Accepted Manuscript Stability indicating HPLC-UV method for detection of Curcumin in Curcuma longa Extract and Emulsion Formulation Haroon Khalid Syed, Kai Bin Liew, Gabriel Onn Kit Loh, Kok Khiang Peh PII: DOI: Reference:

S0308-8146(14)01281-3 http://dx.doi.org/10.1016/j.foodchem.2014.08.066 FOCH 16289

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

30 November 2013 11 April 2014 13 August 2014

Please cite this article as: Syed, H.K., Liew, K.B., Loh, G.O.K., Peh, K.K., Stability indicating HPLC-UV method for detection of Curcumin in Curcuma longa Extract and Emulsion Formulation, Food Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.foodchem.2014.08.066

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Stability indicating HPLC-UV method for detection of Curcumin in Curcuma longa Extract and Emulsion Formulation

Haroon Khalid Syed, Kai Bin Liew, Gabriel Onn Kit Loh and Kok Khiang Peh*

First author: Haroon Khalid Syed, B.Pharm, M.Phil. Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Telephone: 604-6533296. Email: [email protected].

Co-author: Kai Bin, Liew, B.Pharm, MSc. Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Telephone: 604-6533296. Email: [email protected].

Co-author: Gabriel Onn Kit Loh, B.Sc. Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Telephone : 604-6533296. Email: [email protected].

*Corresponding author: Kok-Khiang, Peh, PhD, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Telephone: 604-6532257; Fax: 604-6570017; E-mail: [email protected]; [email protected].

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Abstract A stability-indicating HPLC-UV method for the determination of curcumin in Curcuma longa extract and emulsion was developed. The system suitability parameters, theoretical plates (N), tailing factor (T), capacity factor (  ), height equivalent of a theoretical plate (H) and resolution (Rs) were calculated. Stress degradation studies (acid, base, oxidation, heat and UV light) of curcumin were performed in emulsion. It was found that N > 6500, T < 1.1,   was 2.68 - 3.75, HETP about 37 and Rs was 1.8. The method was linear from 2 – 200 µg/mL with a correlation coefficient of 0.9998. The intra-day precision and accuracy for curcumin were ≤ 0.87% and ≤ 2.0 %, while the inter-day precision and accuracy values were ≤ 2.1% and ≤ -1.92. Curcumin degraded in emulsion under acid, alkali and UV light. In conclusion, the stability-indicating method could be employed to determine curcumin in bulk and emulsions.

Key words: Curcumin; System suitability; Stress degradation study; Stability indicating HPLCUV method.

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1. Introduction Curcuma longa is a famous and important food spice with a rich source of polyphenolic compounds, namely curcuminoids (Saleemulla, Makhija, Khamar & Rani, 2010). This spice has been extensively used in traditional medicine and its demand is increasing in the field of pharmaceutical, food and cosmetic industries. Apart from its daily use in food as a condiment and spice, it has been used in the treatment of a number of ailments such as cough, fever, jaundice, wounds, eczema, inflammatory joints, parasitic skin diseases, cold, liver and urinary diseases (Antony, Elumalai & Benny, 2011). The rhizome has also been found useful in the treatment of anemia, bacterial and viral infections (Nadkarni, 1976; Bakhru, 1997).

Curcuma longa extract has three important compounds called as curcumin (Cur), demethoxycurcumin and bis-demethoxycurcumin, which are present in approximately 70% -77 %, 18% - 20 % and 5% - 10 % (Ahsan, Parveen, Khan & Hadi, 1999). The chemical structures of the three compounds are shown in Fig. 1.

Methods that have been used to quantify curcumin include thin layer chromatography (Ravindranath & Chandrasekhara, 1981), thin layer chromatography-UV and thin layer chromatography- densitometric (Cooray, 1988), high-performance liquid chromatographyultraviolet (HPLC-UV) spectrophotometric and electrochemical detection (Smith & Witowska, 1994), HPLC- UV and LC- MS (Asai & Miyazawa, 2000) and HPLC-UV (Heath, Milagros, Dean & Cherly, 2002). Most of these methods could only detect one single peak of curcumin except a method reported by Asai & Miyazawa (2000), which could simultaneously detect the three

curcumin

peaks

corresponding

to

curcumin,

3

demethoxycurcumin

and

bis-

demethoxycurcumin. Surash &Yadav (2009) reported a sensitive RP-HPLC- UV method for the determination of curcumin but system suitability and stability studies were not performed. Recently, a stability indicating HPLC-UV method was reported by Gugulothu & Patravale (2012) for determination of curcumin and celecoxib in nanoparticle formulation. The result showed that curcumin in solution was not stable and degraded after 1hr of stress studies. Curcumionoids are polyphenols and decomposed when exposed to high temperature (Schieffer, 2002).

The objective of the present study was to develop a simple, rapid and reproducible stability indicating HPLC-UV method for the determination of curcumin in Curcuma longa extract and emulsion. The method could simultaneously detect curcumin, demethoxycurcumin and bisdemethoxycurcumin. Stress degradation studies (acid, base, oxidation, heat and UV light) were carried out on emulsion formulation.

2. Materials and methods 2.1. Materials Curcuma longa extract (≥ 95 % curcuminoids) was purchased from Natural Remedies Pvt Limited, Bangalore, India. Curcumin standard (mixture of curcumin, demethoxycurcumin and bisdemethoxycurcumin, 98+%) was purchased from Acros Organic, New Jersey, USA. Acetonitrile was purchased from J.T. Baker, NJ, USA. Methanol was obtained from Fisher Scientific, UK. Glacial acetic acid was purchased from R & M Marketing, Essex, UK. The organic solvents and chemicals used were either of analytical reagent or high performance liquid chromatography grade. Isopropyl myristate was purchased from Derifats Chemicals Sdn Bhd, Malaysia. Tween 80 and Span 80 were purchased from Sigma-Aldrich, USA. 4

2.2. Instrumentation The HPLC system was comprised of a Shimadzu (VP series, Kyoto, Japan) pump (LC-20AT vp) with solvent cabinet, a degasser (DGU-20A3), a column oven (CTO-10S VP), an auto-injector (SIL-20A HT vp), UV/VIS detector (SPD-20AD vp) and a computer software (LC- solution VP).

2.3. Chromatographic condition The separation was carried out using a reversed phase C-18 Agilent Eclipse Plus (Agilent, USA) column (250 x 4.6 mm ID, 5µm). The flow rate was set at 1.5 ml/min and detection wavelength was 370nm. Sample of 25 µl was injected onto the column.

2.4. Mobile phase optimization Different composition of mobile phase was studied to achieve good resolution and short elution time. The mobile phase was filtered through Whatman filter 0.45 µm (nylon membrane filters 47 mm) and degassed before use.

2.5. Preparation of stock and working standard solutions 50 mg of curcumin working standard was weighed and transferred to a 50mL volumetric flask and dissolved in 30mL of methanol. The volumetric flask was shaken using ultrasonic vibrator for 5 min. The solution was diluted to volume with methanol. The stock standard solution had a concentration of 1mg/mL of curcumin.

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2.6. System suitability studies The chromatographic parameters, such as, theoretical plates (N), the height equivalent of a theoretical plate (HETP), tailing factor (T), capacity factor (  ) and resolution (Rs) were calculated (USP 34, 2010).

The number of theoretical plates (N) is used to describe the quality of chromatographic column.

HETP or H, the height equivalent of a theoretical plate, measures the column efficiency per unit length (L) of the column.

Capacity factor (  ) is an indicator of efficiency of a column to retain sample molecule during an isocratic separation. Literature proposed the acceptable   value ranges from 2-10 (Snyder, Kirkland & Glajch, 1997).

2.7. Limit of detection (LOD) The limit of detection (LOD) of the curcumin was calculated as 3.3 (σ/S), where σ represents standard deviation of intercept and S represents mean slope (Jadhav, Mahadik & Paradkar, 2007).

2.8. Lower limit of quantification (LLOQ) The LLOQ was the lowest point of concentration in the standard calibration curve. The acceptance criteria were RSD of 2% for precision and % Bias of 2% for accuracy.

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2.9. Preparation of curcumin emulsion Briefly, the curcumin o/w emulsion was prepared by a two-step procedure. Span 80 was dissolved in isopropyl myristate and Tween 80 dissolved in distilled water. Curcumin was added in oil phase and mixed thoroughly until completely dissolved. Emulsification was made by adding water into oil phase with the application of high speed Ultra-Turrax (T 25 basic; IKA, Labortechnik, Malaysia).

2.10. Assay of curcumin in Curcuma longa extract 20 mg of curcumin standard was weighed and transferred to a 20 mL volumetric flask and dissolved in 15mL of methanol. The volumetric flask was shaken using ultrasonicator for 5 min. The solution was diluted to volume with methanol. 20 mg of Curcuma longa extract powder was weighed and transferred to a 20 mL volumetric flask and dissolved in 15mL of methanol. The volumetric flask was shaken using ultrasonic vibrator for 5 min. The solution was diluted to volume with methanol. The stock standard solution of curcumin and Curcuma longa extract powder had a concentration of 1mg/mL of curcumin. 0.2 ml of standard and extract powder solution was transferred to 10mL volumetric flask and dissolved in methanol to give a concentration of 20 µg/mL. This sample was injected into HPLC in triplicate to determine the potency of Curcuma longa extract powder. The content of curcumin in Curcuma longa extract powder was calculated by dividing ratio of Curcuma longa extract with ratio of standard times the potency of standard, where the ratio of standard or Curcuma longa extract are calculated by dividing peak area of curcumin in standard or Curcuma longa extract with the total area of three peaks comprising of curcumin, demethoxycurcumin and bisdemethoxycurcumin.

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2.11. Assay of curcumin in emulsion 50 mg of curcumin standard was weighed and transferred to a 50mL volumetric flask and dissolved in 45mL of methanol. The volumetric flask was shaken using ultrasonicator for 5 min. The solution was diluted to volume with methanol. The stock standard solution of curcumin had a concentration of 1mg/mL of curcumin. 1 gram of emulsion (w/w) was transferred into a 50 ml ◦

volumetric flask containing methanol. The flask was heated in water-bath at 60 C until the emulsion melted and diluted to the mark with methanol. The flask was shaken for 15min manually. The flask was then subjected to sonication for 15 minutes. The resulting solution was centrifuged at 4000 rpm for 10 min. 0.2 mL of the supernatant was diluted with methanol in a 10mL volumetric flask to give a concentration of 20µg/ml. The content of curcumin in emulsion was calculated using the following equation,

 % =



× 

  



× 

×

 

     

where x is weight of standard and y is weight of emulsion sample.

2.12. Stress degradation studies Stress degradation study of curcumin in emulsion formulation was performed. Methanolic curcumin stock solution at 1mg/mL was prepared. 5g of emulsion was taken and dissolved in a volumetric flask. Methanol was used as a solvent. 25 µL of the solution was injected into HPLC.

2.13. Acid and alkali degradation studies 1.0 mL of methanolic stock solution of curcumin was added into two flasks. 1.0 mL of 3M HCL or 1.0 mL of 3M NaOH was added separately into each of the flask. The solutions were 8

neutralized immediately and adjusted to volume with methanol. These samples served as zero hour samples. Another set of flasks was left on the bench at room temperature (26˚C / 65% RH) and the same neutralization procedure was repeated after 24 hours. The neutralized solutions were injected in triplicate.

2.14. Oxidative hydrogen peroxide (H2O2) degradation 1.6 mL of methanolic stock solution of curcumin was measured into two flasks. 0.344 mL of 35% H2O2 was added into each of the flask. For the first flask, the solution was adjusted to volume with methanol and served as zero hour sample. Another flask was left on the bench at room temperature (26˚C / 65% RH) and the same procedure was carried out after 24hours. The final solution was injected in triplicate.

2.15. Heat degradation study 1.6mL of methanolic stock solution of curcumin was withdrawn and transferred into a 10 mL volumetric flask. Two flasks were prepared. For the first flask, the solution was adjusted to volume and served as zero hour sample. The other flask was heated in water bath at 80˚C and the samples were injected in triplicate after heating for 2 hours.

2.16. UV light degradation 1.6mL of methanolic stock solution of curcumin was withdrawn and transferred into a 10 mL volumetric flask. The solution was adjusted to volume with methanol and served as zero hour sample. Another flask was stored in UV cabinet (365 nm) and the samples were injected after 24 hours.

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2.17. Linearity Calibration standards of curcumin at concentrations of 2.0, 4.0, 12.5, 25.0, 50.0, 100.0, 200.0 µg/mL in methanol were prepared. The standard calibration curve was constructed using peak area versus known concentrations of curcumin. The linear regression line was used to determine the linearity and concentration of the samples. The linearity of curcumin was determined using six sets of the calibration standards.

2.18. Precision and accuracy Three Quality Control (QC) samples of curcumin at 6, 80 and 160 µg/mL were prepared from the stock solution. The three QC samples and LOQ (2 µg/mL) were used to determine the method precision and accuracy. For intra-day precision and accuracy, six sets of standard solutions were injected on the same day. For inter-day precision and accuracy, six sets of standard solutions were injected over six consecutive days, with one standard curve injected on each day. For precision, the coefficient of variation (%CV) was calculated by dividing standard deviation with mean value. On the other hand, accuracy was presented as the relative percentage error (% bias) which was calculated by dividing the difference between calculated concentration and standard solution with standard solution.

2.19. Stock solution stability The stock solution of curcumin at concentration of 1 mg/mL was kept for 24 hours at room ◦

temperature (26 C). The stock solution was then diluted to a concentration within the standard

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calibration linear range (160µg/mL). The instrumental responses at 24 hours were compared with that of fresh samples at zero hour. 2.20. Recovery of curcumin 1.0 mg of curcumin was added into 50 ml volumetric flask containing emulsion base (total ◦

weight of 1gm) with methanol. The flask was heated in water-bath at 60 C until the emulsion base melted and diluted to the mark with methanol. The flask was shaken for 15 min manually and subjected to sonication for 15 minutes. The resulting solution was centrifuged at 4000 rpm for 10 min. 0.2 mL of supernatant was diluted with methanol in a 10mL volumetric flask to give a concentration of 20µg/ml. 25 µL of sample was injected into the HPLC system. The analysis was repeated three times. The recovery of curcumin was calculated from the slope and the intercept of the calibration curve drawn in the concentration range of 2-200 µg/ml.

3. Results and discussion 3.1. Mobile phase optimization The different compositions of mobile phase, pH and flow rate are presented in Table 1. Mobile phase with composition of acetonitrile, methanol and water at 40:20:40 (v/v/v) and pH 3.0 was found to be the most optimum because the three curcumin peaks, curcumin, demethoxycurcumin and bis-demethoxucurcumin were well resolved and eluted with short retention time of 6.728, 6.166 and 5.666 min. When acetonitrile, methanol and water at ratio of 35:10:55 were used, the peak of curcumin was well separated but quite long retention time (30min). Modification of the composition of mobile phase to 40:15:45 reduced the retention time of the curcumin to 10 min. A shorter retention time of curcumin was achieved with composition at 45:20:35 but separation was very poor.

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3.2. System suitability studies The results of theoretical plates (N), tailing factor (T), height equivalent to theoretical plate (HETP), capacity factor (  ) and resolution (Rs) of the method at three QC concentrations are shown in Table 2. The average theoretical plate was > 2000. No back tailing was observed and tailing factor (< 2) met the requirement stated in United States Pharmacopeia (USP 34, 2010). The capacity factor lied within the range of 2 – 10 (Synder, Kirkland & Glajch, 1997).

3.3. LOD and LLOQ The LOD was 0.305µg/mL for curcumin. The LLOQ was 2µg/mL.

3.4. Assay of curcumin in Curcuma longa extract and emulsion The assay of curcumin in Curcuma longa extract powder and emulsion were found to be 100.20 % and 99.45 %.

3.5. Specificity There was no peak found at the retention time of the analyte in the blank solution. The chromatogram of blank (solvent) is presented in Fig. 2a. The results from the stress testing studies indicated the method was highly specific for curcumin. The degradation products were completely distinguishable from the parent compound. The placebo chromatogram is presented in Fig. 2b.

3.6. Stress degradation studies

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An ideal stability-indicating method is one that can quantify the standard drug and resolve its degradation products (Kadi, Mohammed, Kaseem & Darwish, 2011). The method was able to separate both analyte and degradation product peak. The results of acid, alkali, oxidation, heat and UV degradation are shown in Table 3. Curcumin degraded under acid, alkali and UV light challenges in emulsion. Under alkaline and UV conditions, excipients such as Tween 80 or Span 80 might undergo reactions to form secondary products. These secondary products might interact with curcumin reducing the curcumin concentration in the sample (Zhang, 2009).

3.7. Linearity The standard calibration curve exhibited an excellent linearity and a good correlation coefficient over the given range of 2 – 200 µg/mL of curcumin. The mean linear regression equation from six calibration curves for curcumin was y = 38267.3 (± 764.2) x + 1279.6 (± 3538.3), (x = curcumin concentration, y = average peak area) with a correlation coefficient of 0.9998 (0.0001).

3.8. Precision and accuracy The results of precision and accuracy are shown in Table 4. The chromatogram of curcumin standard solution at 2µg/mL is represented in Fig. 2c. The intra-day precision for curcumin was between 0.11 – 0.86%, whereas the intra-day accuracy was between -1.09 – 1.99%. The interday precision and accuracy was in the range of 1.28 – 2.0% and -1.92 – (-0.20) % respectively. The results were within the ± 2% range recommended by United States Pharmacopeia (USP 34, 2010). Hence, the method indicates good method precision and accuracy.

3.9. Stock solution stability

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The percentage of curcumin remaining after twenty four hours kept at room temperature was 100.41%. The result suggests that the stock solution was stable at least for 24 hours at room temperature.

3.10. Recovery studies of curcumin in emulsion The experiment was conducted to determine the accuracy of the present method for the quantification of curcumin sample. The recovery of curcumin was calculated from the slope and the intercept of the calibration curve drawn in the concentration range of 2-200 µg/ml. The percentage recovery of curcumin ranged between 98.03 % to 98.24 % in emulsion sample.

4. Conclusion It can be concluded that a stability indicating HPLC-UV method for determination of curcumin in Curcuma longa extract and emulsion was successfully developed. The method was rapid, simple, precise, sensitive and specific for the analysis of curcumin in emulsion. Curcumin was stable against oxidation and heat, but susceptible to acid and alkali and very susceptible to UV degradation.

Acknowledgement The authors thanked Universiti Sains Malaysia for the graduate assistant scheme and PRGS grant (1001/PFARMASI/844074) support.

References

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Ahsan, H., Parveen, N., Khan, N. U., & Hadi, S. M. (1999). Pro-oxidant, anti-oxidant and cleavage activities on DNA of curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin. Chem.-Biol. Interact, 163, 161-175.

Antony, S., Elumalai, S., & Benny, M. (2011). Isolation, purification and identification of curcuminoids from turmeric (Curcuma longa) by column chromatography. Journal of Experimental Sciences, 2, 21-25.

Asai, A., & Miyazawa, T. (2000). Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma. J. Life Sciences,67, 2785-2793.

Bakhru, H. K. (1997). Herbs that heal: natural remedies for good health. (1sted.). New Delhi, India: Orient Paperbacks, (Chapter 1).

Cooray, N. F. (1988). Effect of maturity on some chemical constituents of Turmeric. Natl. J. Sci. Coun, Srilanka,16, 39-51.

Gugulothu, D. B., & Patravale, V. B. (2012). A New Stability-Indicating HPLC Method for Simultaneous Determination of Curcumin and Celecoxib at Single Wavelength: an Application to Nanoparticulate Formulation. Pharmaceut Anal Acta, 3, 1-6.

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Heath, D. D., Milagros, A. P., Dean, E. B., & Cherly, L. R. (2002). Curcumin in plasma and urine: quantification by HPLC. Chromatography J. B, 783, 287-295.

Jadhav, B. K., Mahadik, K. R., & Paradkar, A. R. (2007). Development and Validation of Improved Reversed Phase-HPLC Method for Simultaneous Determination of Curcumin, Demethoxycurcumin and Bis-Demethoxycurcumin. Chromatographia, 65, 483-488.

Kadi, A. A., Mohamed, M. S., Kassem, M. G., & Darwish, I. A. (2011).A validated stabilityindicating HPLC method for determination of varenicline in its bulk and tablets. Chemistry Central Journal, 5, 1-6.

Nadkarni, K. M. (1976). Indian material medica. (3rded.) Bombay: Popular Prakashan, (Part 1).

Ravindranath, V., & Chandrasekhara, N. (1981). In vitro studies on the intestinal absorption of curcumin in rats. Toxicology, 20, 251-257.

Saleemulla, K., Makhija, I. K., Khamar, D., & Rani, S. (2010). Development and standardization of turmeric cream by HPTLC. International Journal of Biomedical and Advance Research, 1, 109-116.

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Schieffer, G.W. (2002). Pressurized liquid extraction of curcuminoids and curcuminoid degradation products from turmeric (Curcuma longa) with subsequent HPLC assays. J. Liq. Chromatogr. Related Technol, 25, 3033-3044.

Smith, R. M., & Witowska, B. A. (1994). Comparison of Detectors for the determination of Curcumin in Turmeric by HPLC. Analyst, 109, 259-291.

Snyder, L. R., Kirkland, J. J., & Glajch, J. L. (1997). Practical HPLC Method Development. (2nded.) New York: John Wiley & Sons, (Chapter 1).

United States Pharmacopoeia 34 (2010). The National Formulary USP Convention, Inc. Rockville, Maryland.

Yadav, V. R., & Sarasija, S. (2009). A Sensitive Reversed Phase HPLC Method for the Determination of Curcumin. Pharmacognosy Magazine, 5, 71-74.

Zhang, D. (2009). Polyoxyethylene Sorbitan Fatty Acid Esters.In. C. R. Raymond, J. S. Paul, & E. Q. Marian (6th Ed.), Handbook of Pharmaceutical Excipients (pp. 549- 553). London, Washington DC: Pharmaceutical press, American Pharmacists Association.

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Fig. 1. Chemical structure of Curcuminoids.

Fig. 2a. Chromatogram of solvent (methanol). 18

Fig. 2b. Chromatogram of placebo.

Fig. 2c. Chromatogram of curcumin at 2 µg/mL. 19

Table 1 Mobile phase composition and observation. Composition

pH

(Acetonitrile:Methanol:Water)

Flow rate

Observation

(mL/min)

(v/v/v) 35:10:55

3.0

1.3

Good separation. Long retention time of 30 min.

35:10:55

3.0

1.5

Good separation. Retention time of 24 min.

40:15:45

3.0

1.3

Good separation. Retention time of 13 min.

40:15:45

3.0

1.5

Good separation. Retention time of 10 min.

40:20:40

3.0

1.3

Good separation. Retention time of 9 min.

40:20:40

3.0

1.5

Good separation. Retention time of 7 min.

45:20:35

3.0

1.3

Poor separation. Retention time of 4 – 5 min.

45:20:35

3.0

1.5

Poor separation

Table 2 Results of system suitability studies of quality control samples of curcumin. Mean ± SD, N = 6 20

Conc.

Theoretical plates Tailing

(µg/mL)

HETP

Capacity

factor

Resolution

factor

6

6838.37 ± 318.40

1.01 ± 0.01

36.62 ± 1.62

3.45 ± 0.19

1.79 ± 0.03

80

6755.66 ± 274.49

1.01 ± 0.01

37.05 ± 1.50

3.46 ± 0.20

1.77 ± 0.03

160

6715.00 ± 150.86

1.02 ± 0.01

37.24 ± 0.83

3.47 ± 0.21

1.76 ± 0.03

Table 3 Results of stress degradation studies of curcumin in emulsion. Mean ± SD, N = 3.

Parameters

Exposure time (hr) 0

24

Acid degradation (%)

99.45 ± 0.65

86.33 ± 0.64

Alkali degradation (%)

99.99 ± 0.85

82.21 ± 0.74

Oxidative degradation (%)

96.66 ± 0.08

98.02 ± 0.03

Heat degradation (%)

99.47 ± 0.60

101.06 ± 0.62

UV degradation (%)

98.95 ± 1.31

48.69 ± 0.82

21

Table 4 Results of intra-day and inter-day precision and accuracy. The results are presented as mean, N= 6 Intra-day

Inter-day

Conc.

Precision

Accuracy

Precision

Accuracy

(µg/mL)

(% CV)

(% Bias)

(% CV)

(% Bias)

2

0.86

1.99

2.0

-0.59

6

0.11

0.50

1.28

-0.20

80

0.31

0.48

1.47

-1.92

160

0.36

-1.09

1.30

-1.16

22

Highlights • • •

Determination of curcumin in Curcuma longa extract and emulsion using HPLC method. System suitability studies were performed. Stress degradation studies of curcumin in emulsion formulation.

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Stability indicating HPLC-UV method for detection of curcumin in Curcuma longa extract and emulsion formulation.

A stability-indicating HPLC-UV method for the determination of curcumin in Curcuma longa extract and emulsion was developed. The system suitability pa...
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