A simple dissolution test and standard was developed for Turmeric capsules to replace the less discriminating disintegration test stated in the Supplement to ...
Thai J. Pharm. Sci. 31 (2007) 83-90
83
Original article
An approach to developing dissolution standards for turmeric capsules I: basket rotating method Nantana Sittichai1*, Sirichai Krabesri1, Ekamol Suthison1 and Parkpoom Tengamnuay2 1Bureau of Drug and Narcotic, Department of Medical Sciences, Ministry of Public Health,
Nonthaburi 11000, Thailand 2Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
*Corresponding author: Tel: +66 (0) 2591 0503-14 ext. 99120 Fax: +66 (0) 2580 5733
Abstract: A simple dissolution test and standard was developed for Turmeric capsules to replace the less discriminating disintegration test stated in the Supplement to Thai Herbal Pharmacopoeia 2004. The proposed test used the basket at 100 rpm and the dissolution medium composed of 0.05 M hydrochloric acid with sodium lauryl sulfate at 0.8% w/v. The acceptable Q-value was proposed as 75% in 60 minutes. Keywords: Basket method; Curcumin; Dissolution; Turmeric capsules
84
N. Sittichai et al.
Introduction
Materials and Methods
Turmeric or Khamin Chan Capsules are currently monographed in the Supplement to Thai Herbal Pharmacopoeia (THP) 2004. They contain Khamin Chan in powdered form with the commonly available strength of 250 mg per capsule. THP Turmeric is the dried rhizome of Curcuma longa L. (Family Zingiberaceae). It is indigenous to Thailand, China, India, Indonesia, and Vietnam and extensively cultivated in China, India, Thailand and throughout the tropical regions including Africa [1, 2]. The major chemical constituents are volatile oil and curcuminoids, 50-60% of which are a mixture of curcumin, monodesmethoxycurcumin and bisdesmethoxycurcumin [1]. Volatile oil, as well as curcumin and its derivatives, possess the anti-inflammatory activity. Curcumin also exhibits the anti-oxidant activity in patients with tropical pancreatitis [3]. Clinical trials showed that oral administration of the drug showed a significant response in patients with peptic ulcers and dyspepsia [4]. Its principal use is for the treatment of dyspepsia and stomachic [1, 5, 6]
Chemicals and apparatus Curcumin, Lot 453335/1, Purity ≥ 95.0% (TLC) was from Fluka, Switzerland. Water was purified water. All chemical reagents were analytical-reagent grade and were used as purchased. A Spectronic 1001 Plus UV-Visible double-beam spectrophotometer was from Milton Roy Co., USA and a dissolution apparatus Model 10-1500 was from Vankel Industries Inc., USA. The apparatuses were calibrated as scheduled according to the quality control manual of Bureau of Drug and Narcotic, Department of Medical Sciences. All experiments were carried out at ambient temperature.
It is well accepted that no absorption of the ingredients from the biological fluid is possible unless they are liberated from their dosage forms. The disintegration and dissolution requirements can evaluate the performance of solid oral dosage forms of botanical preparations. In general, the disintegration testing does not assure dissolution of the active ingredients as the dissolution testing does. Yet it seems impossible and unnecessary to measure the dissolution of every known ingredient in a botanical preparation. So it is suggested that the demonstration of only one marker substance in a botanical preparation for which a quantitative determination is specified in the monograph be sufficient [7]. As yet, THP monograph on Turmeric Capsules includes only the disintegration test. It is thus the goal of this study to develop the dissolution standards by employing a rotating basket device with curcumin as a marker substance.
Capsule selection Five different lots (two boxes each) of turmeric capsules with label claims 0.024 g of curcuminoids were purchased from a retail pharmacy. Each box contained 100 capsules. All the capsules were at least one year away from their expiration dates at the time of testing. Before subjecting to the dissolution testing, each product was tested for contents of curcuminoids, water, uniformity of weight, and disintegration, according to the official requirements of THP 2004 [6]. Determination of curcuminoids content Following the procedures described in THP 2004, the UV spectrophotometric method was employed for measuring the absorbance of the sample solution at 420 nm. The content of curcuminoids as curcumin in the sample was calculated on the anhydrous basis, with reference to the standard curcumin curve [6]. The intraday coefficient of variation ranged between 1.9 and 2.98%. Dissolution procedure Preparation of various dissolution media To prepare pH 4.0 Buffer, a 2.86 mL aliquot of glacial acetic acid was added to 900 mL of water in
Thai J. Pharm. Sci. 31 (2007) 83-90 a 1000 mL volumetric flask. An aliquot of a 50% w/v solution of sodium hydroxide was added to this solution to adjust pH to 4.0 ± 0.05, before water was added to make up the final volume. This solution was deaerated by heating to about 45 ÌC and slowly vacuum-degassing for about 5 minutes. A 900 mL portion was transferred to a 1000 mL dissolution vessel and placed into the dissolution testerûs water bath. The medium was equilibrated to 37 ÌC±0.5 ÌC for about 30 minutes prior to starting the dissolution test. To prepare sodium lauryl sulfate (SLS) 0.2% in 0.01 M hydrochloric acid (pH 2.2), a 2.0 g portion of SLS was transferred to a 1000 mL volumetric flask and dissolved into approximately 300 mL of water. A 0.85 mL aliquot of concentrated hydrochloric acid (~12.1M) was added to the solution and then diluted to volume with water. This solution was deaerated under vacuum and a 900 mL portion was transferred to a 1000 mL dissolution vessel and placed into the dissolution testerûs water-bath for equilibration at 37 CÌ ± 0.5 ÌC for about 30 minutes. To prepare SLS 0.3% in 0.05 M hydrochloric acid (pH 1.4), a 3.0 g portion of SLS was transferred to a 1000 mL volumetric flask and dissolved into approximately 300 mL of water. A 4.25 mL aliquot of concentrated hydrochloric acid (~12.1M) was added to the solution and then diluted to volume with water. After the deaeration, 900 mL of the medium was used and equilibrated similar to 0.2% SLS in 0.01 M hydrochloric acid. To prepare SLS 0.4%, 0.6% and 0.8% in 0.05 M hydrochloric acid (pH 1.4), proceed as directed in the preparation of 0.3% SLS in 0.05 M hydrochloric acid but using 4.0 g, 6.0 g and 8.0 g portions of SLS instead of a 3.0 g portion of SLS. Standard curcumin curve and curcumin determination A 400 µg/mL solution of curcumin in methanol was prepared. Aliquots of this solution were diluted with methanol to obtain solutions containing 0.4, 0.8, 1.6, 2.0, 2.4, and 3.2 µg/mL of curcumin. Measure the
85 absorbances of the curcumin solutions relative to the blank at 420 nm, using a 1 cm cell. A 10 mL portion of the dissolution medium was withdrawn from the vessels at 30, 45, and 60 minutes. The dissolution medium withdrawn was not replaced. The sample was filtered using a Whatman filter paper No. 1 discarding the first few mL. Five mL of the filtered solutions of the 6 individual capsules withdrawn were combined as the pooled sample and then diluted to 50.0 mL with methanol. It was further diluted 5 folds with methanol and measured for the absorbances, using methanol as the blank. The average amount of curcuminoids, expressed as curcumin, dissolved at different time was determined by reference to the standard curcumin curve and calculated on the anhydrous basis. Statistical analysis of data The test for normal distribution of data (Kolmogorov-Smirnov One-sample Test) followed by the analysis of variance (ANOVA) were performed using the SPSS program [8]. The significance level (α) was set at 0.05.
Results and Discussion Curcuminoids and water contents, weight variation and disintegration tests Five lots of commercial turmeric capsules of which the labels claimed the curcuminoids content were studied. All products tested met the THP 2004 standard for Khamin Chan Capsules. The contents of curcuminoids, calculated as curcumin in the products tested were found from 7.9 to 10.0% w/w; they were within the THP 2004 officially allowable limit (not less than 5.0% w/w). The tested products passed the specified THP 2004 tests for weight variation and disintegration. The amount of water found in all products ranged between 6.0% and 7.0% v/w; they also complied with the THP 2004 standard (not more than 10.0% v/w).
N. Sittichai et al.
%Drug dissolved
86
100 90 80 70 60 50 40 30 20 10 0
pH 4.0 buffer 0.2% SLS in 0.01 M HCl 0.3% SLS in 0.05 M HCl 0.4% SLS in 0.05 M HCl 0.6% SLS in 0.05 M HCl 0.8% SLS in 0.05 M HCl
20
25
30
35
40 45 Time (min)
50
55
60
65
%Drug dissolved
Figure 1 Dissolution profiles of turmeric capsules containing 0.024 g curcuminoids using the basket method at 100 rpm and 900 mL of various media and at 37 ÌC ± 0.5 ÌC (n = 3)
100 90 80 70 60 50 40 30 20 10 0 0
10
20
30
40
50
60
70
80
90
Time (min) Figure 2 Dissolution profile of turmeric capsules containing 0.024 g curcuminoids using the basket method at 100 rpm and 900 mL of 0.8% SLS in 0.05 M hydrochloric acid and at 37 CÌ ± 0.5 ÌC (n = 3)
Thai J. Pharm. Sci. 31 (2007) 83-90 Dissolution results For the determination of curcumin in the dissolution aliquots, the standard curcumin curve was constructed by the procedure according to THP 2004 except adding one concentration of 0.4 µg/mL. The curve showed high degree of correlation with coefficient of determination (r 2 = 0.9998). The capsule shells did not show any marked interference with the analytical results. The dissolution profiles of turmeric capsules in different dissolution media except water using the basket method at 100 rpm are shown in Figure 1. The results indicated that the product dissolved less than 1% of curcuminoids, calculated as curcumin in 60 minutes in water. It dissolved up to about 4% in 60 minutes in pH 4.0 buffer. A significantly higher dissolution was obtained in the presence of the surfactant, sodium lauryl sulfate (SLS). The lowest amount of surfactant necessary to achieve 75-80% release of curcuminoids in 60 minutes was 0.8% SLS in 0.05 M hydrochloric acid. From the observations, it is recommended that the SLScontaining media be used within 12 hours. The percentage dissolved of curcumin was found to be 20 percent less if the deaerated SLS-containing media used was standed overnight. In this experiment, the rate of agitation was selected as 100 rpm as it provided more dissolution than 75 rpm i.e. 71% release and 57% release in 45 minutes, 78% release and 63% release in 60 minutes, respectively. Figure 2 shows a dissolution-time profile in the chosen 0.8% SLS in 0.05 M hydrochloric acid and it appears that a 60minute collection time point was reasonable for complete and steady dissolution. Detailed observations during the dissolution tests including approximate capsule disintegration time, shape formed by the disintegrated particles (cone-shaped piles or circular-shaped piles) are shown and illustrated in Table 1 and Figure 3. The cone-shape pile was observed after the disintegrated
87 ingredient particles passed through the mesh of the basket. These particles remained unstirred thus affecting the dissolution results. Establishment of acceptable çQé value A comprehensive dissolution profile was obtained for each lot of turmeric capsules using 0.8% SLS in 0.05 M hydrochloric acid and basket at 100 rpm (Table 2). The sampling time points were 30, 45 and 60 minutes. The data in Table 2 shows that the distribution of all values was normal following Kolmogorov-Smirnov One-sample Test (p > 0.05). The United States Pharmacopeia (USP) acceptance criteria for dissolution of botanical dosage forms was followed [9] since these criteria has not yet been included in THP. Table 3 demonstrates that the first stage of dissolution test consists of testing 6 dosage units. If the average amount dissolved (n = 6 capsules) is greater than or equal to Q + 10%, then the dissolution test criteria are met and the test is passed. To establish a Q value that both the regulatory body and the manufacturer deem acceptable, the statistics [10, 11] indicating predicted frequency of Stage 2 testing is shown in Table 4. The data depicted in Table 4 are on the assumption that a Q value of 75 or 80 is the proposed dissolution specification at one or two candidate time points [10, 12, 13]. It is to be noted that the calculations for the percent of average amount dissolved less than Q + 10% have been changed to the percent of average amount dissolved less than Q + 9.5%. This was done to account for the rounding aspect of the dissolution test when comparing the average amount dissolved to the acceptance criteria [9, 10]. Reviewing Table 4, the manufacturer will likely propose Q = 75% at 60 minutes; a specification of Q = 75% at 45 minutes could also be proposed and lead to a low frequency of Stage 2 testing, while a specification of Q = 80% at 30 minutes would lead to a higher frequency of Stage 2 testing.
88
N. Sittichai et al.
Figure 3 Particles from content of turmeric capsules centered under the basket in cone-shaped piles during dissolution study.
Table 1 Observations made during dissolution of turmeric capsules (Basket: 100 rpm; Temp : 37 ÌC ± 0.5 ÌC) Medium pH 4.0 buffer 0.2% SLS in 0.01 M HCl 0.3% SLS in 0.05 M HCl 0.4% SLS in 0.05 M HCl 0.6% SLS in 0.05 M HCl 0.8% SLS in 0.05 M HCl
Observations* Disintegrated within 2 min. into swirling cone-shaped piles of fine particles Disintegrated within 2-3 min. into swirling cone-shaped piles of fine particles centered under the basket on the bottom of the vessel. same as above same as above same as above same as above
* In some batches of product, parts of the disintegrated capsule shell stick to the wire mesh of the basket.
Thai J. Pharm. Sci. 31 (2007) 83-90
89
According to the methodology to set potential dissolution specifications, the allowable percent Stage 2 testing would be proposed [10]. Assuming that 20% Stage 2 testing be desirable. This requires that no more than 4% of the average amount dissolved is less than Q + 10% (or Q + 9.5%, taking into account the normal rounding procedures). This Stage 2 testing frequency would be achieved if the true mean were 1.75 σ above Q + 10% (or Q + 9.5%, taking into account the normal rounding procedures). Then, the Q-value to achieve this Stage 2 testing could be obtained by subtracting the quantity ç10 + 1.75 σé (or the quantity ç9.5 + 1.75 σé) from the overall average. Table 5 summarizes the Q value to yield 20% Stage 2 testing when the above methodology was applied. It demonstrated clearly that the specification that could
perhaps be agreed upon is Q = 75% at 60 minutes since it leads to a moderate frequency of Stage 2 testing (0.68%) as seen in Table 4. The prediction coincides with the dissolution-time profile in Figure 2 that a 60-minute point is the time of choice as the dissolution curve becomes flat.
Conclusion From this experiment, the proposed dissolution test uses the basket device at 100 rpm and requires a release (Q) of 75% in 60 minutes. The proposed dissolution medium is composed of 0.05 M hydrochloric acid with sodium lauryl sulfate at 0.8% w/v. Further investigations employing an alternative device should be carried out to offer a universal specification for dissolution of turmeric capsules.
Table 2 Dissolution profile of five lots of turmeric capsules (Dissolution medium: 0.8% sodium lauryl sulfate in 0.05 M hydrochloric acid; Basket: 100 rpm; Temp : 37 CÌ ± 0.5 ÌC) Turmeric capsule 30 min Lot A Lot A Lot B Lot B Lot C Lot C Lot D Lot D Lot E Lot E
Percentage drug dissolved at time intervals 45 min
76.21 78.74 63.96 76.60 73.68 71.93 69.21 69.80 70.38 62.80 71.33 ± 5.24
Mean ± SD
89.09 84.31 79.15 85.64 75.99 74.82 77.10 76.91 77.30 75.29 79.56 ± 4.97
60 min 95.18 92.68 89.61 91.53 85.69 82.80 90.59 88.32 88.90 85.18 89.05 ± 3.73
Table 3 USP dissolution acceptance criteria for a pooled sample Stage
Number tested
Acceptance criteria
S1 S2 S3
6 6 12
Average amount dissolved is not less than Q + 10% Average amount dissolved (S1 + S2) is equal to or greater than Q + 5% Average amount dissolved (S1 + S2+ S3) is equal to or greater than Q
90
N. Sittichai et al.
Table 4 Summary statistics for potential Q-values Collection time point (minutes)
Number of results
Average (standard deviation)
Potential Q-value
% of Average amount dissolved predicted to be less than Q + 9.5%
Predicted frequency of Stage 2 testing
45 45 60 60
10 10 10 10
79.56 (4.97) 79.56 (4.97) 89.05 (3.73) 89.05 (3.73)
75 80 75 80
0.8389% 0.9772% 0.1131% 0.5478%
4.93% 5.72% 0.68% 3.24%
Table 5 Q-value to yield 20% Stage 2 testing for a pooled sample Collection time point (minutes)
Number of results
Average (standard deviation)
Q-value to achieve Stage 2 testing frequency of 20%
45 60
10 10
79.56 (4.97) 89.05 (3.73)
61.36 73.02
References [1] WHO Monographs on Selected Medicinal Plants, Vol. 1. World Health Organization, Geneva, 1999, pp. 115-124. [2] Standard of ASEAN Herbal Medicine, Vol. I. ASEAN Countries, Jakarta, 1993, pp. 193-206. [3] S. Durgaprasad, C.G. Pai, and J.F. Alvres. A pilot study of the antioxidant effect of curcumin in tropical pancreatitis, Indian J. Med. Res. 122: 273-364 (2005). [4] V. Thamlikitkul, N. Bunyapraphatsara, T. Dechatiwongse, S. Thurapong, C. Chantrakul, T. Thanaverasuwan, S. Nimitnon P. Boonroj, V. Punkrut, V. Gingsungneon, S. Wongkonkatape, W. Ekpalakorn, N. Boontaeng, U. Jesadaporn, S. Taechaiya, S. Petcheroen, A. Riewpaiboon, R. Adthasit, S. Timsard, and E.D. Tenambergen. Randomized double blind study of Curcuma domestica Val. for Dyspepsia, J. Med. Assoc. Thai. 72: 613-620 (1989) [5] Thai Herbal Pharmacopoeia, Vol. I. Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 1995, pp. 24-31. [6] Supplement to Thai Herbal Pharmacopoeia, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 2004, pp. 31-32.
[7] P. Kucera, P.L. Schiff, Jr,. S. W. Page, D.B. Katague, E. J. Staba, E. G. Lovering, E. M. Groom, L. T. Grady, and V. Srini Srinivasan. A practical approach to developing public standards for botanical dosage forms: dissolution protocol for solid oral dosage forms of botanical preparations, Pharmacopeial Forum. 25: 8628-8631 (1999). [8] SPSS 7.5 for Windows, 1998. [9] The United States Pharmacopeia-The National Formulary, 29th ed. Rockville: United Stated Pharmacopeial Convention, Inc. 2006. pp. 3091-3092. [10] J. D. Hofer, and V. A. Gray. Examination of selection of immediate-release dissolution acceptance criteria, Pharmacopeial Forum 29: 335-340 (2003). [11] M. Aliaga, and B. Gunderson. Interactive Statistics, Pearson Education, Inc., USA, 2003, pp. 330-332, 942-943. [12] Anonymous. FIP guidelines for dissolution testing of solid oral products final draft, 1995, Pharmacopeial Forum 21: 1371-1382 (1995). [13] FDA guidance for industry: dissolution testing of immediate release solid oral dosage forms. Cited 4 April 2007. Available from: http://www.fda.gov/cder/guidance/1713 bp1.pdf.
