9.1 OBJECTIVE To monitor the stability of the developed herbal formulations using the limited and specific methods. 9.2 INTRODUCTION The legal definition of stability is aimed at assuring that the drug / drug product remains within specifications established to ensure its identity, strength, quality and purity. Stability is interpreted as length of time under specific conditions and storage that a product will remain within the pre-defined limits for all its important characteristics (ICH Guidelines; 1993). Stability testing of the herbal formulation is also a important task, for acceptability of the product during its shelf life. Measuring chemical stability is very challenging task due to the complexity of a plant extract, which may contain several hundreds of different compounds. The purpose of stability testing is to provide evidence on how the quality of herbal products varies with the time under the influence of environmental factors. Objective of stability testing: Each ingredient, whether therapeutically active or inactive, in a dosage form can affect stability. Environmental factors such as temperature, light, air (specifically oxygen, carbon dioxide and water vapors) and humidity can affect stability. Drug products are subject to decomposition resulting in loss of potency. The purpose of stability testing is to provide evidences on how the quantity and / or quality of a medicinal substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light. Stability testing enables recommended storage conditions, retest periods and shelf lives for the drugs / drug products to be established (ICH Guidelines Q 1 A (R2), 1996) 9.3 LITERATURE REVIEW In-compatibilities between drugs and excipients were quite obvious because of their interactions. Quantization was the most important aspect to determine the presence of substance of interest, it require sensitive methods to determine the same in presence of other components. The same step by step developed from titrimetric to colorimetric reactions, spectrophotometer, TLC, HPLC & GC with different types of detectors are 232
even capable to detect small amounts of impurities and decomposition products. (Cartensen, 1994) Regulatory guidelines for stability: The World Health Organization (WHO) has published a guideline Stability testing of pharmaceutical products containing well established drug substances in conventional dosage forms. A new dosage form is defined as a drug product which is a different pharmaceutical product type, but contains the same active substance as included in the existing drug product approved by the pertinent regulatory authority (ICH Harmonized Tripartite Guideline, 1996). ICH defined well in Stability testing of new drug substances and products the choice of test conditions is based on an analysis of the effects of climatic conditions in the three regions of the EC, Japan and the United States. The mean kinetic temperature in any part of the world can be derived from climatic data, and the world can be divided into four climatic zones, I-IV. The principle has been established that stability information generated in any one of the three regions of the EC, Japan and the United States would be mutually acceptable to the other regions, provided the information is consistent with this guideline and the labeling is in accordance with national/regional requirements (ICH Guidelines Q 1 A (R2), 1996). As per the ICH and WHO guidelines, long-term storage condition for zones III and IV, the intermediate storage condition in the general case for zones I and II in the parent guideline is changed to 30 C ± 2 C/65% RH ± 5% RH. This condition of 30 C ± 2 C/65% RH ± 5% RH can also be a suitable alternative to 25 C ± 2 C/60% RH ± 5% RH as the long-term storage condition for Zones I and II. (ICH guidelines Q1 F step-4, 1996). Storage conditions General case For the General case (as described in the parent guideline), the recommended longterm and accelerated storage conditions for Climatic Zones III and IV are shown below table no 9.1. 233
Table 9.1. Accelerated storage conditions for Climatic Zones III and IV Study Storage condition Long-term 30 C ± 2 C/65% RH ± 5% RH 12 months Accelerated 40 C ± 2 C/75% RH ± 5% RH 6 months Minimum time period covered by data at submission The designated long term testing conditions will be reflected in the labeling and retest date. Long term testing 25 0 C ± 2 0 C / 60% RH ± 5% 12 Months Accelerated Testing 40 0 C ± 2 0 C / 75% RH ± 5% 6 Months No intermediate storage condition for stability studies is recommended for Climatic Zones III and IV. Therefore, the intermediate storage condition is not relevant when the principles of retest period or shelf life extrapolation described in Q1E are applied. (ICH guidelines Q1 F step-4, 1996) Accelerated stability tests In accelerated stability tests, the substance or medicinal product is challenged by a controlled exaggerated stress (storage conditions) over a short time, so that the rate of degradation reaction is enhanced. Therefore, in a relatively short period of time, much information is obtained about the stability of the drug product. Accelerated tests are of particular value in providing information on stability to the formulator in the early stages of product development. The purpose is to determine kinetic parameters to predict the tentative expiry date in a short period of time e. g. 3 6 months. Besides stress testing also helps in validating the stability indicating power of the analytical procedures used. However, accelerated stability is not a substitute for the long-term actual room temperature stability testing. Finally the conditions drawn from accelerated stability testing with respect to extrapolation of shelf life have to be confirmed with the actual ambient room temperature stability testing. The main variables to be considered in a stability testing program are temperature, light and moisture. The effect of light is usually studied in a light cabinet in clear and amber bottles. The effect of moisture is studied by varying the relative humidity and the effect of temperature in constant temperature oven. According to the US-FDA 234
guidelines for stability studies, the samples for the accelerated stability data, samples are stored at room temperature and at 40 0 ± 2 0 C; 75 % ± 5 % relative humidity (abbreviated here in after as RH) for a period of six months. 9.4 MATERIALS AND METHODS The prepared formulations were subjected to observation of some parameters like description, identification, weight per ml, ph, viscosity, assay, heavy metals and microbial purity during the stability studies. (Vijay R Salunkhe, 2009) The herbal formulation was used for the stability studies. The changes in the quality of the product may occur due to the interactions between the product and container / closure, which determine the effects of the container on product quality. Based on the present market strategy the herbal formulations are packed in below described manner. 1. Pet bottles with aluminum cap containing rubber cork seal to protect the formulation from external gaseous environment. 2. Glass bottles with aluminum cap containing rubber cork seal to protect the formulation from external gaseous environment. The formulations were packed in the two above-mentioned sets of containers. One set kept in an incubator maintained at storage temperature 25 0 C ± 2 0 C / 60% RH ± 5%. This served as control sample. Another set kept in an oven maintained at accelerated temperature 40 0 C ± 2 0 C / 75% RH ± 5%. The samples were removed from both control and accelerated storage temperature conditions. These samples were analyzed by HPLC using the specified methodology mentioned in HPLC chapter for their photochemical content, at zero days, one, two and three months. At the specified time intervals the sampling was done, the samples in different types of containers were analyzed as per the methodology mentioned in the chromatography section. The results were tabulated for the one active constituent in table no 9.2. 235
Table 9.2. Specification for Scoparia dulcis Linn. herbal formulation: Code Test Specification T-01 Description Clear Green colored syrup T-02 Identification (By HPLC) T-03 Weight per ml 1.10 to 1.20 gram per ml. T-04 ph 2.50 to 3.50 T-05 Viscosity Between 100 and 210 cps In the Assay test, the retention time of the peaks corresponding to Scopadulcic acid B, Alpha amyrin in the Sample solution matches with the retention time of the peaks corresponding to Scopadulcic acid B, Alpha amyrin obtained with Standard solution T-06 Assay 90.0 % to 110.0 % of label claim Assay of Scopadulcic Acid B, Sod. Benzoate & Sorbic acid 90.0 % to 110.0 % of label claim T-07 Heavy Metals (By AAS) Arsenic : Not more than 2 ppm Mercury : Not more than 1 ppm Lead : Not more than 10 ppm Cadmium : Not more than 0.3ppm T-08 Microbiological purity TAMC : NMT 10 5 (Maximum acceptable count: 500 000) CFU/g or CFU/mL TYMC : NMT 10 4 (Maximum acceptable count: 50 000) CFU/g or CFU/mL Bile tolerant gram-negative bacteria : 10 4 CFU/g or CFU/mL Escherichia coli : Absence (1g or ml) Salmonella : Absence (25g or 25mL) 236
9.5 RESULTS: Table 9.3. Compiled stability data sheet for Scoparia dulcis Linn. herbal formulation (PET bottle) Test Limits Initial 1 Month 2 Month 3 Month Description Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Identification Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Weight per ml Spec* 1.122 1.124 1.124 1.123 1.123 1.122 1.122 ph Spec* 3.21 3.25 3.3 3.24 3.34 3.25 3.28 Viscosity Spec* 130 cps --- --- ---- --- --- 135 cps Scopadulcic Spec* 102.22% 102.41% 99.16% 101.31% 100.84% 100.21% 99.98% acid B Alpha amyrin Spec* 99.02% 98.04% 95.10% 96.08% 95.10% 98.04% 95.10% Sod. Benzoate Spec* 100.62% --- --- ---- --- 100.71% 98.76% Sorbic acid Spec* 101.84% --- --- --- --- 98.92% 94.48% Microbial purity Assay TAMC Spec* 65 cfu/ml --- --- ---- --- 95 cfu/ml 84 cfu/ml TYMC Spec* 33 cfu/ml --- --- --- --- 80 cfu/ml 90 cfu/ml Bile tolerant Spec* Absent --- --- --- --- Absent Absent gram-negative bacteria. Escherichia coli Spec* Absent --- --- --- --- Absent Absent Salmonella Spec* Absent --- --- --- --- Absent Absent Spec*: Refer for specification Complies*: Specification complies, Results obtained are with methods mentioned in chapter 6 and 7. 237
Table 9.4. Compiled stability data sheet for Scoparia dulcis Linn. herbal formulation (Glass bottle) Test Limits Initial 1 Month 2 Month 3 Month Description Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Identification Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Weight per ml Spec* 1.122 1.124 1.124 1.123 1.123 1.122 1.122 ph Spec* 3.24 3.25 3.24 3.24 3.24 3.25 3.28 Viscosity Spec* 130 cps --- --- ---- --- --- 145 cps Scopadulcic Spec* 102.22% 103.53% 99.29% 101.95% 100.91% 101.60% 101.69% acid B Alpha amyrin Spec* 99.02% 99.02% 95.10% 96.08% 95.21% 96.08% 95.10% Sod. Benzoate Spec* 100.62% --- --- ---- --- 99.33% 100.04% Sorbic acid Spec* 101.84% --- --- --- --- 98.48% 95.4% Microbial purity Assay TAMC Spec* 65 cfu/ml --- --- ---- --- 110 cfu/ml 94 cfu/ml TYMC Spec* 33 cfu/ml --- --- --- --- 65 cfu/ml 71 cfu/ml Bile tolerant Spec* Absent --- --- --- --- Absent Absent gram-negative bacteria. Escherichia coli Spec* Absent --- --- --- --- Absent Absent Salmonella Spec* Absent --- --- --- --- Absent Absent Spec*: Refer for specification Complies*: Specification complies, Results obtained are with methods mentioned in chapter 6 and 7. 238
Achyranthes aspera Linn. herbal formulation Table 9.5. Specification for finished product: Code Test Specification 1. Description Clear green colored syrup 2. Identification (BY HPLC) 3. Weight per ml 1.10 to 1.20 gram per ml 4. ph 3.50 to 4.50 In the Assay test, the retention time of the peaks corresponding to Oleanolic acid solution matches with the retention time of the peaks corresponding to Oleanolic acid with Standard solution 5. Viscosity Between 100 and 210 cps 6. Assay 95.0 % to 105.0 % of label claim Assay of Oleanolic acid Sod. Benzoate & Sorbic acid 7. Heavy Metals (By AAS) 8. Microbiological purity 95.0 % to 105.0 % of label claim Arsenic : Not more than 2 ppm Mercury : Not more than 1 ppm Lead : Not more than 10 ppm Cadmium : Not more than 0.3ppm TAMC : NMT 10 5 (Maximum acceptable count: 500 000) CFU/g or CFU/mL TYMC : NMT 10 4 (Maximum acceptable count: 50 000) CFU/g or CFU/mL Bile tolerant gram-negative bacteria : 10 4 CFU/g or CFU/mL Escherichia coli : Absence (1g or ml) Salmonella : Absence (25g or 25mL) 239
Table 9.6. Compiled stability data sheet for Achyranthes aspera Linn. herbal formulation (PET bottle) Test Limits Initial 1 Month 2 Month 3 Month Description Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Identification Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Weight per ml Spec* 1.135 1.134 1.134 1.134 1.134 1.134 1.134 ph Spec* 3.81 3.85 3.83 3.84 3.84 3.85 3.88 Viscosity Spec* 151 cps --- --- ---- --- --- 145 cps Oleanolic acid Spec* 101.25% 101.39% 99.39% 100.44% 99.39% 101.35% 100.20% Sod. Benzoate Spec* 99.91 --- --- ---- --- 98.67% 97.82% Sorbic acid Spec* 102.24 --- --- --- --- 98.04% 94.48% Microbial purity Assay TAMC Spec* 48 cfu/ml --- --- ---- --- 75 cfu/ml 80 cfu/ml TYMC Spec* 36 cfu/ml --- --- --- --- 58 cfu/ml 86 cfu/ml Bile tolerant Spec* Absent --- --- --- --- Absent Absent gram-negative bacteria. Escherichia coli Spec* Absent --- --- --- --- Absent Absent Salmonella Spec* Absent --- --- --- --- Absent Absent Spec*: Refer for specification Complies*: Specification complies, Results obtained are with methods mentioned in chapter 6 and 7. 240
Table 9.7. Compiled stability data sheet for Achyranthes aspera Linn. herbal formulation (Glass bottle) Test Limits Initial 1 Month 2 Month 3 Month Description Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Identification Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Weight per ml Spec* 1.135 1.134 1.134 1.134 1.134 1.134 1.134 ph Spec* 3.81 3.85 3.83 3.84 3.84 3.85 3.88 Viscosity Spec* 151 cps --- --- ---- --- --- 145 cps Oleanolic acid Spec* 101.25% 99.91 102.24 100.57% 98.65% 101.42% 98.89% 100.54% 98.78% Sod. Benzoate Spec* --- --- ---- --- 99.24% 97.33% Sorbic acid Spec* --- --- --- --- 99.52% 95.32% Microbial purity Assay TAMC Spec* 48 cfu/ml --- --- ---- --- 98 cfu/ml 90 cfu/ml TYMC Spec* 36 cfu/ml --- --- --- --- 65 cfu/ml 60 cfu/ml Bile tolerant Spec* Absent --- --- --- --- Absent Absent gram-negative bacteria. Escherichia coli Spec* Absent --- --- --- --- Absent Absent Salmonella Spec* Absent --- --- --- --- Absent Absent Spec*: Refer for specification Complies*: Specification complies, Results obtained are with methods mentioned in chapter 6 and 7. 241
Poly herbal formulation: Table 9.8. Specification for finished product: Sr. No. Tests Specification 1. Description Dark green colored syrupy liquid with characteristic flavor of pineapple, taste sweet. 2. ph Between 4.0 and 6.0 3. Weight per ml Between 1.15 and 1.35 g / ml 4. Viscosity Between 100 and 210 cps 5. Assay of Gallic Acid NLT 65.0 mg per 100 ml 6. Assay of Glycyrrhizic Acid NLT 120.0 mg per 100 ml 7. Assay of Vasicine NLT 2.0 mg per 100 ml 8. Assay of 6-Gingerol Sod. Benzoate & Sorbic acid 9. Heavy Metals (By AAS) 10. Microbiological purity NLT 2.0 mg per 100 ml 90.0 % to 110.0 % of label claim Arsenic : Not more than 2 ppm Mercury : Not more than 1 ppm Lead : Not more than 10 ppm Cadmium : Not more than 0.3ppm TAMC : NMT 10 5 (Maximum acceptable count: 500 000) CFU/g or CFU/mL TYMC : NMT 10 4 (Maximum acceptable count: 50 000) CFU/g or CFU/mL Bile tolerant gram-negative bacteria : 10 4 CFU/g or CFU/mL Escherichia coli : Absence (1g or ml) Salmonella : Absence (25g or 25mL) 9.6 DISCUSSION OF RESULTS IN LIGHT OF OTHERS WORK There is no literature reported for the stability study of Achyranthes aspera Linn., Scoparia dulcis Linn. and poly herbal formulations. Description and identification of the formulations complies up to 3 months for samples maintained at.weight per ml, ph, viscosity and microbial purity results remain unchanged and found compliant for all the three herbal formulations. Scopadulcic acid B and Sodium benzoate results comply with specification. Alpha amyrin and Sorbic acid were slightly reduced from initial values in 3 rd month at 40ºC + and found as 95.1% and 94.5 %. Oleanolic acid content was stable and monitored upto 3 month at, Sodium benzoate and Sorbic acid were slightly reduced to 97.8 %and 94.5 %, but found stable at condition. 242
Table 9.9. Compiled stability data sheet for poly herbal formulation (PET bottle) Test Limits Initial 1 Month 2 Month 3 Month Description Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Identification Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Weight per ml Spec* 1.26 1.264 1.264 1.262 1.262 1.262 1.262 ph Spec* 4.53 4.53 4.6 4.51 4.65 4.54 4.72 Viscosity Spec* 181 cps --- --- ---- --- --- 189 cps Gallic acid Spec* 101.53% 101.11% 102.32% 101.26% 99.53% 102.74% 99.37% Glycyrrhizic Spec* 100.10% 100.38% 98.35% 99.18% 96.75% 97.88% 93.52% acid Vasicine Spec* 99.47% 99.74% 100.53% 100.53% 101.32% 101.32% 99.74% 6-Gingerol Spec* 100.86% 99.14% 99.43% 99.71% 100.29% 98% 100.57% Sod. Benzoate Spec* 102.44% --- --- ---- --- 99.82% 100.53% Sorbic acid Spec* 103.68% --- --- --- --- 98.04 94.36 Microbiological purity Assay TAMC Spec* 92 cfu/ml --- --- ---- --- 48 cfu/ml 30 cfu/ml TYMC Spec* 80 cfu/ml --- --- --- --- 65 cfu/ml 70 cfu/ml Bile tolerant Spec* Absent --- --- --- --- Absent Absent gram-negative bacteria. Escherichia coli Spec* Absent --- --- --- --- Absent Absent Salmonella Spec* Absent --- --- --- --- Absent Absent Spec*: Refer for specification Complies*: Specification complies, Results obtained are with methods mentioned in chapter 6 and 7. 243
Table 9.10. Compiled stability data sheet for poly herbal formulation (Glass bottle) Test Limits Initial 1 Month 2 Month 3 Month Description Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Identification Spec* Complies* Complies* Complies* Complies* Complies* Complies* Complies* Weight per ml Spec* 1.26 1.264 1.264 1.262 1.262 1.262 1.262 ph Spec* 4.63 4.58 4.63 4.55 4.45 4.55 4.65 Viscosity Spec* 181 cps --- --- ---- --- --- 195 cps Gallic acid Spec* 101.53% 101% 100% 101% 98.74% 100.11% 99.16% Glycyrrhizic Spec* 100.10% 98.69% 97.96% 99.26% 97.95% 97.65% 93.04% acid Vasicine Spec* 99.47% 100.0% 101.05% 101.32% 101.58% 100.53% 101.05% 6-Gingerol Spec* 100.86% 100.29 100.86 101.14 99.14 99.71 99.14 Sod. Benzoate Spec* 102.44% --- --- ---- --- 99.69% 98.13% Sorbic acid Spec* 103.68% --- --- --- --- 98.88% 95.08% Microbiological purity Assay TAMC Spec* 92 cfu/ml --- --- ---- --- 48 cfu/ml 35 cfu/ml TYMC Spec* 80 cfu/ml --- --- --- --- 58 cfu/ml 65 cfu/ml Bile tolerant Spec* Absent --- --- --- --- Absent Absent gram-negative bacteria. Escherichia coli Spec* Absent --- --- --- --- Absent Absent Salmonella Spec* Absent --- --- --- --- Absent Absent Spec*: Refer for specification Complies*: Specification complies, Results obtained are with methods mentioned in chapter 6 and 7. 244
Gallic acid, Vasicine, 6-Gingerol and Sodium benzoate are found stable and are monitored up to 3 month at condition. Glycyrrhizic acid and Sorbic acid were considerably reduced to 93.5 %and 94.3 % but found stable at condition. The packing material has no affect on the stability of the product for all the three formulations, glass and PET bottles results are similar, hence PET bottle is recommended for the product storage and transport. 9.7 CONCLUSION All the above formulations are stable up to 3 months at room temperature and accelerated temperatures of measured contents when packed in PET bottles with Aluminum cap containing rubber cork, Glass bottles with Aluminum cap containing rubber cork inside. 9.8 FUTURE PROSPECTS Studies are required to identify all the chemical components present in the formulation and quantification need to be performed to establish exact shelf life of the product, based on the chemical stability of interested molecules. 9.9 REFERENCES ICH Guidelines: Stability testing of New Drug Substances and Products, 27 th October 1993. ICH Harmonized tripartite guideline, Stability testing for new dosage forms 1996. ICH harmonized tripartite guideline, Evolution for stability data, current step 4 version dated 6 February 2003. Cartensen J.T. Drug Stability Principles and Practices, Marcel Dekker Inc. New York, 2nd edition, Chapter 1994; 1: 1 18. The tripartite harmonised ICH guideline was finalised (Step 4) in November 1996. United States Pharmacopoeia, 24 NF (19), The United States Pharmacopoeia Convention Inc. USA, 2000. Vijay R Salunkhe, Journal of Pharmacy Research, 2009; 2: 6. 245