it exhibits broad bioactivity including inducing apoptosis of scar cells   and anti - inflammatory   and anticancer [ 3 , 4 ] properties , which have been used in the traditional chinese medicine for treatment of hypertrophic scar , inflammation , cancer , and other diseases without obvious toxicity or side effect in clinic .
several preparions , hydrogel , lotion , injection , and liposome , for example , have been reported in the literature .
we are further interested in the form of liquid crystalline nanoparticles ( cubosome ) , for cubosome consists of a curved bicontinuous lipid bilayer extending in three dimensions and separating two congruent networks of water channels [ 911 ] , which can enclose hydrophilic , amphiphilic , and hydrophobic substances ranging from low - molecular - weight drugs to proteins , peptides , amino acids , and nucleic acids .
compared to liposomes , cubosomes showed better storing stability at room temperature and could endure heat treatment [ 1315 ] .
cubosomes could exist at almost any dilution level in water and drug leakage was less concerned compared with liposome .
reported that cubosomes had a higher permeability coefficient ( 4.5-fold ) compared to eye drops when dexamethasone was used as a model drug .
therefore , we consider that the cubosome might represent a promising vehicle containing matrine for effective ocular drug delivery .
hplc , lc / ms / ms , and esi - qtof - ms / ms methods have been used to determine matrine in samples at present [ 6 , 17 , 18 ] .
the aim of this study is to establish and validate a simple , sensitive , and accurate hplc method to determine matrine combined in liquid crystalline nanoparticles .
glycerol monooleate ( dimodan mo / d kosher , material number 116703 ) was kindly provided by danisco cultor ( brabrand , denmark ) and used as received .
poloxamer 407 ( peo98pop67peo98 ) was a gift from basf ( ludwigshafen , germany ) .
milli - q - grade water purified through a millipore system ( elga labwater , sartorius , uk ) was used throughout this study .
pbs ( ph 6.8 ) was made according to the chinese pharmacopoeia ( 2010 ) .
liquid crystal nanoparticles were prepared through the fragmentation of glycerol monooleate / poloxamer 407 bulk cubic gels .
glycerol monooleate ( 3  g ) and poloxamer 407 ( 300  mg ) were first melted at 60c in a hot water bath until they were homogeneous , after which matrine was added to dissolve / 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 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 ( jy-96 iin , ningbo scientz biotechology co. , ltd , china ) at 200  w energy input using a pulse mode ( 9-second pulses interrupted by 18-second breaks ) under cooling in a 20c water bath .
the resulting milky coarse dispersion was homogenized using a high - pressure homogenizer ( avestin em - c3 , ottawa , canada ) at certain high pressures and cycles to obtain an opalescent dispersion of the cubic nanoparticles .
the final dispersion of liquid crystal nanoparticles was stored at room temperature for further studies .
the hplc analysis was carried out using a shimadzu system that is equipped with an lc-20at pump , spd-20a uv / vis detector connected to shimadzu spin chrome software .
the chromatographic assay was performed on a reversed - phase ods - bp c18 column ( 5  m , 4.6  mm  250  mm ) at ambient temperature 25c .
the mobile phase under isocratic mode was a mixture of methanol - pbs ( ph 6.8)-triethylamine ( 50  :  50  :  0.1% , v / v ) .
the mobile phase was degassed by an ultrasonic bath and filtered with 0.45  m membrane under vacuum .
all the calculations concerning the quantitative analysis were carried out by an external standard method based on peak areas . to prepare the stock solution , matrine ( 10  mg )
was accurately weighed into 50  ml volumetric flask , made up to volume with methanol , and then the volume was adjusted to 50  ml .
this solution was further diluted with methanol to yield solutions containing 100.0 , 50.0 , 25.0 , 12.5 , 6.3 , 3.1 , and 1.6  g / ml . the chromatogram peak area of each known concentration was calculated .
results from each analysis were subjected to regression analysis . 0.2  ml ( or  200.0  mg ) of the 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 injected ( 10  l ) into the hplc system three times under optimized chromatographic conditions .
the method was validated in terms of parameters of specificity , linearity , sensitivity , accuracy , precision , and reproducibility according to the international conference on harmonisation .
the specificity of the method was assessed by comparing chromatograms of matrine working solution , blank excipients sample without matrine , and equal concentrations samples of compound liquid crystalline nanoparticles made as the previous procedure .
the linearity of the method was studied by injecting seven known concentrations of the standard in the range of 1.6200  g / ml .
the sensitivity of the method was evaluated with limit of detection ( lod ) and limit of quantification ( loq ) .
lod and loq were established at a signal - to - noise ratio ( s / n ) of 3 and 10 , respectively .
the accuracy of the method was tested by comparing the percent analyte recovered by the optimum method at three concentration levels ( 80.0 , 100 , and 120.0  g / ml ) .
intraday precision was determined by injection of standard solutions of matrine at 3 concentration levels ( 50 , 25 , and 12.5  g / ml ) , on the same day .
the specificity was evaluated by analyzing blank excipients sample , matrine standard solution , and liquid crystalline nanoparticles samples .
the retention times of matrine at a flow rate of 1.0  ml / min was 16.3  min .
the calibration curves for matrine were found to be linear within the range of 1.6 to 200.0  g / ml . the regression equation was y = 10706x  2959 ( r = 1.0 ) , where y is peak area and x is the concentration ( g / ml ) of matrine standard solution .
the correlation coefficient indicated a good linear relationship between peak area and concentration over a wide range .
the lod ( signal / noise ratio of 3  :  1 ) was calculated as 1.3  10  g / ml and the loq ( signal / noise ratio of 10  :  1 ) was determined as 3.9  10  g / ml .
mean recovery for matrine at three concentration levels ( 80.0 , 100 , and 120.0  g / ml ) was found to be 102.1  1.9% ( rsd = 1.96% , n = 3 ) , 102.6  1.9% ( rsd = 2.94% , n = 3 ) , 100.5  2.1% ( rsd = 2.12% , n = 3 ) , respectively .
the mean concentration was 9.5  mg / ml ( rsd = 1.4% , n = 3 ) .
several mobile phase systems including methanol - water , ethanol - wate - kh2po4 , and acetonitrile - ethanol - h3po4 systems have been tested in this study .
however , the chromatogram of standard matrine might disappear or appear in a wide range with inaccurate calculation area ( see figure 2 ) .
matrine crystalline has 4 forms , namely ,  ,  ,  , and  matrine .
the incorrect mobile phase may change the matrine solution to nanocrystalline because of the solubility .
the chromatographic method was eventually carried out using an isocratic system with a mobile phase of methanol - pbs ( ph 6.8)-triethylamine ( 50  :  50  :  0.1% ) applied at a flow rate of 1  ml / min with detection wavelength at 220  nm . under these optimum mobile phase conditions ,
elution of analyte was completed in less than 20.0  min and retention time of matrine was 16.3  min .
the method was validated according to ich guidelines with the parameters of specificity , linearity , sensitivity , accuracy , precision , and reproducibility .
a simple , rapid , selective , and sensitive hplc method has been developed and validated for the determination of matrine when formulated in cubosome particles .
the present study is the first report on the matrine determination combined with particle dispersion system .
the method can be used for controlling the quality of the cubosome and helpful for further investigation .
 a reversed - phase high - performance liquid chromatographic method has been developed to quantitatively determine matrine in liquid crystal nanoparticles . 
 the chromatographic method is carried out 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 with spd-20a uv / vis detector and the detection wavelength was at 220  nm . 
 the linearity of matrine is in the range of 1.6 to 200.0  g / ml . the regression equation is y = 10706x  2959 ( r2 = 1.0 ) . 
 the average recovery is 101.7% ; rsd = 2.22%  ( n = 9 ) . 
 this method provides a simple and accurate strategy to determine matrine in liquid crystalline nanoparticle . 