melamine ( mel ) in animal foods and milk products had caused serious accidents , as its metabolism , melamine cyanurate , was easily crystallized in organs and was tending to cause kidney stone and damage [ 13 ] . therefore , establishing a powerful rapid sensitive analytical method for measuring low concentration of mel in kinds of milk products would benefit the routine assay of the related products . several analytical methods , such as gas chromatography - mass spectrometry ( gc - ms ) , high performance liquid chromatography ( hplc ) [ 57 ] gold nanoparticles , colloidal selenium immunoassay , and terahertz time - domain spectroscopy   had been reported .
however , many methods depended on complicated sample treatments . as in gc - ms methods , the sample derivation was strongly demanded as mel contains special label group ; as in hplc methods , liquid - liquid , liquid - solid extraction procedure was needed to cleanse the sample and perform better separation results . in some cases ,
in order to lower the limit of detection ( lod ) or obtain detailed information of the analytes , special detectors , such as mass spectrum ( ms ) , fluorescence detection , surface enhanced raman scattering , electrochemical approach , and online solid - phase extraction , were also used . however , some of these detectors were uncommon or had special demands .
capillary electrophoresis had advantages on separating complicated samples and had been applied to determine mel [ 1214 ] .
moreover , ce online stacking methods , which could easily perform enrichment of low - concentration analytes and achieved lower lods , had also been used for assay of mel in real samples [ 1519 ] .
jin et al .   proposed several stacking methods , such as including large - volume sample stacking - sweeping and selective exhaustive injection - sweeping ( sei - s , in either cation or anion mode ) , for determination of melamine and its derivatives in liquid milk .
lod of mel was around 0.01  ng / ml , however , it 's difficult to achieve better sensitivities for all derivatives in one mode .
combined cation selective exhaustive injection ( csei ) with sweeping - mekc method . in their study , the lod was 23.4  pg / ml for mel ; however , the recovery of mel was unsatisfied ( ca .
wu et al .   developed a mekc sweeping method for mel , face - centred cube central composite design was used for optimization of conditions , and the lod of mel could reach 5  ng / ml with satisfied recovery within 13  min . in our works , acetonitrile - salt stacking ( ass ) [ 2025 ] was used for detecting mel under cation mode in milk products .
the factors that influenced both stacking and separation , such as buffer ph , sample acetonitrile concentration , sample salt concentration , injection time , buffer concentration , stacking and separation voltage , were carefully investigated and optimized .
phosphoric acid , sodium dihydrogen phosphate , disodium hydrogen phosphate , sodium phosphate , sodium chloride , and acetonitrile ( hplc - grade agents ) were purchased from tianjin chemical reagent company ( tianjin , china ) .
water used throughout was purified by olst m6 ( ao lian co , xian , china ) with a conductivity of 18.2   cm and all the solutions and samples were made daily and filtered through 0.22  m filter .
experiments were performed on agilent 3d ce capillary electrophoresis system ( agilent , usa ) equipped with a diode array detector ( dad ) and a temperature controller ( 15~60  0.1c ) .
instrument control and data analysis were carried out by agilent chem station ( rev.a.09.03 , agilent , usa ) on a personal computer .
for ph measurements , a ph meter ( pb-10 , sartorius , germany ) calibrated with a precision of 0.01  ph unit was employed .
all the ce experiments were performed on a fused silica capillary ( yongnian photoconductive fibre factory , hebei , china ) .
the milk samples , obtained from local markets , were deproteinized with acetonitrile at the rate of 6  :  4 ( acetonitrile : milk , v / v ) or as specified .
then , the treated samples were centrifuged for 15  min at 14,200  rpm to eliminate the precipitates .
all supernatants were assayed after being added with 100  mmol / l nacl and filtration .
mmol / l ) were prepared quantitatively with water and stored at 4c until usage .
all working standard samples contained 8  mmol / l phosphate buffer and required concentration of sodium chloride and 60% acetonitrile ( or as specified ) and ~10  mol / l mel ( final concentration ) .
all the separations were performed on a fused - silica capillary ( 75  m i.d .
, effective length of 39  cm , and total length of 50  cm ) at 20c with a constant separation voltage of + 10.0  kv .
for the first usage , the capillary was activated with 1.0  mol / l naoh for 1 hour .
the stacking and separation buffer consisted of 80  mmol / l phosphate buffer ( ph 2.8 ) .
the samples were injected hydrodynamically at + 50  mbar for 40.0  s ( about 20% of the capillary efficient length ) or as specified . before each run , the capillary was rinsed with 0.1  mol / l naoh for 3  min at 1.4  10  mbar .
as the ass method stacked samples via transient pseudoisotachophoresis way and separated samples via capillary zone electrophoresis mode , several factors that affected stacking , such as sample matrix , voltage , injection time , and separation , such as buffer ph , were carefully studied . in general , mel was positively charged under the selected condition .
it was stacked based on its difference of migrating velocity between sample zone ( faster ) and outlet buffer zone ( slower ) .
buffer ph was one of most important factors that would influence separation and stacking . in this section , a ph range of 2.0~5.0
was firstly studied , as the pka values of mel were about 5.0 ; it could only be positively charged when buffer ph was lower than 5.0 .
there was an interferent compound in blank milk sample ( contained no mel , data not shown ) which had similar migration time as mel . regarding this ,
the optimization of ph was mainly based on the resolution ( rs ) between mel and interferent as well as their migration times .
it indicated that , at the lower ph ( < 2.5 ) , the electroosmotic flow ( eof ) was nearly zero , and mel was strongly and positively charged and migrated to the detecting window by itself ; when the ph became higher , the silanol started to dissociate and , as a result , the eof slightly increased and migration times dropped ; ph 3.5 had the slight longer migration time compared with ph 3.0 , which may be caused by the faster decreasement of effective positive charges of mel compared with the slower increasement of eof .
in addition , a peak order change was also observed in ph range of 2.0~2.5 , which was similar to our cze separation works ( data not shown ) .
finally , based on the curve of rs in figure 1 , ph 2.8 , which had the best rs value and relative shorter migration time , was chosen for further usage .
the influence of buffer concentration , ranged from 20.0  mmol / l to 140.0 
mmol / l , on stacking was investigated ( figure 2 ) . theoretically , to some extent ,
higher buffer concentration provided larger conductivity difference between buffer and sample zone , which in turn might perform better primary stacking effect . in our works
mmol / l ) , as the conductance of the buffer was similar to that of the sample zone , the difference of migrating velocity of the mel in buffer and in sample zone was not large enough to initiate stacking .
when buffer concentration was higher than 40.0  mmol / l , stacking phenomena occurred .
as the buffer concentration increased , the theoretical plate number ( n ) increased firstly followed by decreasing when the concentration was higher than 80.0 
mmol / l , which may heat due to the broaden effects of extra joule .
finally , a concentration of 80.0  mmol / l was selected for further studies .
the effect of the acetonitrile concentration in sample on stacking was studied and optimized as organic reagent acted as accelerator for speeding up the velocities of analytes .
it could be seen ( figure 3 ) , as the percent of the acetonitrile was lower than 50% , that the baseline around mel became unsatisfied ( arrow pointed in figure 3 ) for separation and quantification , although the rs value was higher enough between the interferent and mel ( rs > 3.5 ) . when the percentage was larger than 50% , baseline separation and stacking were both achieved . meanwhile , the rs decreased from 3.27 ( 60% ) to 1.53 ( 80% ) as the percentage of acetonitrile increased .
therefore , a percentage of 60% was considered as the better condition that provided less sample dilution and better rs .
nacl concentration was also an important factor that affected stacking , as the na acted as leading ions in ass procedure .
normally , almost all milk products contained na , and the [ na ] varied in larger range ( from 0.18% to 1.5% in mass / volume form or in another form , 3~25  mmol / l ) . in this section
, same real sample was added with different amount of nacl ( in range of 0~150  mmol / l ) and was tested under same condition .
the influences of nacl concentration on rs and migration times were shown in figure 2 .
as the nacl concentration increased , the migration time and rs increased correspondingly ( rs ranged from 1.7 to 3.0 , while migration times ranged from 14.2  min to 20.5  min ) until the concentration of nacl was higher than 100 
all these indicated that the method had a good tolerance on various concentrations of nacl , which made it more suitable for assaying kinds of milk products . finally , to keep the migration times similar , in the later assays ,
100  mmol / l nacl was added to the treated real milk samples before measurements . as the injection time
( presented as percent of the efficient length ) was closely related to sensitivity of the method , the maximum injection time was carefully selected in this section .  
figure 4 showed that migration times increased ( as a result of decreasing eof ) while the rs values decreased ( mainly caused by the decreasing efficient capillary length ) with increasing injection times ( similar to the results of gao 's work ) .
based on the rs value between mel and interferent , 20% of the efficient capillary length ( 50  mbar  40.0  s ) was considered as a best injection percent ( rs = 1.75 ) . in this section , stacking voltage was investigated in detail ( figure 4 ) .
it could be seen that n value appeared as a bell - shaped curve while the voltage increased from 2.5  kv to 25.0  kv .
experimental data showed that neither under lower nor higher voltage could mel be well stacked .
a voltage of + 10.0  kv ( ground at outlet ) was finally decided to be the best condition as it was relatively less time - consuming and of highest n value ( 82,000 ) .
as the status of the inner - wall silanol greatly affected reproducibility of the method , special rinsing step was added before each run .
it could be seen in figure 5 that an additional 0.1  mmol / l naoh rinsing step was crucial for maintaining reproducibility . without naoh rinsing step ( capillary
was only flushed with water and running buffer for 1.0  min and 3.0  min respectively , figure 5-(b ) ) , the separation and stacking were ruined compared with the first run ( figure 5-(a ) ) . when the naoh rinsing step was added before the next assay , better separation and stacking regained ( figure 5-(c ) ) . for the selection of the naoh rinsing time
, our data proved that the 0.1  mmol / l naoh rinsing time should be at least 3.0  min in order to achieve satisfied reproducibility . under the optimized condition
six replicates of the samples were used to test the inter- and intraday reproducibility of the method both for migration times and peak areas .
linearity range , using area as the function of the concentration , was obtained by adding different amount ( final concentration of mel ranged from 0.01  mol / l~50 
mol / l at ten concentration levels ) of mel directly into untreated real milk samples .
recovery was tested with adding known amount of mel to real samples under two final concentration levels ( 0.5  mol / l and 5.0  mol / l ) .
separation and stacking results of two bands of milk products ( yili pure milk and wangzai reconstituted milk ) were shown in figure 6 .
it could be seen that mel was well separated ( from the interferent ,  in figure 6 ) , stacked , and detected in both tested milk samples using the established method within 20  min .
for example , the blank yili sample ( without adding mel , figure 6-(a ) ) showed no mel peak even under stacking mode , while a low concentration of 0.5  mol / l mel could be easily detected ( figure 6-(c ) ) under stacking mode , with a ~20 folds enhancement of sensitivity compared with traditional cze mode ( figure 6-(b ) ) ; for the assay of wangzai reconstituted milk ( with adding mel at a final concentration of 8.0 
an ass method for separation and determination of melamine in milk products was proposed . under the optimum conditions , low concentration of melamine ( 0.03  mol / l ) in milk products
could be successfully determined , which can cope with the maximum tolerable limits set worldwide for melamine and related compounds .
the established method also provided simple sample treatment steps , good reproducibility , and a wider linearity which indicated that it is suitable for routine assay of melamine in milk products .
 melamine was measured in real milk products with capillary electrophoresis ( ce ) based on acetonitrile - salt stacking ( ass ) method . 
 real milk samples were deproteinized with acetonitrile at a final concentration of 60% ( v / v ) and then injected hydrodynamically at 50  mbar for 40.0  s. the optimized buffer contains 80.0  mmol / l ph 2.8 phosphates . 
 melamine could be detected within 20.0  min at + 10  kv with a low limit of detection ( lod ) of 0.03  
 mol / l . 
 satisfactory reproducibility ( inter- and intraday rsd% both for migration time and peak area was lower than 5.0% ) and a wide linearity range of 0.05  mol / l ~ 10.0  mol / l were achieved . 
 the proposed method was suitable for routine assay of mel in real milk samples that was subjected to a simple treatment step . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion