sinomenine ( 7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17-methylmorphinan-6-one , chemical structure is shown in figure 1 ) is an alkaloid isolated from the stem and root of chinese medical plant sinomenium acutum rehd . because of its analgesic and anti - inflammatory effects , sinomenine has been utilized clinically to treat rheumatoid arthritis and neuralgia [ 1 , 2 ] . in order to prevent the negative gastrointestinal adverse reactions and low bioavailability of sinomenine oral preparations , for example , microemulsion based gel , liposome , and ethosomes   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 . cubosome could also enhance the drug deposition in the skin and showed excellent skin - targeted characteristics . 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 glycerol monooleate ( dimodan mo / d kosher , material number 116703 ) was kindly provided by danisco cultor ( brabrand , denmark ) . poloxamer 407 ( peo98pop67peo98 ) was a gift from basf ( ludwigshafen , germany ) . ( guangdong , china ) . methanol ( chromatographic grade ) was purchased from tianjin fuyu chemical co. , ltd . water purified through a flow water purification system ( qingdao , china ) was used throughout this study . pbs ( ph 6.8 ) was made according to the chinese pharmacopoeia ( 2010 ) . analyses were performed on uv-6000s spectrophotometer ( shanghai metash instruments co. , ltd . ) and lc-20at high performance liquid chromatograph with spd-20a uv - detector ( shimadzu ( suzhou ) instruments co. , ltd ) . firstly , glycerol monooleate ( 2.7  g ) and poloxamer 407 ( 0.3  g ) were first melted at 60c 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 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 . 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 . 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 . 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 25c . the mean z - average diameter and polydispersity indices ( pdi ) 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 ) . 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 , the response was high and the retention time was less than 10  min with good reproducibility . 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 ) . obviously , the peaks of the sinomenine were well separated and were not affected by the excipients . 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 a = 10835c + 1058  ( r = 1.0 ) , where a is peak area and c 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 . mean recovery for sinomenine at different concentration levels was found to be 100.3  2.2% ( rsd = 2.19% , n = 3 ) , 103.1  2.2% ( rsd = 2.2% , n = 3 ) , and 103.3  1.7% ( rsd = 1.6% , n the low values of rsd revealed the present method was accurate , reliable , and reproducible . the lod ( signal / noise ratio of 3  :  1 ) and loq ( signal / noise ratio of  10  :  1 ) were determined as 7.8  10  g / ml and 2.6  10  g / ml , respectively . the mean concentration was 8.0  mg / ml ( rsd = 1.9% , n = 3 ) . the stress testing showed the cubosome was physically stable under high temperature ( 60c ) 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 [ 911 ] . that also provided the base for the practicability and scientificalness of the formulations and process parameters . in this work , we used hplc method to separate and determine the sinomenine in cubosome nanoparticles . calculated by samples , the intra- and interassay precision ( rsd ) were less than 2% . the separation time was 8.53  min and the excipients did not interfere with the detection of sinomenine .
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 ; r2 = 1.0 ) and perfect recovery ( 102.2% ) . 
 the chromatograph technique was proper for quality control of sinomenine in cubosome nanoparticles .