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Application of validated RP–HPLC–PDA method for the simultaneous estimation of curcumin and piperine in Eudragit E 100 nanoparticles
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 2 4 e2 2 9
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Application of validated RPeHPLCePDA method for the simultaneous estimation of curcumin and piperine in Eudragit E 100 nanoparticles C. Moorthi a,*, C. Senthil Kumar b, S. Mohan b, Kiran Krishnan c, K. Kathiresan a a
Department of Pharmacy, Annamalai University, Annamalai Nagar 608 002, Chidambaram, Tamil Nadu, India Saraswati Institute of Pharmaceutical Sciences, Dhanap Post, Gandhi Nagar 382355, Gujarat, India c Apotex Advancing Generics, Apotex Corporation, Suite 400, 2400 North Commerce Parkway, Weston, FL 33326, United States b
article info
abstract
Article history:
Background: The primary aim of the study was to develop an HPLC method for the simul-
Received 10 December 2012
taneous estimation of curcumin and piperine with adequate separation and to validate the
Accepted 8 March 2013
developed method as per ICH guideline and implement the developed method for the
Available online 4 April 2013
simultaneous estimation of curcumin and piperine which are encapsulated in the Eudragit E 100 nanoparticles.
Keywords:
Method: Chromatographic conditions were optimized and the best chromatographic con-
Analytical method development
ditions was achieved when the chromatographic separation was carried out on a Luna C18
Curcumin
column (Reversed phase, 150 mm 4.6 mm with 5 m particle size, Phenomenax) using a
Eudragit E 100
mobile phase combination of 0.1% ortho phosphoric acid aqueous solution and acetonitrile
Nanosuspension
(45:55, v/v) in an isocratic mode elution with a flow rate of 1.2 mL min1 at the column oven
Piperine
temperature of 35 C. The detection was monitored at a wavelength of 262 nm. Results: The retention time of curcumin and piperine was 8.6 and 5.9 min, respectively. The developed method was validated for system suitability, accuracy, precision, limit of detection, limit of quantitation, linearity, range, robustness and all the validated parameters were within the acceptable criteria. The method was linear over the range (10e150 mg mL1) for both drugs with correlation coefficients of greater than 0.999. Limit of detection were 0.3 ppm for curcumin and 0.1 ppm for piperine and limit of quantification were 0.4 ppm for curcumin and 0.9 ppm for piperine. Conclusions: The developed method was successfully implemented in the estimation of curcumin and piperine encapsulated in Eudragit E 100 nanoparticles and this method is also suitable for use in routine analysis of curcumin and piperine in active pharmaceutical ingredients and in pharmaceutical dosage forms. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
* Corresponding author. E-mail address: [email protected] (C. Moorthi). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.03.006
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 2 4 e2 2 9
1.
Introduction
Curcumin is an orangeeyellow crystalline phytochemical isolated from Curcuma longa and classified as a functional food, as it possess wide spectrum of pharmacological activities including anti-cancer activity due to its diverse molecular targets. Curcumin is extremely safe and can be well tolerated at high doses and has also been declared as “generally regarded as safe” by US FDA. In spite of its efficacy and safety, the clinical usefulness of curcumin in the treatment of cancer is limited due to certain limitations including lack of aqueous solubility, rapid clearance from the systemic circulation, intestinal metabolism, hepatic metabolism, lack of cancer cell targeting and multidrug resistance. Hence, to overcome these limitations, we have proposed a dual drug loaded Eudragit E 100 nanosuspension containing curcumin and piperine.1e4 However, the total amount of curcumin and piperine encapsulated in the Eudragit E 100 polymer matrix determines the efficacy of the nanosuspension. Analytical techniques for the simultaneous estimation of curcumin and piperine have been reported.5 In the reported high performance liquid chromatography (HPLC) method, separation between curcumin and piperine was 9 and 9.5, respectively.5 However, this narrow separation (0.5 min) may not be sufficient enough to estimate curcumin and piperine which are encapsulated in polymer matrix as the polymer and other excipients in the formulation may interfere in the chromatographic separation of curcumin and piperine. Hence, an analytical technique with adequate separation between curcumin and piperine is essential. The primary aim of the study was to develop an HPLC method for the simultaneous estimation of curcumin and piperine with adequate separation and to validate the developed method as per ICH guideline and implement the developed method for the simultaneous estimation of curcumin and piperine which are encapsulated in the Eudragit E 100 nanoparticles.
225
250 mg of beta-cyclodextrin in 20 ml of distilled water was prepared and emulsified with organic phase under sonication (Lark, India) for 10 min to form nanoparticles. However, the sonication process was continued for another 50 min to evaporate any residual solvent present in the nanosuspension. Average particle size, polydispersity index and zeta potential were measured using Zetasizer (Malvern, UK).
2.3. Fabrication of Eudragit E 100 nanosuspension using mechanical stirring About 5 mg of curcumin, 5 mg of piperine and 250 mg of Eudragit E 100 were dissolved in 20 ml organic solvent (mixture of 12 ml of ethanol and 8 ml of distilled water). An aqueous phase containing 125 mg of poloxamer 188 and 50 mg of beta-cyclodextrin in 25 ml of distilled water was prepared and emulsified with organic phase under mechanical stirring (Remi, India) at 500 rpm for 10 min to form nanoparticles. However, the stirring process was continued for another 3 h to evaporate any residual solvent present in the formulation. Average particle size, polydispersity index and zeta potential were measured using Zetasizer (Malvern, UK).
2.4.
Instrumentation
Analyses were performed using an Alliance HPLC (Waters Corp.) equipped with pump, degasser, photodiode array detector and autosampler. The generated analytical signals were monitored and integrated using Empower chromatography data software.
2.5.
Method development
Method development for the simultaneous estimation of curcumin and piperine was carried out with different flow rates, different columns, different elution modes, different mobile phase and buffer ratio.
2.
Material and methods
2.6.
2.1.
Reagents and solvents
The developed method was validated in compliance with ICH guideline for system suitability, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), linearity, range and robustness.6e8
Curcumin (97%) and beta-cyclodextrin were purchased from Himedia Laboratories, India. Piperine (97%) and poloxamer 188 were purchased from SigmaeAldrich, India. Eudragit E 100 was obtained from Degussa, India. HPLC grade ethanol was purchased from Brampton, Canada. HPLC grade methanol, acetonitrile and water were purchased from Merck, India. Analytical grade ortho phosphoric acid was purchased from Rankem, India.
2.6.1.
Method validation
System suitability
Six replicate injections containing curcumin and piperine were analysed using the developed method. Theoretical plate count more than 3000, tailing less than 1.5 and percentage relative standard deviation (% R.S.D) of peak area and the retention time less than 2% were set as acceptance criteria.
2.2. Fabrication of Eudragit E 100 nanosuspension using sonication
2.6.2.
About 5 mg of curcumin, 5 mg of piperine and 250 mg of Eudragit E 100 were dissolved in 10 ml organic solvent (mixture of 6 ml of ethanol and 4 ml of distilled water). An aqueous phase containing 250 mg of poloxamer 188 and
Three replicate injections containing known amount of curcumin and piperine at 50, 100 and 150% were added to the preanalysed samples and its recovery was estimated using the developed method. Percentage recovery within 100 2% and % R.S.D of assay less than 2% were set as acceptance criteria.
Accuracy
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2.6.6.
Robustness
The robustness of a developed analytical method refers to its ability to remain unaffected by small but deliberate change of the chromatographic condition which provides an indication of its reliability during normal usage. Assay was carried out using the developed method with slight change in the column oven temperature (30 C & 40 C) and pH of the mobile (2.8 & 3.2).
2.7. Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles was determined by an indirect method by measuring the free curcumin and piperine in the nanosuspension. Prepared Eudragit E 100 nanosuspension was subjected to centrifugation (Remi, India) at 19,000 rpm for about 45 min at 20 C. About 1 mL of supernatant was withdrawn and mixed with 1 ml of methanol and the solution was then filtered through a 0.22 mm membrane. Six replicate injections were analysed using the developed method to estimate the curcumin and piperine.
Fig. 1 e Typical chromatogram of curcumin (150 mg mLL1) and piperine (150 mg mLL1) using 0.1% ortho phosphoric acid and acetonitrile (45:55 v/v) in an isocratic mode elution with a flow rate of 1.2 mL minL1 and the column oven temperature of 35 C. The detection was monitored at a wavelength of 262 nm.
3. 2.6.3.
3.1. Fabrication of Eudragit E 100 nanosuspension containing curcumin and piperine
Six replicate injections containing curcumin and piperine were analysed using the developed method within a short period of time on the same day. The % R.S.D of peak area, assay and tailing less than 2% were set as acceptance criteria.
2.6.4.
Eudragit E 100 nanosuspension prepared using sonication has shown an average particle size of 140 nm with a polydispersity index of 0.254 and zeta potential of 28.8 mV. Whereas, Eudragit E 100 nanosuspension prepared using mechanical stirring has shown an average particle size of 87 nm with a polydispersity index of 0.239 and zeta potential of 22 mV.
Limit of detection and limit of quantitation
LOD and LOQ of curcumin and piperine were estimated from the signal-to-noise ratio. Signal-to-noise ratio of three for estimating LOD and 10 for estimating LOQ were set as acceptance criteria.
3.2. 2.6.5.
Results and discussions
Precision
Method development
Linearity and range
Linearity was evaluated at five concentration levels at 10%, 25%, 50%, 100% and 150% of the targeted assay concentration of curcumin and piperine. The linearity was then determined by least square regression analysis from the peak area against drug concentration plot. The analytical range was established by the highest and lowest concentrations of analyte where acceptable linearity, accuracy and precision were obtained.
Method development for the simultaneous estimation of curcumin and piperine was carried out with different columns but Luna C18 column has shown higher theoretical plate count and lesser tailing. Different ratio of mobile phase and buffer have been tried but the mixture of 0.1% v/v ortho phosphoric acid and acetonitrile at 45:55 proportions has shown adequate separation of curcumin and piperine. However, further increase or
Table 1 e System suitability of the developed method. Samples
1 2 3 4 5 6 Average S.D R.S.D (%)
Peak area
Plate counts
Tailing
Retention time
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
2,467,475 2,455,932 2,475,781 2,478,884 2,487,645 2,484,449 2,475,027.67 11,705.42 0.47
3,740,512 3,711,958 3,732,022 3,744,973 3,749,042 3,746,015 3,737,420.33 13,805.83 0.37
7973 7848 7786 8053 7751 7823 7872.33 116.60 1.48
7570 7697 7818 8102 7645 7688 7753.33 188.97 2.44
1.11 1.12 1.12 1.11 1.11 1.11 1.11 0.01 0.46
1.15 1.15 1.15 1.14 1.15 1.15 1.15 0.00 0.36
8.68 8.41 8.40 8.36 8.37 8.40 8.44 0.12 1.43
5.96 5.85 5.85 5.83 5.81 5.83 5.86 0.05 0.91
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Table 2 e Recovery studies of the developed method. Analyte
Level
Sample
Spiked (mg mL1)
Recovered (mg mL1)
Recovery (%)
R.S.D (%)
Curcumin
50%
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
75 75 75 150 150 150 225 225 225 75 75 75 150 150 150 225 225 225
75.50 76.41 75.80 148.94 148.15 149.44 225.16 225.94 226.62 74.61 74.39 74.48 150.44 148.66 150.80 222.42 223.28 229.09
100.67 101.89 101.07 99.29 98.76 99.63 100.07 100.42 100.72 99.49 99.18 99.31 100.29 99.11 100.53 98.85 99.23 101.81
0.61
100%
150%
Piperine
50%
100%
150%
0.44
0.32
0.15
0.76
1.61
Table 3 e Precision of the developed method. Samples
1 2 3 4 5 6 Average SD R.S.D (%)
Peak area
Assay
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
2,449,608 2,497,458 2,450,028 2,483,098 2,478,822 2,488,832 2,474,641 20,216.09 0.82
3,772,888 3,756,286 3,793,774 3,730,058 3,715,387 3,752,275 3,753,444 28,317.00 0.75
98.97 100.90 98.98 100.32 100.15 100.55 99.98 0.82 0.82
100.94 100.50 101.50 99.80 99.41 100.37 100.42 0.76 0.75
1.11 1.11 1.11 1.12 1.10 1.10 1.11 0.01 0.68
1.14 1.14 1.16 1.14 1.15 1.14 1.15 0.01 0.73
decrease in proportion of 0.1% v/v ortho phosphoric acid does not exhibit adequate separation between curcumin and piperine. Initially, 0.8 ml flow rate was used but increase in flow rate from 0.8 to 1.2 ml has shown adequate separation and high theoretical plates. Similarly, isocratic elution mode has shown better separation in comparison with gradient elution mode.
Table 4 e Linearity parameter and range of the developed method. Calibration samples (mg mL1)
Tailing
Peak area of curcumin
Peak area of piperine
10 195,616 286,543 25 439,284 703,279 50 936,366 1,367,606 100 1,717,340 2,646,426 150 2,553,086 4,007,604 Slope 16,938 26,525 Intercept 27,932 20,952 Linearity equation Y ¼ 16,938x þ 27,932 Y ¼ 26,525x þ 20,952 Correlation coefficient (R2) 0.999,491 0.999,915 10e150 10e150 Range (mg mL1)
After extensive preliminary experimental trials, the best chromatographic conditions to obtain adequate separation, resolution and symmetrical peak of curcumin and piperine were achieved only when the chromatographic separation was carried out on a Luna C18 column (Reversed phase, 150 mm 4.6 mm with 5 m particle size, Phenomenax) using a mobile phase combination of 0.1% ortho phosphoric acid aqueous solution and acetonitrile (45:55, v/v) in an isocratic mode elution with a flow rate of 1.2 mL min1 at the column oven temperature of 35 C. The detection was monitored at a wavelength of 262 nm. Fig. 1 shows a typical chromatogram of curcumin and piperine indicating complete resolution of curcumin at 8.685 min and piperine at 5.969 min.
3.3.
Method validation
3.3.1.
System suitability
Six replicate injections containing curcumin (150 mg mL1) and piperine (150 mg mL1) and the results are summarized in Table 1. The developed method satisfies the acceptance criteria of the system suitability parameters and ensures the validity of the developed method.
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Table 5 e Robustness of the developed method. Robustness parameters
Level
Assay (%) Curcumin
Piperine
Curcumin
Piperine
100.34 99.01 100.88 99.02
99.71 98.80 99.75 97.17
0.46 0.48 0.84 0.47
0.26 1.81 0.23 2.00
Column oven temperature
30 C 40 C 2.8 3.2
pH of the mobile phase
R.S.D (%)
Table 6 e Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles. Sample
Amount found (mg) in nanoparticles Sonication method
Encapsulation efficiency (%)
Stirring method
Sonication method
Stirring method
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
4.7734 4.7771 4.7778 4.7705 4.7733 4.7733 4.7742 0.0027 0.0573
4.5992 4.5887 4.5885 4.5966 4.5925 4.5956 4.5935 0.0044 0.0954
4.7417 4.7611 4.7527 4.7557 4.7435 4.7398 4.7491 0.0086 0.1818
4.6846 4.7052 4.6929 4.7001 4.6880 4.6809 4.6919 0.0093 0.1985
95.4687 95.5428 95.5552 95.4098 95.4650 95.4650 95.4844 0.0548 0.0574
91.9849 91.7737 91.7706 91.9319 91.8498 91.9127 91.8706 0.0876 0.0954
94.8349 95.2217 95.0544 95.1134 94.8702 94.7951 94.9816 0.1726 0.1818
93.6921 94.1031 93.8574 94.0019 93.7593 93.6175 93.8385 0.1863 0.1986
1 2 3 4 5 6 Average S.D R.S.D (%)
Label claim: 5 mg of curcumin and 5 mg of piperine.
3.3.2.
Accuracy
Three replicate injections containing known amount of curcumin and piperine at 50%, 100% and 150% were added to the pre-analysed samples (150 mg mL1 of curcumin and 150 mg mL1 of piperine) and analysed using the developed method. The results are summarized in Table 2. The developed method satisfies the acceptance criteria of the recovery study and ensure accuracy of the developed method.
3.3.3.
Precision
Six replicate injections containing curcumin (150 mg mL1) and piperine (150 mg mL1) and the results are summarized in Table 3. The % R.S.D of the assay, peak area and tailing were less than 1% which denoted very good repeatability of the measurement. Hence the developed method displayed a good precision.
3.3.4.
Limit of detection and limit of quantitation
The LOD were 0.3 ppm for curcumin and 0.1 ppm for piperine at a signal-to-noise ratio of 3:1. Similarly, LOQ were 0.4 ppm for curcumin and 0.9 ppm for piperine at a signal-to-noise ratio of 10:1.
3.3.5.
Linearity and range
Calibration standard solutions of 10, 25, 50, 100 and 150 mg mL1 were prepared and analysed using the developed method. Obtained peak areas were plotted against the concentration and the linearity was calculated by least square regression method. The results are summarized in Table 4.
3.3.6.
Robustness
The robustness of the developed method was investigated with slight change in the column oven temperature (30 C &
40 C) and pH of the mobile phase (2.8e3.2) and the results are summarized in Table 5. However, these changes had an influence on the assay but not considered significant as the % R.S.D was 2%.
3.4. Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles The developed method was successfully implemented to determine the encapsulation efficiency of curcumin and piperine in the Eudragit E 100 nanoparticles. The results are summarized in Table 6. Both methods have shown lesser standard deviation and % R.S.D was less than 2% which ensures the precision of the developed method.
4.
Conclusion
In this study, a simple, accurate and precise analytical method for simultaneous estimation of curcumin and piperine was developed with adequate separation and validated as per ICH guideline. The developed method was successfully implemented in the estimation of curcumin and piperine encapsulated in Eudragit E 100 nanoparticles and this method is also suitable for use in routine analysis of curcumin and piperine in active pharmaceutical ingredient and in pharmaceutical dosage forms.
Conflicts of interest All authors have none to declare.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 2 4 e2 2 9
references
1. Moorthi C, Kiran K, Manavalan R, Kathiresan K. Preparation and characterization of curcumin-piperine dual drug loaded nanoparticles. Asian Pac J Trop Biomed. 2012;2:841e848. 2. Moorthi C, Kathiresan K. Curcuminepiperine/ curcuminequercetin/curcuminesilibinin dual drug loaded nanoparticulate combination therapy: a novel approach to target and treat multidrug resistant cancers. J Med Hypotheses Ideas; 2012. http://dx.doi.org/10.1016/J.Jmhi.2012.10.005. 3. Moorthi C, Manavalan R, Kathiresan K. Nanotherapeutics to overcome conventional cancer chemotherapy limitations. J Pharm Pharm Sci. 2011;14:67e77. 4. Moorthi C, Kathiresan K. Nanotoxicology: toxicity of engineered nanoparticles and approaches to produce safer nanotherapeutics. Int J Pharm Sci. 2012;2:117e124.
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5. Nagappan KV, Meyyanathan SN, Raja RB, Kannan E. A liquid chromatography method for the simultaneous determination of curcumin and piperine in food products using diode array detection. Asian J Res Chem. 2009;2:115e118. 6. Chen W, Shen Y, Rong H, Lei L, Guo S. Development and application of a validated gradient elution HPLC method for simultaneous determination of 5-fluorouracil and paclitaxel in dissolution samples of 5-fluorouracil/paclitaxel-co-eluting stents. J Pharm Biomed Anal. 2012;59:179e183. 7. Venishetty VK, Parikh N, Sistla R, Ahmed FJ, Diwan PV. Application of validated RPeHPLC method for simultaneous determination of docetaxel and ketoconazole in solid lipid nanoparticles. J Chromatogr Sci. 2011;49:136e141. 8. Shaikh KA, Patil SD, Devkhile AB. Development and validation of a reversed-phase HPLC method for simultaneous estimation of ambroxol hydrochloride and azithromycin in tablet dosage form. J Pharm Biomed Anal. 2008;48:1481e1484.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Application of validated RPeHPLCePDA method for the simultaneous estimation of curcumin and piperine in Eudragit E 100 nanoparticles C. Moorthi a,*, C. Senthil Kumar b, S. Mohan b, Kiran Krishnan c, K. Kathiresan a a
Department of Pharmacy, Annamalai University, Annamalai Nagar 608 002, Chidambaram, Tamil Nadu, India Saraswati Institute of Pharmaceutical Sciences, Dhanap Post, Gandhi Nagar 382355, Gujarat, India c Apotex Advancing Generics, Apotex Corporation, Suite 400, 2400 North Commerce Parkway, Weston, FL 33326, United States b
article info
abstract
Article history:
Background: The primary aim of the study was to develop an HPLC method for the simul-
Received 10 December 2012
taneous estimation of curcumin and piperine with adequate separation and to validate the
Accepted 8 March 2013
developed method as per ICH guideline and implement the developed method for the
Available online 4 April 2013
simultaneous estimation of curcumin and piperine which are encapsulated in the Eudragit E 100 nanoparticles.
Keywords:
Method: Chromatographic conditions were optimized and the best chromatographic con-
Analytical method development
ditions was achieved when the chromatographic separation was carried out on a Luna C18
Curcumin
column (Reversed phase, 150 mm 4.6 mm with 5 m particle size, Phenomenax) using a
Eudragit E 100
mobile phase combination of 0.1% ortho phosphoric acid aqueous solution and acetonitrile
Nanosuspension
(45:55, v/v) in an isocratic mode elution with a flow rate of 1.2 mL min1 at the column oven
Piperine
temperature of 35 C. The detection was monitored at a wavelength of 262 nm. Results: The retention time of curcumin and piperine was 8.6 and 5.9 min, respectively. The developed method was validated for system suitability, accuracy, precision, limit of detection, limit of quantitation, linearity, range, robustness and all the validated parameters were within the acceptable criteria. The method was linear over the range (10e150 mg mL1) for both drugs with correlation coefficients of greater than 0.999. Limit of detection were 0.3 ppm for curcumin and 0.1 ppm for piperine and limit of quantification were 0.4 ppm for curcumin and 0.9 ppm for piperine. Conclusions: The developed method was successfully implemented in the estimation of curcumin and piperine encapsulated in Eudragit E 100 nanoparticles and this method is also suitable for use in routine analysis of curcumin and piperine in active pharmaceutical ingredients and in pharmaceutical dosage forms. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
* Corresponding author. E-mail address: [email protected] (C. Moorthi). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.03.006
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 2 4 e2 2 9
1.
Introduction
Curcumin is an orangeeyellow crystalline phytochemical isolated from Curcuma longa and classified as a functional food, as it possess wide spectrum of pharmacological activities including anti-cancer activity due to its diverse molecular targets. Curcumin is extremely safe and can be well tolerated at high doses and has also been declared as “generally regarded as safe” by US FDA. In spite of its efficacy and safety, the clinical usefulness of curcumin in the treatment of cancer is limited due to certain limitations including lack of aqueous solubility, rapid clearance from the systemic circulation, intestinal metabolism, hepatic metabolism, lack of cancer cell targeting and multidrug resistance. Hence, to overcome these limitations, we have proposed a dual drug loaded Eudragit E 100 nanosuspension containing curcumin and piperine.1e4 However, the total amount of curcumin and piperine encapsulated in the Eudragit E 100 polymer matrix determines the efficacy of the nanosuspension. Analytical techniques for the simultaneous estimation of curcumin and piperine have been reported.5 In the reported high performance liquid chromatography (HPLC) method, separation between curcumin and piperine was 9 and 9.5, respectively.5 However, this narrow separation (0.5 min) may not be sufficient enough to estimate curcumin and piperine which are encapsulated in polymer matrix as the polymer and other excipients in the formulation may interfere in the chromatographic separation of curcumin and piperine. Hence, an analytical technique with adequate separation between curcumin and piperine is essential. The primary aim of the study was to develop an HPLC method for the simultaneous estimation of curcumin and piperine with adequate separation and to validate the developed method as per ICH guideline and implement the developed method for the simultaneous estimation of curcumin and piperine which are encapsulated in the Eudragit E 100 nanoparticles.
225
250 mg of beta-cyclodextrin in 20 ml of distilled water was prepared and emulsified with organic phase under sonication (Lark, India) for 10 min to form nanoparticles. However, the sonication process was continued for another 50 min to evaporate any residual solvent present in the nanosuspension. Average particle size, polydispersity index and zeta potential were measured using Zetasizer (Malvern, UK).
2.3. Fabrication of Eudragit E 100 nanosuspension using mechanical stirring About 5 mg of curcumin, 5 mg of piperine and 250 mg of Eudragit E 100 were dissolved in 20 ml organic solvent (mixture of 12 ml of ethanol and 8 ml of distilled water). An aqueous phase containing 125 mg of poloxamer 188 and 50 mg of beta-cyclodextrin in 25 ml of distilled water was prepared and emulsified with organic phase under mechanical stirring (Remi, India) at 500 rpm for 10 min to form nanoparticles. However, the stirring process was continued for another 3 h to evaporate any residual solvent present in the formulation. Average particle size, polydispersity index and zeta potential were measured using Zetasizer (Malvern, UK).
2.4.
Instrumentation
Analyses were performed using an Alliance HPLC (Waters Corp.) equipped with pump, degasser, photodiode array detector and autosampler. The generated analytical signals were monitored and integrated using Empower chromatography data software.
2.5.
Method development
Method development for the simultaneous estimation of curcumin and piperine was carried out with different flow rates, different columns, different elution modes, different mobile phase and buffer ratio.
2.
Material and methods
2.6.
2.1.
Reagents and solvents
The developed method was validated in compliance with ICH guideline for system suitability, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), linearity, range and robustness.6e8
Curcumin (97%) and beta-cyclodextrin were purchased from Himedia Laboratories, India. Piperine (97%) and poloxamer 188 were purchased from SigmaeAldrich, India. Eudragit E 100 was obtained from Degussa, India. HPLC grade ethanol was purchased from Brampton, Canada. HPLC grade methanol, acetonitrile and water were purchased from Merck, India. Analytical grade ortho phosphoric acid was purchased from Rankem, India.
2.6.1.
Method validation
System suitability
Six replicate injections containing curcumin and piperine were analysed using the developed method. Theoretical plate count more than 3000, tailing less than 1.5 and percentage relative standard deviation (% R.S.D) of peak area and the retention time less than 2% were set as acceptance criteria.
2.2. Fabrication of Eudragit E 100 nanosuspension using sonication
2.6.2.
About 5 mg of curcumin, 5 mg of piperine and 250 mg of Eudragit E 100 were dissolved in 10 ml organic solvent (mixture of 6 ml of ethanol and 4 ml of distilled water). An aqueous phase containing 250 mg of poloxamer 188 and
Three replicate injections containing known amount of curcumin and piperine at 50, 100 and 150% were added to the preanalysed samples and its recovery was estimated using the developed method. Percentage recovery within 100 2% and % R.S.D of assay less than 2% were set as acceptance criteria.
Accuracy
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2.6.6.
Robustness
The robustness of a developed analytical method refers to its ability to remain unaffected by small but deliberate change of the chromatographic condition which provides an indication of its reliability during normal usage. Assay was carried out using the developed method with slight change in the column oven temperature (30 C & 40 C) and pH of the mobile (2.8 & 3.2).
2.7. Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles was determined by an indirect method by measuring the free curcumin and piperine in the nanosuspension. Prepared Eudragit E 100 nanosuspension was subjected to centrifugation (Remi, India) at 19,000 rpm for about 45 min at 20 C. About 1 mL of supernatant was withdrawn and mixed with 1 ml of methanol and the solution was then filtered through a 0.22 mm membrane. Six replicate injections were analysed using the developed method to estimate the curcumin and piperine.
Fig. 1 e Typical chromatogram of curcumin (150 mg mLL1) and piperine (150 mg mLL1) using 0.1% ortho phosphoric acid and acetonitrile (45:55 v/v) in an isocratic mode elution with a flow rate of 1.2 mL minL1 and the column oven temperature of 35 C. The detection was monitored at a wavelength of 262 nm.
3. 2.6.3.
3.1. Fabrication of Eudragit E 100 nanosuspension containing curcumin and piperine
Six replicate injections containing curcumin and piperine were analysed using the developed method within a short period of time on the same day. The % R.S.D of peak area, assay and tailing less than 2% were set as acceptance criteria.
2.6.4.
Eudragit E 100 nanosuspension prepared using sonication has shown an average particle size of 140 nm with a polydispersity index of 0.254 and zeta potential of 28.8 mV. Whereas, Eudragit E 100 nanosuspension prepared using mechanical stirring has shown an average particle size of 87 nm with a polydispersity index of 0.239 and zeta potential of 22 mV.
Limit of detection and limit of quantitation
LOD and LOQ of curcumin and piperine were estimated from the signal-to-noise ratio. Signal-to-noise ratio of three for estimating LOD and 10 for estimating LOQ were set as acceptance criteria.
3.2. 2.6.5.
Results and discussions
Precision
Method development
Linearity and range
Linearity was evaluated at five concentration levels at 10%, 25%, 50%, 100% and 150% of the targeted assay concentration of curcumin and piperine. The linearity was then determined by least square regression analysis from the peak area against drug concentration plot. The analytical range was established by the highest and lowest concentrations of analyte where acceptable linearity, accuracy and precision were obtained.
Method development for the simultaneous estimation of curcumin and piperine was carried out with different columns but Luna C18 column has shown higher theoretical plate count and lesser tailing. Different ratio of mobile phase and buffer have been tried but the mixture of 0.1% v/v ortho phosphoric acid and acetonitrile at 45:55 proportions has shown adequate separation of curcumin and piperine. However, further increase or
Table 1 e System suitability of the developed method. Samples
1 2 3 4 5 6 Average S.D R.S.D (%)
Peak area
Plate counts
Tailing
Retention time
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
2,467,475 2,455,932 2,475,781 2,478,884 2,487,645 2,484,449 2,475,027.67 11,705.42 0.47
3,740,512 3,711,958 3,732,022 3,744,973 3,749,042 3,746,015 3,737,420.33 13,805.83 0.37
7973 7848 7786 8053 7751 7823 7872.33 116.60 1.48
7570 7697 7818 8102 7645 7688 7753.33 188.97 2.44
1.11 1.12 1.12 1.11 1.11 1.11 1.11 0.01 0.46
1.15 1.15 1.15 1.14 1.15 1.15 1.15 0.00 0.36
8.68 8.41 8.40 8.36 8.37 8.40 8.44 0.12 1.43
5.96 5.85 5.85 5.83 5.81 5.83 5.86 0.05 0.91
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Table 2 e Recovery studies of the developed method. Analyte
Level
Sample
Spiked (mg mL1)
Recovered (mg mL1)
Recovery (%)
R.S.D (%)
Curcumin
50%
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
75 75 75 150 150 150 225 225 225 75 75 75 150 150 150 225 225 225
75.50 76.41 75.80 148.94 148.15 149.44 225.16 225.94 226.62 74.61 74.39 74.48 150.44 148.66 150.80 222.42 223.28 229.09
100.67 101.89 101.07 99.29 98.76 99.63 100.07 100.42 100.72 99.49 99.18 99.31 100.29 99.11 100.53 98.85 99.23 101.81
0.61
100%
150%
Piperine
50%
100%
150%
0.44
0.32
0.15
0.76
1.61
Table 3 e Precision of the developed method. Samples
1 2 3 4 5 6 Average SD R.S.D (%)
Peak area
Assay
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
2,449,608 2,497,458 2,450,028 2,483,098 2,478,822 2,488,832 2,474,641 20,216.09 0.82
3,772,888 3,756,286 3,793,774 3,730,058 3,715,387 3,752,275 3,753,444 28,317.00 0.75
98.97 100.90 98.98 100.32 100.15 100.55 99.98 0.82 0.82
100.94 100.50 101.50 99.80 99.41 100.37 100.42 0.76 0.75
1.11 1.11 1.11 1.12 1.10 1.10 1.11 0.01 0.68
1.14 1.14 1.16 1.14 1.15 1.14 1.15 0.01 0.73
decrease in proportion of 0.1% v/v ortho phosphoric acid does not exhibit adequate separation between curcumin and piperine. Initially, 0.8 ml flow rate was used but increase in flow rate from 0.8 to 1.2 ml has shown adequate separation and high theoretical plates. Similarly, isocratic elution mode has shown better separation in comparison with gradient elution mode.
Table 4 e Linearity parameter and range of the developed method. Calibration samples (mg mL1)
Tailing
Peak area of curcumin
Peak area of piperine
10 195,616 286,543 25 439,284 703,279 50 936,366 1,367,606 100 1,717,340 2,646,426 150 2,553,086 4,007,604 Slope 16,938 26,525 Intercept 27,932 20,952 Linearity equation Y ¼ 16,938x þ 27,932 Y ¼ 26,525x þ 20,952 Correlation coefficient (R2) 0.999,491 0.999,915 10e150 10e150 Range (mg mL1)
After extensive preliminary experimental trials, the best chromatographic conditions to obtain adequate separation, resolution and symmetrical peak of curcumin and piperine were achieved only when the chromatographic separation was carried out on a Luna C18 column (Reversed phase, 150 mm 4.6 mm with 5 m particle size, Phenomenax) using a mobile phase combination of 0.1% ortho phosphoric acid aqueous solution and acetonitrile (45:55, v/v) in an isocratic mode elution with a flow rate of 1.2 mL min1 at the column oven temperature of 35 C. The detection was monitored at a wavelength of 262 nm. Fig. 1 shows a typical chromatogram of curcumin and piperine indicating complete resolution of curcumin at 8.685 min and piperine at 5.969 min.
3.3.
Method validation
3.3.1.
System suitability
Six replicate injections containing curcumin (150 mg mL1) and piperine (150 mg mL1) and the results are summarized in Table 1. The developed method satisfies the acceptance criteria of the system suitability parameters and ensures the validity of the developed method.
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Table 5 e Robustness of the developed method. Robustness parameters
Level
Assay (%) Curcumin
Piperine
Curcumin
Piperine
100.34 99.01 100.88 99.02
99.71 98.80 99.75 97.17
0.46 0.48 0.84 0.47
0.26 1.81 0.23 2.00
Column oven temperature
30 C 40 C 2.8 3.2
pH of the mobile phase
R.S.D (%)
Table 6 e Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles. Sample
Amount found (mg) in nanoparticles Sonication method
Encapsulation efficiency (%)
Stirring method
Sonication method
Stirring method
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
Curcumin
Piperine
4.7734 4.7771 4.7778 4.7705 4.7733 4.7733 4.7742 0.0027 0.0573
4.5992 4.5887 4.5885 4.5966 4.5925 4.5956 4.5935 0.0044 0.0954
4.7417 4.7611 4.7527 4.7557 4.7435 4.7398 4.7491 0.0086 0.1818
4.6846 4.7052 4.6929 4.7001 4.6880 4.6809 4.6919 0.0093 0.1985
95.4687 95.5428 95.5552 95.4098 95.4650 95.4650 95.4844 0.0548 0.0574
91.9849 91.7737 91.7706 91.9319 91.8498 91.9127 91.8706 0.0876 0.0954
94.8349 95.2217 95.0544 95.1134 94.8702 94.7951 94.9816 0.1726 0.1818
93.6921 94.1031 93.8574 94.0019 93.7593 93.6175 93.8385 0.1863 0.1986
1 2 3 4 5 6 Average S.D R.S.D (%)
Label claim: 5 mg of curcumin and 5 mg of piperine.
3.3.2.
Accuracy
Three replicate injections containing known amount of curcumin and piperine at 50%, 100% and 150% were added to the pre-analysed samples (150 mg mL1 of curcumin and 150 mg mL1 of piperine) and analysed using the developed method. The results are summarized in Table 2. The developed method satisfies the acceptance criteria of the recovery study and ensure accuracy of the developed method.
3.3.3.
Precision
Six replicate injections containing curcumin (150 mg mL1) and piperine (150 mg mL1) and the results are summarized in Table 3. The % R.S.D of the assay, peak area and tailing were less than 1% which denoted very good repeatability of the measurement. Hence the developed method displayed a good precision.
3.3.4.
Limit of detection and limit of quantitation
The LOD were 0.3 ppm for curcumin and 0.1 ppm for piperine at a signal-to-noise ratio of 3:1. Similarly, LOQ were 0.4 ppm for curcumin and 0.9 ppm for piperine at a signal-to-noise ratio of 10:1.
3.3.5.
Linearity and range
Calibration standard solutions of 10, 25, 50, 100 and 150 mg mL1 were prepared and analysed using the developed method. Obtained peak areas were plotted against the concentration and the linearity was calculated by least square regression method. The results are summarized in Table 4.
3.3.6.
Robustness
The robustness of the developed method was investigated with slight change in the column oven temperature (30 C &
40 C) and pH of the mobile phase (2.8e3.2) and the results are summarized in Table 5. However, these changes had an influence on the assay but not considered significant as the % R.S.D was 2%.
3.4. Encapsulation efficiency of curcumin and piperine in Eudragit E 100 nanoparticles The developed method was successfully implemented to determine the encapsulation efficiency of curcumin and piperine in the Eudragit E 100 nanoparticles. The results are summarized in Table 6. Both methods have shown lesser standard deviation and % R.S.D was less than 2% which ensures the precision of the developed method.
4.
Conclusion
In this study, a simple, accurate and precise analytical method for simultaneous estimation of curcumin and piperine was developed with adequate separation and validated as per ICH guideline. The developed method was successfully implemented in the estimation of curcumin and piperine encapsulated in Eudragit E 100 nanoparticles and this method is also suitable for use in routine analysis of curcumin and piperine in active pharmaceutical ingredient and in pharmaceutical dosage forms.
Conflicts of interest All authors have none to declare.
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