A Validated High Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Berberine Chloride and Guggulsterone Z in Herbal Formulation

Hemorrhoids, also known as piles, are swollen veins located around the anus or in the lower rectum. It can either be internal or external. The symptoms of hemorrhoids include extreme itching around the anus, irritation and pain around the anus, itchy or painful fecal leakage, painful bowel movement, blood on tissue after having a bowel movement. Commiphora wightii (Guggulu) is known for its active ingredients guggulsterones (GE& GZ). It has astringent, antiseptic with anti-inflammatory and demulcent properties that helps to reduce the pain and inflammation. Berberisaristata (Daruharidra) is known for its isoquinoline alkaloid, berberine (BER). It is found to have antibacterial, anti-inflammatory and laxative properties and is useful in hemorrhoids [4]. Combination of these drugs are used in the treatment of hemorrhoids. Literature review revealed that UV, HPLC, HPTLC methods have been reported for the estimation of individual phytoconstituents, BER and GZ in crude and herbal formulation & LC-MS, LC-MS-MS have been reported for the estimation in biological fluid [2, 3]. Any method has not been reported for the simultaneous estimation of both the phytoconstituents from herbal formulation. Thus, it was a thought of interest to develop and validate analytical method for simultaneous estimation of BER and GZ. 2. Experimental


Introduction
Hemorrhoids, also known as piles, are swollen veins located around the anus or in the lower rectum. It can either be internal or external. The symptoms of hemorrhoids include extreme itching around the anus, irritation and pain around the anus, itchy or painful fecal leakage, painful bowel movement, blood on tissue after having a bowel movement. Commiphora wightii (Guggulu) is known for its active ingredients guggulsterones (GE& GZ). It has astringent, antiseptic with anti-inflammatory and demulcent properties that helps to reduce the pain and inflammation. Berberisaristata (Daruharidra) is known for its isoquinoline alkaloid, berberine (BER). It is found to have antibacterial, anti-inflammatory and laxative properties and is useful in hemorrhoids [4]. Combination of these drugs are used in the treatment of hemorrhoids. Literature review revealed that UV, HPLC, HPTLC methods have been reported for the estimation of individual phytoconstituents, BER and GZ in crude and herbal formulation & LC-MS, LC-MS-MS have been reported for the estimation in biological fluid [2,3]. Any method has not been reported for the simultaneous estimation of both the phytoconstituents from herbal formulation. Thus, it was a thought of interest to develop and validate analytical method for simultaneous estimation of BER and GZ.
flask to obtain BER stock solution containing 100 µg/ mL of berberine chloride. Guggulsterone Z (5 mg) was accurately weighed and transferred to 50 mL capacity volumetric flask and volume was made up to mark with methanol to obtain GZ stock solution containing 100 µg/ mL stock solution.

Preparation of Sample solution
Twenty tablets were weighed and powdered. 5 gram tablet powder was weighed. It was extracted thrice with 25 mL petroleum ether. Residue was collected and again extracted with 25 mL chloroform thrice to obtain GZ. The chloroform layer was combined, reduced to 20 mL and transferred in to 25 mL volumetric flask; volume made up to mark with chloroform (solution A). Residue was collected and again extracted with 25 mL methanol thrice to obtain BER. Methanol layer was combined. 5 mL of 1% HCl was added to it, reduced to 20 mL and transferred to 25 mL volumetric flask; volume made up to mark with methanol (solution B).

Chromatographic Separation
Appropriate volumes of standard solutions or sample solutions were spotted on the TLC plate at constant application rate of 159 nL/sec in the form of bands with bandlength of 8 mm. The chromatogram was developed in ascending mode in a twin-trough chamber previously saturated with the mobile phase (toluene-acetonitrile-formic acid, 5: 3: 0.5 v/v/v) for 20 min. The chromatogram was allowed to migrate to a distance of 8 cm. The developed TLC plate was dried and densitometric scanning was performed at 264 nm on Linomat Scanner IV in the absorbancereflectance mode using a slit-dimension of 6.00 × 0.20 mm and a scanning speed of 20 mm/s.

Validation of HPTLC method
The developed method was validated as per ICH Q2(R1) guideline.

Specificity
The spots of BER and GZ were confirmed by comparing the R f and absorbance-reflectance spectrum of the sample with those of the standard. The peak purity of BER and GZ were assessed by comparing the spectra at three different levels, i.e., peak start (s), peak apex (m) and peak end (e) positions and correlations between them [r 2 (s,m) and r 2 (m,e)] were considered for the determination of peak purity.

Linearity
The linearity of BER and GZ were assessed by analysis of five different concentrations in the range of 100-500 ng/ spot for BER and 200-1000 ng/spot for GZ in terms of correlation co-efficient values.

Repeatability of Sample Application
BER standard solution containing 300 ng/spot of BER and 6 µl of GZ containing 600 ng/spot of GZ (3 µl) was spotted seven times on TLC plate. The plate was developed, dried and photometrically analyzed as described under chromatographic separation. The areas of seven spots were measured and relative standard deviation (%RSD) was calculated.

Repeatability of measurement of peak area and R f
BER standard solution containing 300 ng/spot of BER and 6 µl of GZ containing 600 ng/spot of GZ(3 µl) was spotted seven times on TLC plate. The plate was developed, dried and photometrically analyzed as described under chromatographic separation. Area and R f of spot was measured seven times without changing the position of plate and %RSD of obtained data was calculated.

Intraday reproducibility
Variation of results within same day is called as intraday variation. It (%RSD) was determined by spotting three middle concentrations (2, 3, 4 µl of BER and 2, 4, 6 µl of GZ) of calibration curve three times on the same day on TLC plate.

Interday reproducibility
It (%RSD) was determined by spotting three middle concentrations (2, 3, 4 µl of BER and 2, 4, 6 µl of GZ) of calibration curve for three times on 3 different days on TLC plate.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD and LOQ were separately determined at a signal-to-noise ratio of 3 and 10. LOD and LOQ were experimentally verified by diluting known concentrations of BER and GZ until the average responses were 3 or 10 times of the standard deviation of the responses for 6 replicate determinations.

Accuracy
Accuracy (% recovery) was determined by analysis of previously analyzed sample spiked with the standard solution of BER and GZ at three levels (80, 100, 120 %). Three replicates from each sample were performed and mean recovery (%) was calculated.

Robustness
Robustness testing was carried out using experimental design approach (Design Expert 9). Plackett-Burman design for testing seven factors was selected for robustness testing. Table  1 depicts the factors selected and their levels for robustness testing of the proposed method. The peak area and % recovery of BER and GZ were observed at each experiment designed. The experiments designed were repeated three times in random order. The significance of factors effects was determined graphically by means of Pareto chart.

Analysis of Sample Solutions
Sample solution (10 µl) was spotted on the TLC plate and analyzed as described under chromatographic condition. The experiment was repeated 3 times. The % content was calculated by fitting peak area values into calibration equation of BER and GZ.

Method Optimization
For the estimation of BER and GZ, different solvent systems in varying ratios of toluene, ethyl acetate, water, acetonitrile, formic acid were tried and photometrically scanned at 264 nm (iso-absorptive point of BER and GZ). Finally, suitable separation and symmetric peak shape were achieved using a mobile phase system consisting of toluene-acetonitrile-formic acid (5: 3: 0.5 v/v/v) previously saturated for 20 min at 25°C. The R f value of BER was found to be 0.40 ± 0.02 and 0.68 ± 0.02 ( Fig. 1 and 2).

Specificity
Comparison of chromatograms of standard solution and sample solution from formulation showed identical R f values i.e. 0.40 ± 0.02 for BER and 0.68 ± 0.2 for GZ. Comparison of the spectra scanned at peak start (S), middle (M) and end (E) showed high degree of correlation (above 0.990). This confirmed the purity of the corresponding spots. Also, the spectrum of individual sample was compared with the spectrum of standard BER and GZ. The correlation obtained was 0.9996 for BER and 0.9992 for GZ (Fig. 3), this confirmed the identity of spots.

Linearity
BER showed good correlation coefficient in the concentration range of 100-500 ng/spot and GZ in the concentration range of 200-1000 ng/spot. The linear regression analysis obtained by plotting the peak areas of analyteversus concentration showed good correlation coefficients (correlation coefficient greater than 0.995). The overlain linearity chromatogram is shown in Fig.4. The regression equation for BER and GZ was found to be y = 9.8151x + 812.48 and y = 9.8016x + 1566.1, respectively.

Precision
The % RSD for repeatability of peak area measurement of BER and GZ were 0.38 and 0.31, respectively ( Table  2) and repeatability of sample application were 0.32 and 0.25, respectively (Table 3). The% RSD for intraday reproducibility was 1.23 for BER and 1.7 for GZ; % RSD for interday reproducibility was 0.58 for BER and 1.42 for GZ (Table 4 and 5).

LOD and LOQ
LOD and LOQ value for BER was 35 ng/spot and 100 ng/ spot, respectively and GZ was 80 ng/spot and 200 ng/spot, respectively.

Accuracy
Accuracy of the developed method was calculated by performing recovery studies. Proposed method was employed for estimation of amount of BER and GZ from pre analyzed sample solutions spiked at three different levels of standard. Mean % recovery were found out to be 99.06 -108.89 % for BER and 93.79 -99.11 % for GZ (Table 6).

Robustness
The proposed HPTLC method was tested for robustness using PB design with 8 experiments. Seven HPTLC factors were analyzed (Table 7). The different levels for each factor were selected symmetrically around the nominal value of the corresponding factor. The limits of the factors studied were selected according to error ranges which would be typically encountered in an analytical laboratory. Pareto charts were used for the evaluation of robustness data for R f value and % recovery (Fig.5). In all the situations, the % recovery and R f value for BER and GZ were not significantly affected by factor changes (Table 8). Hence, the method was found to be robust.

Analysis of marketed formulation containing BER and GZ
The spots at R f value 0.40 (for BER) and R f 0.68 (for GZ) were observed in the densitogram of the drug samples extracted from tablets. Amounts of BER and GZ were calculated using equation y = 9.8151x + 812.48 for BER and y = 9.8016x + 1566.1 for GZ. The % content was 0.088 % for BER and 0.132 % for GZ.

Conclusion
The proposed HPTLC method for simultaneous estimation of BER and GZ from marketed formulation was validated as per ICH Q2(R1) guideline [1]. The method was found to be linear in the range of 100-500 ng/spot for BER and 200-1000 ng/spot for GZ. Moreover, the method was found to be precise, accurate, robust and sensitive during validation performances. The method was successfully applied on commercially available Pilex tablet from Himalaya Herbals.