of the peak was observed with respect to mobile phase composition and calculated the percent relative standard deviation (%RSD). A low value of %RSD indicated that method was withstanding small changes, and so the method was robust [Table 2]. The LOD and LOQ can be determined based on the technique of signal-to-noise ratio. The concentration of sample giving the signal to noise ratio of three was fixed as the LOD. The concentration of the sample giving the signal to noise ratio of ten was fixed as LOQ. It is found that 18 ng of colchicine standard gave the signal to noise ratio three and 50 ng of colchicine standard gave the signal to noise ratio ten. Hence, 18 and 50 ng was fixed as the LOD and LOQ, respectively. The specificity of the proposed method was determined by comparing the sample and standard peak for its Rf and UV spectra [Figure 2].

Figure 1

High performance thin layer chromatographic chromatogram of colchicine in standard (a), and in crude drug suranjan talkh (b) suranjan shirin (c) tablet (d) capsules (e) at 350 nm showing Rf value 0.068±0.02

Table 1

Accuracy of the method (n=6)

Table 2

Robustness of the method (n=6)

Figure 2

Superimposed ultraviolet spectra of colchicine in all tracks indicating specificity of the method

Analysis of colchicine in formulation and crude drugs

A spot at Rf value 0.68 ± 0.02 was observed in the samples [Figure 1b–e]. The peak obtained was pure and there were no interference from the excipients, which were used in the formulations. The colchicine content in the crude drug was found to be 56.8 and 58 µg/g and in tablets 25.6 µg/g, whereas 38.6 µg/g in capsule formulation with %RSD of 0.26. The low %RSD showed that the method can be adopted for the quantitative analysis of colchicine in crude colchicum and its different formulations.

Conclusion

A simple HPTLC method was developed for the estimation of colchicine in different herbal samples. The developed method was validated as per the ICH guidelines and found simple, accurate, précise and specific for the analysis and quality control of crude and herbal formulations containing colchicum or colchicine as an ingredient to check its safe limit.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.