.1. INTRODUCTION.1.1 STABILITY INDICATING ASSAY The stability - indicating assay is a method that is employed for the analysis of stability samples in pharmaceutical industry. It is essential to validate an assay with regards to its precision, accuracy, reproducibility, selectivity and robustness. In pharmaceutical research, in addition to these requirements, an assay is often required to be proven beyond doubt to be stability - indicating. A stability indicating assay is one that can accurately and selectively differentiate an intact drug from its potential decomposition products 425. The United States Food and Drug Administration draft guidelines of 1998 define stability indicating assays as validated quantitative analytical methods that can detect the changes with time in the chemical, physical or microbiological properties of the drug substance and drug product and that are specific so that the contents of active ingredient, degradation products and other components of interest can be accurately measured without interference. Thus according to definition, the discriminating nature of the method indicates the method to be stability indicating as well as stability - specific. The ICH guidelines require establishment of stability-indicating assays by conducting forced degradation studies or stress testing under a variety of conditions like ph, light, oxidation, dry heat etc. and separation of drug from degradation products. It is important to note that the necessity for stability - indicating capability applies to the complete testing regimen used for a given material and may not be necessary or appropriate for specific individual methods 426. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 305
.1.1.1 Need for performing stability indicating assays 426 To differentiate the active ingredient from closely related impurities and degradation products. To provide assurance on detection of changes in identity, quantity and potency of the drug substance or drug product. To monitor the stability of a given drug in a finished product. To establish and confirm the shelf life of drug substances and drug products. To perform various other applications like cleaning, validation testing, performance testing i.e. dissolution testing..1.1.2 Regulatory status of stability indicating assays The current guidelines available for stability testing of drug substances and products requires the conduct of stress testing or forced degradation studies for performing stability indicating assays but none of the guidelines specify how these studies emphasize that the testing of those features which are susceptible to change during storage and are likely to influence quality; safety and/or efficacy must be done by validated stability indicating testing methods (ICH guidelines Q1A on Stability Testing of New Drug Substances and Products) 427.The ICH guidelines Q1B on Photostability Testing of New Drug Substances and products mentions that the intrinsic photostability characteristics of new drug substances and products should be evaluated to demonstrated that an appropriate light exposure does not result in inappropriate change. The ICH guidelines Q6A on note for guidelines on specifications and Q5C on Stability Testing of Biotechnological/Biological Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 306
products also mention the requirement of stability-indicating assays 428. The requirements are also listed in the following- United States Food and Drug Administration stability guidelines and draft guidelines. World Health organization {WHO}. European Committee for Proprietary medicinal products. Canadian Therapeutic products Directorate s guidelines on stability testing. United States Pharmacopoeia {USP}. Current ICH guidelines Q7A on Good Manufacturing practices for Active Pharmaceutical Ingredients. Code of Federal Regulation{21CFR}..1.1.3 Design of Stability Studies 429 A minimum of four samples should be generated for every stress condition, 1. The blank solution stored under normal conditions. 2. The blank subjected to stress in the same manner as the drug solution. 3. Zero time sample containing the drug which is stored under normal conditions. 4. Drug solution subjected to stress treatment. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 307
The comparison of the results of these provides real assessment of changes. It is advised to withdraw the samples at different time periods for each reaction condition. The information obtained from this is essential in establishment of Stability indicating analytical methods. Stability indicating analytical methods well established for active pharmaceutical ingredient and thus the allopathic medicine can be tested for it quality standards with respect to the degradation of the products. But the picture is not same with the plant based formulations. The basic standardization of the herbal products with the bioactive content is not well employed and the stability indicating methods are far away from the list of testing methods of their routine analysis. The fact remains the same that how much an allopathic medicine needs the stringent analytical protocol, the same is required for the plant based formulations. Thus, considering the need for development of stability indicating analytical methods for herbal medicine the objective of this study is as follows: To apply the developed and validated HPTLC method (as described in chapter 9, 10) for biomarkers namely; for the detection of forced degradation products of Conessine, Rutin and Quercetin. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 308
.2 STABILITY STUDIES OF RUTIN.2.1 Chromatographic conditions used for the developed and validated HPTLC method for rutin (Chapter 10) The following densitometric conditions were used for HPTLC studies: Stationary phase : : Precoated plates of Silica Gel 60 GF 254 (Merck) Mobile phase : Chloroform: methanol: formic acid (8.2:1.5:1) Saturation time Development time Wavelength Lamp Band width Length of : 15 min :15 min : 254 nm : Deuterium : 7 mm : 8 cm chromatogram Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 309
.2.2 Forced degradation studies of Rutin.2.2.1 Acid degradation 1 M of hydrochloric acid (HCl) was prepared by diluting 8.5 ml of concentrated HCl to 100ml of distilled water. 1mg/ml solutions were prepared of rutin. 1 ml of rutin solution and 4 ml of 1N HCl were mixed and mixture was refluxed on water bath for 3 hours at 60 C. The refluxed solution of rutin and HCl was allowed to attend ambient temperature and then the refluxed solution was neutralized by 1 N NaOH to ph 7 and the volume was made up to 10 ml with methanol. Then the final solution was applied on to the TLC plates. Total degradation was found when the rutin was refluxed 1 N HCl for 3 hr, therefore the exposure time was reduced to 1 hr with the same concentration of HCl. Then the stressed sample was analyzed. The chromatogram of 1 hr refluxed sample showed the same pattern of degradation as that of 3 hr refluxed sample. There were total six peaks including peak of rutin at R f 0.19 and other peaks were detected at R f 0., 0.17, 0.34, 0.36, 0.49 (Fig..1). Thus, exposure time of the rutin to HCl was kept for 1 hr and the concentration of HCl was decreased to 0.1N. Further on analysis the stressed sample when analyzed showed almost no change as compared to the pervious conditions. Hence it was concluded that the rutin was not stable under any stressed acidic conditions tested. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 310
Fig..1: HPTLC Chromatogram of rutin after acid degradation.2.2.2. Base degradation 1M of NaOH was prepared by dissolving 4 g of sodium hydroxide pellets in 100 ml of distilled water. 1 ml of rutin solution (1 mg/ml) and 4 ml of 1N NaOH were mixed and the mixture was refluxed on water bath for 3 hours at 60 C. The solution was allowed to attend ambient temperature then the solution was neutralized by 1 N HCl to ph 7 and the volume was made up to 10 ml with methanol. Then the final solution was applied on to the TLC plates. Total degradation was found when the rutin was refluxed 1 N NaOH for 3 hr, therefore the exposure time was reduced to 1 hr with the same concentration of NaOH. Then the stressed sample was analyzed. The chromatogram of 1 hr refluxed sample showed the same pattern of degradation as that of 3 hr Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 3
refluxed sample. There were total six peaks including peak of rutin at R f 0.22 were detected and other peaks at R f 0.09, 0.14, 0.23, 0.35, 0.54 (Fig..2). Thus, exposure time of the rutin to NaOH was kept for 1 hr and the concentration of NaOH was decreased to 0.1N. Further on analysis the stressed sample when analyzed showed almost no change as compared to the pervious conditions. Hence it was concluded that the rutin was not stable under any stressed alkaline conditions tested. Fig..2: HPTLC Chromatogram of rutin after base degradation Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 312
.2.2.3. Oxidative degradation Oxidative degradation of rutin was studied using 3% of H 2 O 2 for 3 hr. 1 ml of rutin solution (1 mg/ml) and 9 ml 3 % H 2 O 2 solution were mixed and the mixture was refluxed on water bath for 3 hr at 60 C.The solution was allowed to attend ambient temperature and applied on to the TLC plates. When the sample was analyzed, three additional peaks were obtained of degradants. There were no additional peaks at same R f when untreated sample of rutin was analyzed confirming the formation of three degradation products (Fig..3). Comparison of the peak area of rutin in stressed condition with that of untreated sample revealed a 35.66% decrease. The UV spectra confirmed the peak of rutin in the stressed sample (Fig..4) Fig..3: HPTLC Chromatogram of rutin after oxidative stress Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 313
Degradant (254 nm, 366 nm) Rutin (standard) (254 nm, 366 nm Fig..4: UV spectrum of rutin (standard) and degraded product after oxidative stress.2.2.4. Wet degradation 10 ml of aqueous rutin solution (1 mg/ml) were refluxed on water bath for 3 hr at 60 C. The solution was allowed to attend ambient temperature and applied on to the TLC plates. There were four peaks of degradants beside the rutin peak with the R f values 0.06, 0.36, 0.61, 0.80(Fig..5). Rutin peak at R f 0.21 was confirmed by comparing the UV spectrum with the untreated standard rutin. The λ max of the tested rutin (365) and even of the standard rutin λ max was 365 nm (Fig..6). Thus it was confirmed that only 22.76% of rutin was degraded. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 314
Fig..5: HPTLC Chromatogram of rutin after wet degradation Degradant (254 nm, 366 nm) Rutin (standard) (254 nm, 366 nm Fig.6: UV spectrum of rutin (standard) and degradation product after wet degradation Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 315
.2.2.5. Dry heat 5mg of rutin was placed in oven for 3 hr at 100 C and then the heated sample of rutin was dissolved in 5ml of methanol. 5mg of rutin was placed in oven for 24 hr at 60 C and then the heated sample was dissolved in 5ml of methanol. The solution was allowed to attend ambient temperature and applied on to the TLC plates. When the stressed sample was analyzed, no degradation was found at both the conditions. Hence the exposure time was increased up to 48 hr and the temperature was kept constant at 60 C. When the stressed sample was analyzed, there were no additional peaks. The comparison between the peaks areas of stressed sample of rutin with that of untreated sample showed no difference (Fig..7). Hence it was concluded that drug was stable under the tested conditions. Fig..7: HPTLC Chromatogram of rutin after dry degradation Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 316
.2.2.6. Photo stability study 5mg of rutin was exposed to UV short (254 nm) light for 24 hr. Then the exposed sample of rutin was dissolved in 5ml of methanol and applied on to the TLC plates. The stress study under short UV showed two additional peaks with R f 0.12, 0.64 (Fig..8). There were no additional peaks at the same R f of standard when the untreated sample was analyzed confirming the formation of two degradation products. Comparison of the peak area of rutin in stressed condition with that of untreated sample revealed 18.47 % decrease in peak area of rutin under short UV. The UV spectra of the peak with R f 0.12 in stressed sample matched with the untreated rutin (Fig..9) and confirmed the presence of rutin. Thus it was concluded that 18.47% of rutin was degraded upon UV exposure. Fig..8: HPTLC Chromatogram of rutin after UV exposure Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 317
Rutin (standard) (254 nm, 366 nm) Degradant (254 nm, 366 nm) Fig..9: UV spectrum of rutin (standard) and degradation product after UV exposure Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 318
.3 STABILITY STUDIES OF QUERCETIN.3.1 Chromatographic conditions used for the developed and validated HPTLC method for quercetin (Chapter 10) The following densitometric conditions were used for HPTLC studies: Stationary phase : : Precoated plates of Silica Gel 60 GF 254 (Merck) Mobile phase : Chloroform: methanol: formic acid (8.2:1.5:1) Saturation time Development time Wavelength Lamp Band width Length of : 15 min :15 min : 254 nm : Deuterium : 7 mm : 8 cm chromatogram Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 319
.3.2 Forced degradation of Quercetin.3.2.1 Acid degradation The hydrochloric acid (HCl) was prepared by diluting 8.5 ml of concentrated HCl to 100ml of distilled water. 1mg/ml solution was prepared of quercetin. 1 ml of quercetin solution and 4 ml of 1N HCl were mixed and the mixture was refluxed on water bath for 3 hours at 60 C. The refluxed solution of quercetin and HCl was allowed to attend ambient temperature and then the refluxed solution was neutralized by 1 N NaOH to ph 7 and the volume was made up to 10 ml with methanol. Then the final solution was applied on to the TLC plates. Total degradation was found when the quercetin was refluxed 1 N HCl for 3 hr, therefore the exposure time was reduced to 1 hr with the same concentration of HCl. Then the stressed sample was analyzed. The chromatogram of 1 hr refluxed sample showed the same pattern of degradation as that of 3 hr refluxed sample. There were six peaks which were degradants as none of the peak showed similar R f as that of standard. Among the all degradants peak at R f 0.29 was in highest percent (64.99%) (Fig..10) as compare to other degradation compounds. Thus, exposure time of the quercetin to HCl was kept for 1 hr and the concentration of HCl was decreased to 0.1N. Further on analysis the stressed sample when analyzed showed almost no change as compared to the pervious conditions. Hence it was concluded that the quercetin was not stable under any stressed acidic conditions tested. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 320
Fig..10: HPTLC Chromatogram of Quercetin after acid degradation.3.2.2 Base degradation 1M of NaOH was prepared by dissolving 4 g of sodium hydroxide pellets in 100 ml of distilled water. 1 ml of quercetin solution (1 mg/ml) and 4 ml of 1N NaOH were mixed and refluxed on water bath for 3 hours at 60 C. The solution was allowed to attend ambient temperature and then the solution was neutralized by 1 N HCl to ph 7 and the volume was made up to 10 ml with methanol. Then the final solution was applied on to the TLC plates. Total degradation was found when the quercetin was refluxed 1 N NaOH for 3 hr, therefore the exposure time was reduced to 1 hr with the same concentration of NaOH. Then the stressed sample was analyzed. The chromatogram of 1 hr refluxed sample showed the same pattern of degradation as that of 3 hr refluxed sample. Thus, exposure time of the Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 321
quercetin to NaOH was kept for 1 hr and the concentration of NaOH was decreased to 0.1N. Further on analysis the stressed sample when analyzed showed, degradants peak at R f 0.22, 0.41, 0.59, 0.78. The peak at R f 0.22 was in higher concentration with 84.47% (Fig..). Hence it was concluded that the quercetin was not stable under any stressed alkaline conditions tested. Fig..: HPTLC Chromatogram of Quercetin after base degradation.3.2.3 Oxidative degradation 1 ml of quercetin solution (1 mg/ml) and 9 ml 3 % H 2 0 2 solution were mixed and the mixture was refluxed on water bath for 3 hr at 60 C.The solution was allowed to attend ambient temperature and applied on to the TLC plates. There was no oxidative degradation quercetin found when studied using 3% of H 2 O 2 for 3 hr. The exposure time to oxidative condition was increased gradually up to 8 hr. When the stressed sample was analyzed, there were no additional peaks. There was no difference in peak area of stressed sample and Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 322
untreated sample of quercetin. Thus, it indicates that there was no degradation due to oxidative stress (Fig..12). Hence it was concluded that quercetin was stable under the conditions tested. Fig..12: HPTLC Chromatogram of quercetin after oxidative stress.3.2.4 Wet degradation 10 ml of aqueous quercetin solution (1 mg/ml) were refluxed on water bath for 3 hr at 60 C. The solution was allowed to attend ambient temperature and then applied on to the TLC plates. There were three peaks of degradants beside the quercetin peak with the R f values 0.34, 0.56, 0.59, 0.76 (Fig..13). The R f of quercetin was shifted from 0.76 to 0.92. At R f 0.76 there was a degradant peak was observed with higher percentage of peak area (51.81%). Quercetin peak at R f 0.92 was confirmed by comparing the UV spectrum with the untreated standard Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 323
quercetin (Fig..14). The λ max of the tested quercetin and of the standard quercetin λ max was 254 nm and 385 nm. Thus it was confirmed that 86.15% of quercetin was degraded. Fig..13: HPTLC Chromatogram of quercetin after wet degradation Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 324
Degradant (254 nm, 385 nm) Quercetin (standard) (254 nm, 385 nm) Fig.14: UV spectrum of quercetin (standard) and degradant (UV exposed condition).3.2.5 Dry Heat Degradation 5mg of quercetin was placed in oven for 3 hr at 100 C and then the hated sample of quercetin was dissolved in 5ml of methanol. 5mg of quercetin was placed in oven for 24 hr at 60 C and then the heated sample was dissolved in 5ml of methanol. The both the solutions were allowed to attend ambient temperature and applied on to the TLC plates. When the stressed sample of 3 hr at 100 C was analyzed, no degradation was found (Fig..15). But the sample of 24 hr at 60 C showed total three peaks out which one with R f 0.77 was of quercetin which was confirmed by the UV spectrum (Fig..16). The other two were (with R f 0.88, 0.96) of degradants and the total percent of degradation of quercetin was found to be 5.22 % (Fig..17 ). Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 325
Fig..15: HPTLC Chromatogram of quercetin after dry degradation for 3 hr at 100 C Fig..16: HPTLC Chromatogram of quercetin after dry degradation for 24 hr at 60 C Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 326
Degradant (254 nm, 385 nm) Quercetin (standard) (254 nm, 385 nm) Fig..17: UV spectrum of quercetin (standard) and degradant (Dry degradation for 24 hr at 60 C ).3.2.6 Photostability study 5mg of quercetin was exposed to UV short (254 nm) light for 24 hr to study the UV degradation. Then the exposed sample of quercetin was dissolved in 5ml of methanol and applied on to the TLC plates. When the stressed sample was analyzed there was no degradation observed of the quercetin sample (Fig..18). The sample was again exposed for 48 hr. Further chromatographic studies showed no degradation. Hence, it was concluded that the quercetin sample was stable under tested conditions. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 327
Fig..18: HPTLC Chromatogram of quercetin after UV exposure Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 328
.4 STABILITY STUDIES OF CONESSINE.4.1 Chromatographic conditions used for the developed and validated HPTLC method for conessine (Chapter 9) The following densitometric conditions were used for HPTLC studies: Stationary phase : : Precoated plates of Silica Gel 60 GF 254 (Merck) Mobile phase : Toluene: ethyl acetate: diethylamine (2.5:6.5:1) Saturation time Development time Wavelength Lamp Band width Length of : 15 min :15 min : 247 nm : Deuterium : 7 mm : 8 cm chromatogram Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 329
.4.2 Forced degradation of Conessine.4.2.1 Acid degradation 1 M of hydrochloric acid (HCl) was prepared by diluting 8.5 ml of concentrated HCl to 100ml of distilled water.1mg/ml solutions was prepared of standard conessine. 1 ml of conessine solution and 4 ml of 1N HCL were mixed and the mixture was refluxed on water bath for 3 hours at 60 C. The solution was allowed to attend ambient temperature then the solution was neutralized by 1 N NaOH to ph 7 and the volume was made up to 10 ml with methanol. The final solution was applied on to the TLC plates. Total degradation was found when the conessine was refluxed 1 N HCl for 3 hr, therefore the exposure time was reduced to 1 hr with the same concentration of HCl. Then the stressed sample was analyzed. The chromatogram of 1 hr refluxed sample showed the same pattern of degradation as that of 3 hr refluxed sample. Thus, exposure time of the conessine to HCl was kept for 1 hr and the concentration of HCl was decreased to 0.1N. Further the stressed sample when analyzed showed no change as compare to pervious conditions. There were four peaks which were degradants (Fig..19). Among the all degradants, peak at R f 0.19 was in highest percent (35.43%) as compare to other degradation compounds. R f value of none of the peaks matched with the R f of treated sample. Hence it was concluded that the conessine was not stable under any stressed acidic conditions tested. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 330
Fig..19: HPTLC Chromatogram of conessine after acid degradation.4.2.2 Base degradation 1M of NaOH was prepared by dissolving 4 g of sodium hydroxide pellets in 100 ml of distilled water. 1 ml of conessine solution (1 mg/ml) and 4 ml of 1N NaOH were mixed and the mixture was refluxed on water bath for 3 hours at 60 C. The solution was allowed to attend ambient temperature then the solution was neutralized by 1N HCl to ph 7 and the volume was made up to 10 ml with methanol. The final solution was applied on to the TLC plates. Total degradation was found when the conessine was refluxed 1 N NaOH for 3 hr, therefore the exposure time was reduced to 1 hr with the same concentration of NaOH. Then the stressed sample was analyzed. The chromatogram of 1 hr refluxed sample showed the same pattern of degradation as that of 3 hr refluxed sample. The degradants showed peak at R f Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 331
0.07 0.09, 0.95. The peak at R f 0.09 was in higher concentration with 69.22 % (Fig..20). Thus, exposure time of the conessine to NaOH was kept for 1 hr and the concentration of NaOH was decreased to 0.1N. Further on analysis the stressed sample when analyzed showed almost no change as compared to the pervious conditions. Hence it was concluded that the conessine was not stable under any stressed alkaline conditions tested. Fig..20: HPTLC Chromatogram of conessine after base degradation Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 332
.4.2.3 Oxidative degradation 1 ml of conessine solution (1 mg/ml) and 9 ml 3 % H 2 0 2 solution were mixed and the mixture was refluxed on water bath for 3 hr at 60 C.The solution was allowed to attend ambient temperature and applied on to the TLC plates. There was no oxidative degradation conessine found when studied using 3% of H 2 O 2 for 3 hr. The exposure time to oxidative condition was increased gradually up to 8 hr. When the stressed sample was analyzed, there were no additional peaks. Also the comparison between the peak areas of stressed sample of conessine with that of untreated sample showed no difference, indicating that there was no degradation (Fig..21). Hence it was concluded that conessine was stable under the conditions tested. Fig..21: HPTLC Chromatogram of conessine after oxidative stress Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 333
.4.2.4 Wet degradation 10 ml of aqueous conessine solution (1 mg/ml) was refluxed on water bath for 3 hr at 60 C. The solution was allowed to attend ambient temperature and applied on to the TLC plates. There was no degradation found when conessine was refluxed with water for 3hr, so the exposure time to wet condition was increased gradually up to 8 hr. When the stressed sample was analyzed, there were no additional peaks. The comparison between the peak areas of stressed sample of conessine with that of untreated sample showed no difference, indicating that there was no degradation (Fig..22). Hence it was concluded that the drug was stable under the conditions tested. Fig..22: HPTLC Chromatogram of conessine after wet degradation Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 334
.4.2.5 Dry heat 5mg of conessine was placed in oven for 3 hr at 100 C and then the heated sample was dissolved in 5ml of methanol. 5mg of standard was placed in oven for 24 hr at 60 C and then the heated sample was dissolved in 5ml of methanol. The both the solutions were allowed to attend ambient temperature and were applied on to the TLC plates. When the stressed sample of 3 hr was analyzed, no degradation was found (Fig..23). But the sample of 24 hr showed total two peaks (Fig..24) out which one with R f 0.81 was of conessine which was confirmed by the UV spectrum (Fig..25). The other one was with R f 0.92 of degradant and the total percent of degradation of conessine was found to be 8.52 %. Fig..23: HPTLC Chromatogram of conessine after dry degradation for 3 hr at 100 C Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 335
Fig.. 24: HPTLC Chromatogram of conessine after dry degradation for 24 hr at 60 C Degradant (247 nm) Conessine (standard) (247 nm) Fig..25: UV spectrum of conessine(standard) and degradant (dry degradation for 3 hr at 100 C) Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 336
.4.2.6 Photostability study 5mg of conessine was exposed to UV short (254 nm) light for 24 hr. Then the exposed sample was dissolved in 5ml of methanol and applied on to the TLC plates. When the stressed sample was analyzed there was no degradation observed of the conessine sample. The sample was again exposed to the wavelength for 48 hr. Further chromatographic studies showed no degradation (Fig..26). Hence, it was concluded that the conessine sample was stable under tested conditions. Fig..26: HPTLC Chromatogram of conessine after UV exposure Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 337
.5 CONCLUSION The application of developed and validated HPTLC methods for rutin, quercetin and conessine as stability indicating methods was successfully employed. It was observed that rutin was totally stable only under dry condition but the others conditions had altered the concentration of rutin. Quercetin was stable under oxidative stress and UV exposure but the peak area was lowered with presence of degradants peaks in all other stress conditions. Dry, wet, oxidative and UV exposure stress conditions could not affect the conessine marker. But conessine had degraded in acidic and basic conditions. Rutin and quercetin are biomarkers with very good anti-oxidant and many other therapeutic activities. They are even present in many medicinal plants. Thus the stability indicating method can very well adapted for the evaluations of many different formulations containing these two biomarkers. On other hand conessine is very specific biomarker to species Holarrhena with well proven pharmacological activities. Thus even the stability indicating method of this marker is essential and beneficial to have a check on the stability of formulations containing conessine. Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 338