Development of an isocratic HPLC method for catechin quantification and its application to formulation studies

Fitoterapia 83 (2012) 1267–1274

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Development of an isocratic HPLC method for catechin quantification and its application to formulation studies Danhui Li, Nataly Martini, Zimei Wu, Jingyuan Wen ⁎ School of Pharmacy, Faculty of Medical and Health Sciences, The University of Auckland, Building 505, Grafton Campus, 85 Park Road, 1010 Auckland, New Zealand

a r t i c l e

i n f o

Article history: Received 30 April 2012 Accepted in revised form 13 June 2012 Available online 23 June 2012 Keywords: Catechin Isocratic HPLC Validation Pharmaceutical studies In vitro release

a b s t r a c t The aim of this study was to develop a simple, rapid and accurate isocratic HPLC analytical method to qualify and quantify five catechin derivatives, namely (+)-catechin (C), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG). To validate the analytical method, linearity, repeatability, intermediate precision, sensitivity, selectivity and recovery were investigated. The five catechin derivatives were completely separated by HPLC using a mobile phase containing 0.1% TFA in Milli-Q water (pH 2.0) mixed with methanol at the volume ratio of 75:25 at a flow rate of 0.8ml/min. The method was shown to be linear (r2 >0.99), repeatable with instrumental precisionb2.0 and intra-assay precisionb2.5 (%CV, percent coefficient of variation), precise with intra-day variationb1 and inter-day variationb2.5 (%CV, percent coefficient of variation) and sensitive (LODb1μg/mL and LOQb3μg/mL) over the calibration range for all five derivatives. Derivatives could be fully recovered in the presence of niosomal formulation (recovery rates>91%). Selectivity of the method was proven by the forced degradation studies, which showed that under acidic, basic, oxidation temperature and photolysis stresses, the parent drug can be separated from the degradation products by means of this analytical method. The described method was successfully applied in the in vitro release studies of catechin-loaded niosomes to manifest its utility in formulation characterization. Obtained results indicated that the drug release from niosomal formulations was a biphasic process and a diffusion mechanism regulated the permeation of catechin niosomes. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Flavonoids, being polyphenolic compounds constituting of two benzene rings joined by a linear three-carbon chain (represented as C6-C3-C6), are a class of secondary plant metabolites with a broad spectrum of pharmacological properties [1,2]. Up until now, over 4000 flavonoids have been identified and they are widely found in leaves, seeds, bark and flowers of plants [3]. As member of the flavonoid

⁎ Corresponding author at: School of Pharmacy, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel.: +64 9 923 2762; fax: +64 9 367 7192. E-mail addresses: [email protected] (D. Li), [email protected] (N. Martini), [email protected] (Z. Wu), [email protected] (J. Wen). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.06.006

family, catechins are polyphenolic compounds naturally presenting in tea, grape seeds and other plants [4]. Five primary catechin derivatives, (+)-catechin (C), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG), as listed in Fig. 1, are often investigated due to their health-promoting properties including antimutagenicity [5], anti-obesity [6], antibacterial [7], lipid lowering [8] and bowel modulating action [9]. Most of these functions have been attributed to the antioxidation and free radical scavenging activities of catechins [10,11]. Catechins are described to be potent antioxidants and are able to scavenge different reactive oxygen radicals by virtue of reducing properties of the multiple hydroxyl group attached to the aromatic rings [12]. As a result, catechin derivatives are considered to be promising drug candidates in the pharmaceutical and nutritional fields. In the area of pharmaceutical science, using formulation

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Fig. 1. Chemical structures of five catechin derivatives.

strategy to enhance the effectiveness of catechin derivatives is recognized as a reasonable approach due to the fact that catechins are poorly absorbed when orally administered at a dietary level and is readily degraded [13]. There is extensive literature reporting catechins in various formulations. Batchelder et al. have developed catechin transdermal patch [14]; liposomal system have been applied to enhance C and EGCG skin permeation [15] and Lee et al. have incorporated catechin into microparticles for a sustained release profile [16]. To conduct pharmaceutical formulation research of catechin derivatives, such as to carry out characterization and pharmacokinetic studies, an accurate, effective and reliable analytical method is required. High performance liquid chromatography (HPLC) is one of the most popular analytical techniques used to determine catechin derivatives [17,18]. However, most of these methods employ gradient elution which causes base line shifting and is time-consuming; in addition, a more sensitive analytical method is of necessity in the in vitro and in vivo study of formulation characterization. Therefore there is a demand for a faster, easier and more precise quantitative HPLC method to determine catechin derivatives from variable formulations. In this study, a novel isocratic HPLC method with UV detection was developed and validated in light of International Conference on Harmonisation (ICH) guidelines. Niosomes are nonionic surfactant vesicles that are formed from self-assembly of nonionic surfactant in aqueous surrounding resulting in a closed bilayer structure. Evolved from liposomes, niosomes recognized as promising drug carriers as they possess greater stability and lack of many disadvantages associate with liposomes including hydration, oxidation and aggregation [19]. Since this isocratic

HPLC method was designed to be used in the formulation characterization and pharmacological studies, the application of the method in the in vitro release of catechin-loaded noisome was conducted to manifest its utility and effectiveness. 2. Experimental methods 2.1. Chemicals and reagents Catechin derivatives, sorbitan monostearate (Span 60), cholesterol (CH) and dihexadecyl phosphate (DCP) were purchased from Sigma-Aldrich (Sigma, USA). Methanol and acetonitrile (ACN) of analytical reagent grade were purchased from Merck (Merck, Germany). Trifluoroacetic acid (TFA) was purchased from Fluka (Fluka, Germany). Milli-Q water was available from the Pharmaceutics Laboratory at University of Auckland (Auckland, New Zealand). Other chemicals were of analytical grade. 2.2. Chromatographic conditions A HP 1100 series liquid chromatographic system comprising of vacuum degasser, quaternary pump, autosampler, thermastatted column compartment and diode array detector were used, with data acquisition by Chemstation software (Agilent Corporation, Germany). Chromatography was performed on a Jupiter C18 column (250×4.6 mm, 5 μm; Phenomenex, USA) fitted with a C18 guard column (10×3.0mm). The drug elution was performed at 25°C at a flow rate of 0.8mL/min and detected at the UV wavelength of 280nm. All samples were analyzed by HPLC using an injection volume of 20μL.

Fig. 2. HPLC profile of five catechin derivatives. C-(+)-catechin, EGC-(−)-epigallocatechin, ECG-(epicatechin gallate), EC-(−)-epicatechin, and EGCG-(−)-epigallocatechin gallate. (RT: Retention Time).

D. Li et al. / Fitoterapia 83 (2012) 1267–1274

2.3. Preparation of stock solution A stock solution of catechin derivatives was prepared by dissolving the known amount of C, EC, ECG, EGC, and EGCG in the mixture of 20% methanol in water (v/v). As a result, the stock solution contained five derivatives at difference concentrations, namely C: 301μg/mL, EC: 306μg/mL, EGC: 285μg/mL, ECG: 300μg/mL and EGCG: 290μg/mL. Different concentrations of standard solution were prepared by dilution of the stock using the same solvent. Samples were stored at 4°C and protected from light before use. 2.4. Preparation of blank, C- and EGCG-loaded niosomes Niosomes were prepared by the method from Azmin with certain modifications [20–22]. Briefly, Span 60 and cholesterol at a molar ratio of 4:1 with a certain amount of DCP (1.1 mg) were dissolved in a mixture of chloroform and methanol (4:1, v/v) in a 50 mL round bottom flask. The resulting solution was then rotary evaporated to form a thin film. The dried lipid film was purged with a stream of nitrogen for 5 min in order to get rid of residual traces of the organic solvent. For the drug loaded niosomes, the dry film was then hydrated with 10 mL of 15% ethanol in water containing C (2 mg) and EGCG (1 mg) respectively using a swirl-evaporator at 100 rpm, 60 °C for 2 h, while 10 mL of 15% ethanol water without drug was used as hydration solution for the blank niosomes. The resultant niosomal suspension was set aside for at least 2 h at room temperature to allow for the vesicle's membrane to anneal before being kept in a fridge. 2.5. Mobile phase optimization To achieve satisfactory resolution of the five derivatives and their degradation products different mobile phases such as the combination of methanol, ACN and Milli-Q water at volume ratio of 55:40:5; water and methanol at volume ratio of 80:20, 60:40 and 75:25 in the absence or the presence of TFA were investigated in this study. The mobile phases were filtered through a 0.45 μm membrane and degassed prior to use. Satisfactory retention times for all the eluting components were set within 20 min. It generally suffices if the peak resolution (Rs) is greater than 2 and the run time is acceptably short [23], where Rs is defined as: RS ¼ 2ðRta −Rtb Þ=ðWa þ Wb Þ

ð1Þ

where Wa and Wb are the widths of the two peaks at the baseline, obtained from the chromatograms; whereas Rta and Rtb are the retention times. 2.6. Method validation The HPLC assay of the five catechin derivatives was validated in terms of linearity, repeatability, intermediate precision, sensitivity, selectivity and recovery. Linearity: Eight standard solutions were prepared by dilution of the stock solution with the same solvent. The calibration curve was constructed by plotting the peak area

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against each catechin concentration. The slope, y-intercept and linearity of the curve were determined by linear regression. Repeatability [23]: (1) Instrumental precision (system precision) was determined by analyzing three different concentrations of each catechin in five replicate injections. (2) Intra-assay precision (method precision) was determined by analysis of five independent standard solutions of three different concentrations for each catechin. Intermediate precision [24]: Intra-day repeatability was obtained by analyzing three concentrations of each catechin at four different times in one day. Inter-day repeatability was determined by analyzing the three concentrations of each catechin four times on three consecutive days. All injections were carried out in triplicate. The precision of the assays was calculated in terms of percentage of relative standard deviation (% R.S.D) of determinations. Sensitivity [24]: Sensitivity of the HPLC method was determined by the estimation of limit of detection (LOD) and limit of quantification (LOQ) based on the standard deviation of the response (σ) and the slope (S) of the standard curve using the following equations: LOD ¼ 3:3σ=S and LOQ ¼ 10σ=S:

ð2Þ

Recovery: Blank niosomes prepared by the above method were spiked with a mixture of five catechins. The recoveries of each catechin from niosome at three concentrations levels were determined by measuring the percentages of detected concentrations over added concentrations. Selectivity: A forced degradation study was performed to generate degradation products that were used to demonstrate selectivity of the method. The selectivity was assessed by subjecting the C and EGCG solutions to five different stress conditions. Since some of the stress conditions described by ICH guidelines were too extreme and resulted in complete degradation within few minutes, changes of strength of some stresses were made to achieve 10 to 30% degradation in sufficient time. The adjusted stress conditions included acid hydrolysis (0.1 M HCl), base hydrolysis (0.5 mM NaOH), oxidation (0.01% v/v H2O2), photolysis (artificial daylight illumination of 10,000 lx) at room temperature and heat (70 °C). Samples were analyzed in comparison to the initial samples after 10%–30% degradation had been achieved. The peak purity was assessed by examining the similarity of the UV spectra obtained at the five points by the HPLC-PDA detector. If the degradation products coelute with the drug peak, the five UV spectra are different. The peak purity analysis was conducted throughout the selectivity test. 2.7. In vitro release studies from niosomes The developed HPLC method was applied in in vitro release studies after validation. In vitro release studies for up to 24h were carried out using Franz diffusion cells to obtain the release profile of C- and EGCG-niosomes. The C- and EGCG-niosomes were prepared by the aforementioned method. Prior to the studies, the noisome samples were freeze-dried overnight to prevent degradation with manitol as lyoprotectant. The freeze-dried samples were reconstituted with phosphate buffer solution (PBS) (pH 5.5, 2.5 mL) and the niosomal dispersion

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Table 1 Calibration curves of catechins (n=3). Catechins

Concentration range (μg/mL)

Equationa

Correlation coefficient (r2)

C EC EGC ECG EGCG

0.08–150.1 0.25–153.0 2.9–142.5 1.0–150.0 0.15–145.0

y=15.45x+4.85 y=13.74x+1.77 y=4.01x+1.92 y=38.42x−0.10 y=27.37x−20.25

0.99 0.99 0.99 0.99 0.99

a

y is the peak area (mAU∗min) and x is the concentration of catechin (μg/mL).

was added to the upper donor chambers of the Franz diffusion cells, respectively. A 12mL aliquot of PBS (pH 5.5) was filled in the downer receptor chamber, of which the temperature was maintained at 37°C to mimic the condition of normal human skin. The upper and downer chambers were divided by a single layer of synthetic cellulose membranes (12000–14000wt cut-off, pre-soaked in release medium overnight). At time intervals (15min, 30 min, 1, 2, 3, 6, 9, 12, and 24 h), the aliquot of 500 μL samples was taken from the receptor chambers and replaced with the buffer. The samples were then analyzed using the validated HPLC method. The release kinetics of C and EGCG from niosomal formulations was investigated putting data through the Higuchi model [25]. The Higuchi model is described by the following equation: Mt =M∞ ¼ Kt

1=2

ð3Þ

where Mt/M∞ denotes the fraction of drug released at time t and K is the Higuchi release rate constant. The release data were fitted to the model using linear regression analysis by MS Excel 2007 version 3.0.

good linearity over the range tested with the correlation coefficients r 2 all above 0.99. Repeatability: Instrumental precision was measured by repetitive injection of the same homogeneous sample of three concentrations. The relative standard deviation (R.S.D.) was determined to assess instrumental precision. Intra-assay precision was determined by preparing five independent standard solutions of three different concentrations and measuring the R.S.D. values (Table 2). The R.S.D. values of both instrumental precision and intra-assay precision of five catechins were below 2.5%, indicating the HPLC analytical method for catechin was precise. Intermediate precision: Intermediate precision of the method was determined by assessing intra-day and inter-day repeatability. The results of intra-day and inter-day repeatability studies are listed in Table 3. The R.S.D. values in all tested groups were approximately or below 3%, which is acceptable. Sensitivity: The sensitivity of HPLC method was determined by LOD and LOQ. LOD is the lowest concentration of the analyte the method can detect and LOQ is the lowest concentration that can be quantified accurately by the method. The LOD and LOQ of catechin derivatives are shown in Table 4, indicating that the method was sensitive to detect all five catechins. The rank order for LOD of five derivatives was CbECGbECbEGCGbEGC and the same order was applied for LOQ. Compared to other reported HPLC methods, the sensitivity of the described method was considerably improved. Recovery: The recovery measures the closeness between the theoretically added amount and the experimental value and was performed by spiking the empty niosomes with a known amount of catechin derivatives. The recovery of all catechin derivatives at different concentrations was above 90% with R.S.D. values below 3% (Table 5). The results indicate that all five catechin derivatives can be fully recovered in the presence of niosomal components.

3. Results and discussion Retention time and separation of catechin derivatives highly depended on the ratio of organic content and pH of the mobile phase. Acceptable resolution of catechins was not obtained with the mixture of water, methanol and acetonitrile at volume ratio of 55:40:5 or water:methanol at 80:20 or 60:40, nor with the mobile phase of water:methanol at the volume ratio of 75:25. Nevertheless addition of 0.1% TFA into the mobile phase resulted in satisfactory peak separation. The optimum separation of five catechins was achieved by HPLC using 0.1% TFA in Milli-Q water (pH 2.0) mixed with methanol at the volume ratio of 75:25. The retention time of EGC, C, EGCG, EC and ECG was 6.19, 6.75, 8.58, 10.51, 18.25 min, respectively as shown in Fig. 2. All the Rs were above 2.

Table 2 Instrumental precision and intra-assay precision of the method (means±SD). Concentration (μg/mL)

C

EC

EGC

3.1. Validation ECG

Linearity: The calibration curves were obtained by plotting peak areas against concentrations. The results of regression analysis are shown in Table 1. Equations in the table represent the relationship between response areas and concentrations of five catechins, where y stands for peak area and x for concentration. All catechin derivatives exhibited

EGCG

a b

10.0 50.0 100.0 10.2 51.0 102.0 9.5 47.5 95.0 10.0 50.0 100.0 9.7 48.3 96.7

Intra-assay precisionb

Instrumental precisiona Peak area (mAU * s)

R.S.D. (%)

Peak area (mAU * s)

R.S.D. (%)

166.9±1.1 796.0±9.0 1569.3±1.6 146.9±0.3 713.4±3.4 1439.3±1.4 41.0±0.7 193.4±2.2 406.7±0.1 396.6±0.5 1928.9±1.2 3927.9±2.8 244.8±1.3 1265.7±9.3 2813.9±2.6

0.7 1.1 0.1 0.2 0.5 0.1 1.8 1.1 0.0 0.1 0.1 0.1 0.5 0.7 0.1

160.9±2.8 783.4±10.9 1555.2±20.8 142.5±2.6 705.0±4.3 1416.8±15.7 40.9±0.7 189.4±4.4 392.4±6.8 387.2±6.5 1918.6±18.6 3858.9±43.7 249.1±4.3 1262.3±9.4 1416.4±15.0

1.8 1.4 1.3 1.8 0.6 1.1 1.8 2.3 1.7 1.7 1.0 1.1 1.7 0.8 1.1

Five replicate injections of each sample. Three injections of each sample.

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Table 3 Intra-day and inter-day repeatability of the assay (means±SD, n=3). Concentration (μg/mL)

C

EC

EGC

ECG

EGCG

Intra-day repeatability

10.0 50.0 100.0 10.2 51.0 102.0 9.5 47.5 95.0 10.0 50.0 100.0 9.7 48.3 96.7

Inter-day repeatability

Peak area (mAU * s)

R.S.D. (%)

Peak area (mAU * s)

R.S.D. (%)

155.6±0.4 772.9±2.3 1526.2±2.3 140.9±0.4 709.9±2.3 1401.3±2.1 30.2±0.0 194.5±0.7 364.2±0.3 373.1±1.0 1949.2±9.3 3862.4±10.8 237.5±1.9 1439.8±5.3 2724.7±1.3

0.3 0.3 0.2 0.3 0.3 0.2 0.1 0.4 0.1 0.3 0.5 0.3 0.8 0.4 0.1

153.6±1.7 763.0±9.8 1512.0±12.3 139.4±1.3 701.0±8.9 1390.3±10.3 31.3±0.9 196.3±2.1 381.2±8.9 373.1±0.7 1928.5±21.7 3842.2±21.0 256.1±6.2 1436.5±4.4 2787.0±64.0

1.1 1.3 0.8 0.9 1.3 0.7 3.0 1.1 2.3 0.2 1.1 0.6 2.4 0.3 2.3

Selectivity: Forced degradation studies were performed to demonstrate the selectivity of the developed method. The chromatograms of C and EGCG resolved after exposure to base hydrolysis, acid hydrolysis, oxidation, temperature and light illumination until 10%–30% degradation has been found are shown in Figs. 3 and 4. The results demonstrate that the chemical stability of both C and EGCG was pH-dependent, which supports the results of Y. L. Su [26] and K. Dvorakova [27]. The two catechin derivatives underwent extensive degradation when subjected to the basic condition and around 30% degradation of either derivative was found after 1 h's exposure while they were quite stable under acidic condition where only 10% of the drug was decomposed respectively after 7 days. Oxidation and temperature both caused extensive degradation. Under oxidation stress, for both C and EGCG, 30% degradation was found by day 3. Under 70 °C condition, around 30% of C and EGCG had degraded by day 3. The stress of light led to less degradation in comparison to other four stresses and only 10% degradation was detected by day 9. In both C and EGCG degradation studies, the peaks of degradation products were separated from the peak of the parent drug and the peak purity assessment indicated that UV spectra of the drug were similar across the peak. 3.2. In vitro release profiles from niosomes In vitro release testing is a regulatory requirement of formulation characterization and quality control in pharmaceutical research to assure that the dosage form will release the active ingredients at an ideal rate and extent. For the Table 4 LOD and LOQ of the method (means±SD, n=3).

purpose of obtaining release profile and studying release kinetics, a sensitive and accurate analytical method is of necessity in sample testing and HPLC method is often used due to its effectiveness in the determination of sample of small volume. Thus following the method validation, the developed HPLC method was applied in the analysis of the release samples. Release performances of C and EGCG from prepared niosomes were studied using Franz diffusion cells for up to 24 h with drug aqueous solution as the control group. The release profiles of C and EGCG from niosomes and from aqueous solution are shown in Fig. 5. From the release profiles, it appears that the diffusion of free drug from solution was fast and nearly complete (>90%) within 2 h. In comparison to the free drug solution, the efflux of C and EGCG from the niosomal formulation was a biphasic process containing an initial fast phase followed by a sustained release phase. About 20 and 40% of C and EGCG were released from niosomes over a period of 3 h respectively. In both cases, the initial rapid release phase was followed by a prolonged release of up to more than 20 h and around 62 and 92% cumulative release of C and EGCG were observed

Table 5 Recoveries of five catechin derivatives from niosomes (means±SD, n=3). Concentration spiked (μg/mL)

Concentration detected (μg/mL)

Recovery (%)

R.S.D.(%)

EGC

17.3±0.1 46.8±0.6 113.4±0.1 19.3±0.5 48.4±1.2 115.5±0.5 20.6±0.3 52.8±0.9 100.8±0.1 19.8±0.2 51.1±0.6 136.2±0.5 19.2±0.1 50.5±0.6 142.7±0.3

91.1 98.5 94.5 96.5 96.7 96.3 106.7 109.3 94.5 97.0 100.0 96.4 96.0 101.0 93.1

0.7 1.3 0.1 2.5 2.4 0.4 1.7 1.7 0.1 0.8 1.1 0.4 0.7 1.2 0.2

C

EGCG

Catechins

LOD (μg/mL)

LOQ (μg/mL)

C EC EGC EGCG ECG

0.02 0.08 0.96 0.33 0.05

0.06 0.23 2.90 0.98 0.16

EC

ECG

19.0 47.5 120.4 20.0 50.0 120.0 19.3 48.33 106.7 20.4 51.0 141.3 20.0 50.0 153.3

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Fig. 3. Representative HPLC chromatograms of C under different stress conditions: sample degraded in 0.1M HCL (A), sample degraded in 0.5mM NaOH (B), sample degraded 0.01% v/v H2O2 (C), sample degraded at 70°C (D), and sample subjected to photolytic degradation (E). Blank aqueous C solution (1mg/mL) (inset) as the control.

respectively at the end of 24 h. The similar release pattern has been observed for insulin delivery and also for caffeine from niosomal formulation [28,29]. The initial rapid release phase may be attributed by the permeation and desorption of free drug from the surface of niosomes and the sustained release

phase relates to the diffusion of the drug through bilayers, which may lead to high retention of the drug. This kind of release pattern is of interest for dermal application in the view that the initial fast release improves drug penetration, while the further sustained release provides the drug over a prolonged

Fig. 4. Representative HPLC chromatograms of EGCG under different stress conditions: sample degraded in 0.1M HCL (A), sample degraded in 0.5mM NaOH (B), sample degraded 0.01% v/v H2O2 (C), sample degraded at 70°C (D), and sample subjected to photolytic degradation (E). Blank aqueous EGCG solution (1mg/mL) (inset) as the control.

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A Cumulative C released (%)

110.0 100.0 90.0 80.0 70.0 60.0 50.0

C niosome

40.0 30.0 C aqueous solution

20.0 10.0 0.0 0

200

400

600

800

1000

1200

1400

Time (min)

Cumulative EGCG released (%)

B 110 100 90 80 70 60 50 40 30 20 10 0

EGCG niosome

EGCG aqueous solution

0

200

400

600

800

1000

1200

1400

Time (min) Fig. 5. Release profiles of a) C and b) EGCG through cellulose acetate membrane after application of C-loaded niosomes in comparison with the free drug solutions (mean±SD, n=3).

period, which may maintain a therapeutic concentration in the skin without the need for frequent re-application [30]. The release curve was fitted into Higuchi model to find the release mechanism and the release profiles of both C- and EGCG-niosomes fitted well with the Higuchi equation with regression coefficient (r 2) of 0.990 and 0.993 respectively, indicating that both niosomal formulations showed diffusion mechanism for the release of drug. 4. Conclusion In this study an isocratic HPLC method has been developed and validated for the simultaneous quantitative determination of five catechin derivatives. It has the merits of being time-saving and highly sensitive compared to the gradient HPLC methods. The present method has been shown to be efficient in separating and qualifying five catechin derivatives. It gives satisfactory resolution of all five catechin derivatives and is linear, precise, accurate and reasonably sensitive. The method was successfully applied in the in vitro release studies of C- and EGCG-loaded niosomes providing insights on the release mechanism from the carrier system. Therefore, the described method can be regarded as a reliable, rapid and simple

analytical method to qualify and quantify catechin derivatives and catechin formulations.

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