Amelioration and Affirmation for the Assessment of Curcumin in API and Ayurvedic Herbal Formulation Haridra Capsule by UFLC Discrete Method

 

Y. Thejeswari¹*, S. Rubesh Kumar²

Department of Pharmaceutical Analysis, JNTUA - Oil Technological Research Institute, Anantapuram -515001, Andhra Pradesh, India.

*Corresponding Author E-mail: vyshu.teja@gmail.com

 

Curcumin is an ancient medicine to present clinical trials. Curcuminoids natural yellow pigments, the principle constituents of Curcuma longa of Zingiberaceae family comprises of curcumin, demethoxycurcumin and bisdemethoxycurcumin. In the present study, a fast, simple and reliable method for the determination of curcumin present in the API and Ayurvedic formulation haridra capsule by using a single mobile phase for a cost effective method. The effluent was monitored at 419nm. The method was validated according to ICH  guidelines various parameters like Specificity, Linearity, Precision, Accuracy, Limit of detection, Limit of quantification, Robustness and System suitability. The method was selected for the determination of curcumin. The system was linear with a correlation coefficient (r2) of 0.9998.  RSDs% of intraday and interday precisions were determined to be 0.40 and 0.67 for curcumin. Limit of detection and limit of quantification was found to be  as 2.97 and 9 for curcumin. The percentage recovery studies was found to be 99.47 to 102.2%w/v. Stress degradation studies proved that compound showed degradation in conditions like hydrolytic under acidic, alkaline, oxidative, photolytic and dry heat degradation studies for the API as per the ICH guidelines. The simplicity of the method makes it highly valuable for quantification of curcumin in API and Ayurvedic formulations.

 

 

 

INTRODUCTION:

Turmeric (Curcuma longa Linn.) is a perennial of Zingiberaceae family.  Other names for turmeric include Indian saffron, turmeric root and yellow root, Haldi and curcuma longa [1]. Curcumin, a polyphenol compound, is responsible for the yellow colour of turmeric and is the most active pharmacological agent. The three principal components of curcumin that are present in various proportions are all dicinnamoylmethane derivatives [2] in Fig.1.

 

Fig 1: Chemical structure of curcuminoids of C. longa

 

1) R₁ = R₂ = OCH3: curcumin

2) R₁ = OCH₃, R₂ = H: demethoxycurcumin

3) R₁ = R₂ = H: bisdemethoxycurcumin

Accepted on 05.08.2013         © RJPT All right reserved

 

Curcumin Molecular Formula C₂₁H₂₀O₆, Formula weight: 308.

Curcumin is chemically described as diferuloylmethane or 1, 7-bi's-(4-hydroxy-3-methoxy-phenyl) -hepta-1, 6-diene-3, 5-Dione. Curcumin (diferuloylmethane) is a low molecular weight poly phenol [3].  Curcumin is insoluble in water and ether, but soluble in ethanol, dimethylsulfoxide, and other organic solvents [4].  Natural curcumin, isolated from curcuma longa, contains Curcumin I (diferuloylmethane as the major constituent, 77%), as well as Curcumin II (bisdemethoxycurcumin, 6%) and Curcumin III (dimethoxycurcumin, 0.3%) [5]. Curcumin and its related compounds called Curcuminoids having anti-inflammatory, antiviral, antibacterial [6], anti carcinogenic  [7-9], antifungal properties [10], antioxidant [11], with potential activity against cancer, diabetes, arthritis, Alzheimer's disease [12] and other chronic maladies".  It is also known for its potential use in the treatment of human immunodeficiency virus (HIV) [13]^.

 

Curcumin is official in British, European and United States of  pharmacopoeias [14, 15].  For the estimation of Haridra many analytical methods such as UV [16], HPLC [17], Thin layer chromatography (TLC) [18], Gas chromatography (GC) [19], High performance thin layer chromatography (HPTLC) [20] Gas chromatography-Mass spectroscopy (GCMS) [21], Capillary electrophoresis [22], Fourier transform infrared spectroscopy (FT/IR) [23] and Scanning electron microscopy (SEM) [23] methods were reported. In the present investigation a UFLC  method was developed for curcumin in both API and Ayurvedic formulations by using single solvent methanol as a mobile phase and the method was validated as per International Conference on Harmonization (ICH) guidelines and also performed stability indicating studies for Active pharmaceutical ingredient (API) and herbal Ayurvedic formulations.

 

The objective of the present study was  developed and validated as a simple, accurate and  rapid new method for the estimation of Curcumin  in capsule dosage form by Ultra Fast Liquid Chromatography.

 

MATERIALS AND METHODS:

Drug Material

The drug Curcumin was obtained from HIMEDIA (RM 1449), Mumbai and HPLC grade methanol from Merck. All chemicals were of analytical grade such as Hydrogen peroxide (SD Fine Chem Limited), Sodium Hydroxide (Mio Chem Pvt. Ltd), and Hydrochloric acid (Merck Chemicals). Haridra capsule (Himalaya group of company) containing 400 mg of Curcuma longa from the local drug store.

 

Instrumentation

The Shimadzu LC 20 AD UFLC system with 20 µL loops, LC 20 AD pump, SP-20AD, photo- diode array detector and Waters Symmetry C8, 350µm, 4.6 x 150mm column was used. LC solutions software was utilized for instrument control, data collection and data processing using a Pentium (R) Dual Core Processor.

 

Chromatographic Conditions

The analysis was carried out under isocratic conditions using methanol as mobile phase at a flow rate of 1.0 ml/min. Chromatograms were recorded in 419nm.

 

Preparation of standard solution

Accurately weighed 10 mg quantity of API was transferred into the 10ml volumetric flask and dissolved  in 10 ml of methanol to give a concentration of 1000µg/ml. To 1ml of standard solution, 5ml of methanol was added, sonicated for 10 to 15min.  The solution was filtered through a membrane filter and the volume made up to 10ml with methanol.  The stock solution was further diluted to a final concentration of 10µg/ml by using methanol. This solution was used for further studies.

 

Method development

Working standard of various concentrations was prepared by taking aliquots of standard solution and diluted to the required concentration for calibration plot and which was injected into the chromatographic system.

 

 

Assay preparation for Ayurvedic formulation

An equivalent weight of 10mg of Haridra capsule was accurately weighed and transferred in 10ml volumetric flask, suspended in 5ml of methanol and ultrasonicated for 10 to 15min. The solution was then filtered through a membrane filter, made up the volume with methanol and mixed well. 

 

Validation

The reliability of the Ultra Fast Liquid Chromatography method for the analysis of curcumin was validated for various parameters like specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness and system suitability as per guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH Guidelines) Q2 R1 [24,25,26].

 

Specificity

For the chromatographic method, developing a separation involves demonstrating specificity, which is the ability of the method to accurately measure the analyte response in the presence of all interferences. Therefore, the sample preparation was analyzed and the analyte peak was evaluated for peak purity and resolution.

 

Linearity

Due to the verification of the normal distribution of results, linearity was evaluated through the relationship between the concentration and the area obtained from the UFLC Photo diode array detector (PDA). The determination of correlation coefficient (r²) was calculated by means of the least-square analysis. The calibration line were achieved by each concentration of curcumin (10 to 90 µg/ml), to identify the extent of the total variability of the response that could be explained by the linear regression model.

 

Precision

The precision of each method indicates the degree of dispersion within a series on the determination of the same sample. Six real samples were analyzed on the same day (Intraday) and three on consecutive days (interday), and then the relative standard deviations (RSDs%) were calculated. Each sample was injected to Ultra fast liquid chromatography thrice times.

 

Accuracy

This parameter shows the proximity between the experimental values and the real ones. It ensures that no loss or uptake occurred during the process. The determination of this parameter was performed during the method by studying the recovery using a standard addition procedures with two additional levels. Three replicate amounts of sample were weighed and each of them was divided into three equal portions.  Adding known quantities of standard drug to the excipients. In each additional level, three determinations were carried out and the recovery percentage was calculated in every case. Each sample was injected into Ultra Fast Liquid Chromatography three times.

 

Limit of Detection

The limit of detection is the smallest concentration of the analyte that gives a measurable response that can be accurately detected (signal to noise ratio of 3).

 

LOD = (3.3 x standard deviation)/ Slope of calibration curve

 

Limit of Quantification

The limit of quantification is the smallest concentration of the analyte, which gives a response that can be accurately quantified (signal to noise ratio of 10). 

 

LOQ = (10 x standard deviation) / Slope of calibration curve.

 

Robustness

The robustness of an analytical procedure is defined as a measure of its capacity to obtain comparable and acceptable results when perturbed by small but deliberate variations in specified experimental conditions. Robustness provides an indication of the test method’s suitability and reliability during normal use.

 

System suitability

System suitability tests are an integral part of  UFLC methods  and are used to verify that the accuracy and precision of the system are adequate for the analysis to be performed. Parameters such as plate count, tailing factor,  resolution and repeatability (RSD of retention time and area for six repetitions)  are determined and compared against the specifications set for the method. In the present study, the system suitability test was performed on an UFLC system to determine the accuracy and precision of the system, by injecting six injections of a solution containing 20 μl of curcumin. RSD for peak area and retention time < 1%, tailing factor (T) < 2 and theoretical plate (N) were > 5000 for both UFLC systems.

 

Stress Degradation Studies

The stress degradation studies such as hydrolytic (in acidic and alkali medium), oxidative, photolytic and dry heat induced degradation studies were performed for API as per International Conference on Harmonization guidelines Q1A (R2) [27]_.

 

1. Hydrolytic degradation under acidic conditions

Hydrolytic degradation study was performed by taking 0.05g of the drug was dissolved in 50ml of 0.1N methanolic hydrochloric acid (1mg/ml) and 25ml of  it was refluxed in round bottomed flask on a boiling water bath for 8hrs.  The remaining solution was kept at room temperature.

 

2. Hydrolytic degradation under alkaline condition

Hydrolytic degradation study was performed by taking 0.05g of the drug dissolved in 50ml of 0.1N methanolic sodium hydroxide (1mg/ml) and 25ml of  it was refluxed in round bottomed flask on a boiling water bath for  8hrs.  The remaining solution was kept at room temperature.

 

3. Oxidative degradation

Hydrolytic degradation study was performed by taking 0.05g of the drug was dissolved in 50ml of 3%hydrogen peroxide (1mg/ml) and 25ml of it was refluxed in round bottomed flask on a boiling water bath for 8hrs.  The remaining solution was kept at room temperature.

 

UFLC Sample Preparation

From the  above solutions in each degradation study 1ml of each was withdrawn  at a  time interval of 1,3,5 and 8hrs. This solution was injected into the system and the degradation of the drug was analyzed.

 

4. Dry heat induced degradation

Dry heat induced degradation study was performed by taking 0.01g of drug in different weighing bottles were kept at 70⁰C and 25⁰C  for different time intervals i.e., 7,14,30 days.

 

5. Photolytic degradation 

A photolytic degradation study was performed by exposing 0.01g of the drug was evenly spread on a petri dish and kept under sunlight and blank in dark condition for different time intervals i.e., 7,14,30 days.

 

Figure 2a, 2b UFLC Chromatogram of Curcumin API and Ayurvedic Haridra capsule

 

RESULTS AND DISCUSSIONS:

In the present investigation, the study was carried out on Haridra capsule for the estimation of curcumin.  An assay method was developed for the API and the marketed Ayurvedic formulation haridra showed 98.98% w/v percentage purity. Specificity showed that there is no interference or overlapping of the peaks either due to excepients or diluents with the main peak of curcumin. The assay was linear over the concentration range 10 µg/ml - 90 µg/ml for Curcumin. Accuracy determined through recovery studies by adding known quantities of standard drug to the sample solution of haridra capsule  was found to be within 98.82% – 101.17%. The interday and intraday precision and the robustness %RSD was found within the limits. Limit of detection and limit of quantification was 2.97 and 9 respectively. All the stress degradation studies proved  that the degradation of the drug in various conditions like acidic, alkali and oxidative induced hydrolytic degradation of the drug which increased gradually from 1and 3 hr and  photolytic,  dry heat degradation of the drug were tested at 1 and 5 days showed a progressive degradation of the drug.

 

Method development and validation

All compounds with similar structures especially in the case of curcumin were shown in Figure1; it was difficult to separate all components simultaneously. After comparison between the different columns such as C8, C18 long, C18 short, the best separation efficiency was obtained by using the C8 column. The mobile phase investigations showed that the methanol was the key to a good separation. According to this, the best separation was achieved by using methanol. The isocratic mode of the instrument was used to obtain the best resolution and the shortest run time. Curcumin peak was resolved from the neighbouring peak and displayed excellent peak symmetry and separation efficiency as seen in Figure 2a,2b. The results obtained from the method validation for various parameters like linearity, specificity, accuracy, precision, limit of detection, limit of quantification, robustness and system suitability showed that the proposed method was suitable for the analysis of curcumin.

 

Table1: Calibration values of Curcumin in standard drug

Concentration (µg/ml)	Peak area (mV.s)
10	12797
20	22959
30	32429
40	42124
50	51477
60	60827
70	71006
80	80896
90	89586

 

Fig 3: Linearity graph of Curcumin

 

The LC Solutions software showed that the method was specific for curcumin as the reported peaks were completely separated from the other interfering compounds. So this method because of reaching suitable recovery and good precision can be recommended for the quantification of curcumin in Ayurvedic formulation.

 

Fig 4: Linearity overlay of Curcumin

Table 2: Intra-and inter-day Precision

 

Table 3: Recovery of curcuminoids analysis in Curcumin

S. No.	Concentration (µg/ml)	Amount of standard added(gm)	Area	Recovery(gm)	Amount recovery (gm)	%Recovery	Mean	SD	RSD
1	0.8	0.01 0.01 0.01	4208 4230 4258	0.7612 0.7652 0.7703	0.776 0.776 0.776	98.10% 98.60% 99.26%	98.65%	0.0058	0.000589
2	1	0.01 0.01 0.01	6612 6623 6619	0.97 0.95856 0.95798	0.97 0.97 0.97	98.65% 98.82% 98.76%	98.74%	0.0008	0.00087
3	1.2	0.01 0.01 0.01	9742 9756 9792	1.17499 1.17612 1.181	1.164 1.164 1.164	100.94% 101.08% 101.45%	101.16%	0.0026	0.00266

 

Linearity Study

The linear relationship between the detector response and different concentrations of curcumin were confirmed as it was shown in figure 3, Table 1 and overlay of the linearity curve in figure4.

 

Precision Study

The relative standard deviations (RSDs %) of the Intra-day and inter-day precision was shown in Table 2. After these validation studies, the method’s ability to provide good quantization in our laboratory was confirmed. Measurement of precision (reproducibility), which focused more on the bias in the results, rather than on determining the differences in precision alone, as inter laboratory crossover studies and repeatability (RSD of retention time and area for six repetitions) are determined and compared against the specifications set for the method.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ) for Curcumin was found to be 12.00 μg/ml, 36.38μg/ml. The typical chromatogram of Curcumin was shown in Figure 2. A mobile phase of methanol was found to be most suitable to obtain a peak well defined and free from tailing.

 

Accuracy Study

Accuracy, which was evaluated as recovery, after spiked the samples with standards at three concentration levels has been shown in Table 3. 

 

Robustness

The robustness of an analytical procedure were defined as a measure of its capacity to obtain comparable and acceptable results when perturbed by small but deliberate variations in specified experimental conditions in Table 4.

 

Table 4: Robustness of curcuminoids analysis in Curcumin

Table 5: System suitability of curcuminoids analysis in Curcumin

 

System suitability

System suitability tests are an integral part of UFLC method parameters, such as plate count, tailing factor, area, retention time and resolution. The parameters measured and their recommended limits obtained from the analysis of the system suitability sample are shown in Table5. 

 

Fig 5: Acid degradation of standard at 1hr and 3hr

 

 

Fig 6: Alkali degradation of standard at 1hr and 3hr

 

 

Fig 7: Photolytic Degradation of Standard and Blank

 

 

Fig 8: Oxidative  Degradation of Standard at 1hr and 3hr

 

 

Stress Degradation Studies

All the stress degradation studies proved that the degradation of the drug in various conditions like acidic, alkali and oxidative induced hydrolytic degradation of the drug which increased gradually from 1and 3 hr and  photolytic,  dry heat degradation of the drug were tested at 1 and 5 days showed a progressive degradation of the drug were reported in fig.5-9.

 

 

 

CONCLUSION:

A simple and novel UFLC method for separating and determining  curcumin in API and Ayurvedic formulation was developed to reduce the time required for analysis. It was a simple, fast, accurate and reliable technique in chromatographic conditions with minimum use of solvents. The coefficient of variance was satisfactory low and recovery was close to 100% indicating reproducibility of the method.  The linearity was observed within the limit hence method  can be used for the routine market analysis of haridra capsule for the determination curcumin.

 

Fig 9: Dry heat Degradation of Standard at 1^st day and 5^th day

 

 

ACKNOWLEDGEMENTS:

Author is thankful to the Director, Oil Technological Research Institute, Jawaharlal Nehru Technological University Anantapuram,  for providing necessary facility for the work.

 

REFERENCES:

1.     B. B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa. Curcumin: the Indian solid gold. Adv Exp Med Biol 2007;595: 1-75.

2.     Ohshiro, M., Kuroyanagi M, et al. Structures of sesquiterpenes from Curcuma longa. Phytochem 1990; 29 (7): 2201-2206.

3.     Guddadarangavvanahally K, et al. Evaluation of Antioxidant activities and antimutagenicity of turmeric oil: A byproduct of curcumin production. Z Naturforsch 2002; 57c: 828-835.

4.     Phan TT, et al. Protective effects of curcumin against oxidative damage on skin cells in vitro: Its implication for wound healing. J Trauma 2001; 51: 927-931.

5.     Kolev, Tsonko M, Velcheva, Evelina A, Stamboliyska, Bistra A, Spiteller, Michael. "DFT and experimental studies of the structure and vibrational spectra of curcumin". International Journal of Quantum Chemistry 2005; 102 (6): 1069–79.

6.     R. Singh, R. Chandra, M. Bose, P.M. Luthra. Antibacterial activity of Curcuma longa rhizome extract on pathogenic bacteria. Current Science 2002; 83 (6): 737-740.

7.     L. A. F. Ferreira, O. B. Henriques, A. A. S. Andreoni, G. R. F. Vital, M. M. C. Campos, G. G. Habermehl and V. L. G. DeMoraes. Antivenom and bio- logical effects of ar-turmerone isolated from Curcuma longa (Zingiberaceae), Toxicon 1992; 30: 1211-1218.

8.     Kunnumakkara AB, Anand P, Aggarwal BB. Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signalling proteins. Cancer Lett 2008; 269(2): 199-225.

9.     Piper JT, Singhal SS, Salameh MS, Torman RT, Awasthi YC, Awasthi S. Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver. Int J Biochem Cell Biol 1998; 30:  445-456.

10.   Aggarwal BB, et al. Anticancer potential of curcumin: Preclinical and clinical studies. Anticancer Res 2003; 23: 363- 398.

11.   M. T. Barrat, H. J. D. Dorman, S. G. Deans, C. Figueiredo, J. G. Barroso, G. Roberto. Antimicrobial and antioxidant properties of some commercial essential oils. Flavour and Fragrance Journal., 1998; 13: 235-244.

12.   Park SY, Kim D. Discovery of natural products from Curcuma longa that protect cells from beta-amyloid insult: a drug discovery effort against Alzheimer’s disease. J Nat Prod 2002; 65: 1227-1231.

13.   Ammon HP, Wahl MA: Pharmacology of Curcuma longa. Planta Med 1991; 57: 1–7.

14.   British Pharmacopoeia, Medicines and Health Care Products Regulatory Agency, 2 nd ed. (MHRA), London; 2008.

15.   United States Pharmacopoeia and National Formulary, Asian Edition, the United States Pharmacopoeia Convention Inc, U.S.A; 2009.

16.   Almedia LP, Cherubino, APF, Alves RJ, Dufosse L, Gloria MBA. Separation and determination of the physico-chemical characteristics of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Res Int, 2005; 38: 1039-1044.

17.   Das U, Kawase M, Sakagami H, Ideo A, Shimada J, Molnar J, Barath Z, Bata Z, Dimmock JR. Bioorg Med Chem, 2007;15:3373. 

18.   Soares, P.K., and Scarminio, I.S. Phytochem. Anal, 2008;19(1): 78–85.

19.   Miquel J., Bernd A., Sempere J. M. and Diaz-Alperi R. A. The curcuma antioxidants: pharmacological effects and prospects future clinical use. A review. Arch. Gerontol. Geriatr, 2002; 34: 37-46.

20.   Paramasivam M, Aktar MW, Poi R, Banerjee H, Bandyopadhyay A: Occurrence of curcuminoids in curcuma longa: a quality  standardization by HPTLC. Bangladesh J Pharmacol, 2008; 3: 55-58.

21.   Kohli K, Ali J, Ansari MJ, Raheman Z. Curcumin: a natural anti-inflammatory agent. Ind J Pharmacol, 2005; 37:141-147.

22.   Chattopadhyay, K. Biswas, U., Bandyopadhyay and Banerjee, R.K. Turmeric and curcumin: biological actions and medicinal applications. Current Science, 2004; 87: 44–50.

23.   Parize, A. L.; Heller, M.; Fávere, V. T.; Laranjeira, M. C. M.; Brighente, I. M. C.; Micke, G. A.; Souza, T. C. R.; Latin Am. J. Pharm, 2009; 28: 19.

24.   L.I., Ho, Y.S., Hsieh, C.Y., and Lin, J.K. Stability of curcumin in buffer solutions and characterization of its degradation products. J. Pharm. Biomed. Anal 1997; 15(12): 1867-1876.

25.   ICH Guideline Q2 (R1), Validation of analytical procedures: text and methodology, November 2005.

26.   Validation of Analytical Procedures: Methodology (Q2B), ICH Harmonized, Tripartite Guidelines, Geneva; 1996.

27.   ICH Guideline Q1A (R2),   “Stability Testing of New Dosage Forms”,   February 2003.

 

 

 

Accepted on 20.07.2013                                © RJPT All right reserved