Determination of Sinomenine in Cubosome Nanoparticles by HPLC Technique

We applied HPLC technique to quantitatively analyze sinomenine in cubosome nanoparticles. The chromatographic method was performed by using an isocratic system. The mobile phase was composed of methanol-PBS(pH6.8)-triethylamine (50 : 50 : 0.1%) with a flow rate of 1 mL/min; the detection wavelength was at 265 nm. Sinomenine can be successfully separated with good linearity (the regression equation is A = 10835C + 1058; R 2 = 1.0) and perfect recovery (102.2%). The chromatograph technique was proper for quality control of sinomenine in cubosome nanoparticles.

In order to prevent the negative gastrointestinal adverse reactions and low bioavailability of sinomenine oral preparations, researchers have been trying to use transdermal drug delivery system to overcome these shortcomings. For example, microemulsion based gel [3], liposome [4], and ethosomes [5] had been documented in the literature. We concerned the development of sinomenine preparations for a long time and found cubosome, a novel kind of transdermal drug delivery system, could be very well suitable to the sinomenine. Cubosome and liposome both had the bilipids membrane, but cubosome showed better storing stability compared to liposomes [6]. Cubosome could also enhance the drug deposition in the skin and showed excellent skintargeted characteristics [7]. Our previous experiment has proved cubosome could enhance sinomenine skin permeation (7-fold compared to gel) and a patent was applied in China (ZL 201310087778.6). Here we describe the determination of sinomenine in cubosome nanoparticles by HPLC technique.  and LC-20AT High Performance Liquid Chromatograph with SPD-20A UV-detector (Shimadzu (Suzhou) Instruments Co., Ltd). [7,8]. Firstly, glycerol monooleate (2.7 g) and poloxamer 407 (0.3 g) were first melted at 60 ∘ C in a hot water bath until they were homogeneous, after which sinomenine (0.3 g) was added to blend under continuous stirring. Water (6.7 mL) was then added gradually and the mixture was vortex-mixed to achieve a homogeneous state. After equilibration for at least 48 hours at room temperature, the cubic phase gel was formed. By adding 20 mL of water, the cubic gel was disrupted by mechanical stirring. Subsequently, the crude dispersion was fragmented for 10 min by intermittent probe sonication. The final dispersion of cubic nanoparticles was stored at room temperature for later studies.

Chromatographic Conditions.
The analytical column was an Hichrom C18 column (4.6 × 250 mm, 5 m, inner diameter, 5 m). The mobile phase was methanol-PBS(pH6.8)triethylamine (50 : 50 : 0.1%) with a flow rate of 1 mL/min, and the detection wavelength was at 265 nm. The sample injection volume was 10 L. The column temperature was maintained at 25 ∘ C.

Preparation of Standard Solutions.
Sinomenine stock solution was prepared in 50% methanol solution and sonicated for 5 min to obtain stock solution concentrations of 320 g/mL. This solution was further diluted with 50% methanol solution to yield solutions containing 160.0, 80.0, 40.0, 20.0, 10.0, 5.0, 2.5, and 1.25 g/mL.
2.6. Sample Preparation Procedure. 0.2 mL (approximately 200 mg) of cubosome nanoparticles was accurately transferred into a 10 mL volumetric flask, dissolved, and made up to volume with methanol. Then, the sample solutions were filtered using a 0.45 m filter membrane and 10 L aliquot was injected into the HPLC system.

Preparation of Sinomenine
Cubosome. The milky coarse dispersions of phytantriol-based sinomenine cubosomes  were obtained (Figure 2). The cubosomal particle size was determined by photon correlation spectroscopy using a ZetasizerNano ZS90 (Malvern Instruments Malvern, UK) at 25 ∘ C. The mean -average diameter and polydispersity indices (PDI) were obtained by cumulative analysis using the MALVERN software. The mean diameter of different cubosome dispersions was in an approximate range of 177.8 nm with the polydispersity indices (PDI) value of 0.158 (Figure 3).

Chromatographic
Conditions. UV spectrum of sinomenine showed maximum absorbance at 265 nm wavelength ( Figure 4). Therefore, 265 nm was selected as the detection wavelength. In selected spectrometry conditions, sinomenine chromatography's symmetry was with higher degrees. The  response was high and the retention time was less than 10 min with good reproducibility.

Specificity.
Blank cubosome sample was prepared according to Section 2.3 (no sinomenine), and the chromatogram was shown in Figure 5(a). A certain concentration of sinomenine standard solution and sinomenine cubosome sample solution were injected into the HPLC system with the same operation, and the chromatogram was shown in Figures 5(b) and 5(c). The retention time was 8.53 min.
Obviously, the peaks of the sinomenine were well separated and were not affected by the excipients.

Linearity.
The calibration curves for sinomenine were found to be linear within the range of 1.25 to 320.0 g/mL. The regression equation was = 10835 + 1058 where is peak area and is the concentration ( g/mL) of sinomenine standard solution. The correlation coefficient indicated a good linear relationship between peak area and concentration over a wide range.

Precision.
Precision was demonstrated at 3 concentration levels in intraday and interday studies. Intraday precision was determined by injection of sinomenine standard solutions on the same day. Interday precision was checked by repeating the studies on two different days. The intraday and the interday precisions of sinomenine are summarized in Table 1. The RSD is found to be acceptable (RSD < 2%). 103.3 ± 1.7% (RSD = 1.6%, = 3), respectively. The low values of RSD revealed the present method was accurate, reliable, and reproducible.
3.9. The Stability Experiment. The stability experiment was aimed at testing the effects during storage. The stress testing showed the cubosome was physically stable under high temperature (60 ∘ C) within 10 days ( Figure 6). It was similar to the literatures in which cubosomes showed better storing stability at room temperature and could endure heat treatment compared to liposome [9][10][11]. That also provided the base for the practicability and scientificalness of the formulations and process parameters.

Conclusions
In this work, we used HPLC method to separate and determine the sinomenine in cubosome nanoparticles. The linear range of sinomenine concentration was 1.25 to 320.0 g/mL. Calculated by samples, the intra-and interassay precision (RSD) were less than 2%. The average recovery rate was 102.3%. Sinomenine peaks were well resolved and free from tailing (<1.5). This method was sensitive with good precision and accuracy. The separation time was 8.53 min and the excipients did not interfere with the detection of sinomenine. This method was applied to sinomenine cubosome researches.