a dietary supplement is a product , which contains a  dietary ingredient  intended to supplement the diet . in general , the natural dietary supplements for male sexual potency consist of different herbal extracts such as ginseng root , lychee seed , barbary wolfberry fruit , longan aril , and aweto .
these dietary supplements could improve male sexual potency without causing danger , even when overdose occurred .
recently , chemically synthetic phosphodiesterase-5 ( pde-5 ) inhibitors have been used to improve erectile dysfunction ( ed ) , one of the most popularly used is vardenafil hydrochloride   ( levitra ) , 2-[2-ethoxy-5-(4-ethyl - piperazine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3h - imidazo[5,1-f]-triazin-4-one monohydrochloride , trihydrate ( figure 1 ) .
vardenafil , approved by the united states food and drug administration ( fda ) in 2003 , is a new oral , potent , highly selective phosphodiesterase-5 ( pde5 ) inhibitor marketed for the improvement of ed in man .
studies have shown that doses of vardenafil ( 1040  mg ) are rapidly absorbed following oral administration , reaching maximum plasma concentration in some men within 15 minutes .
nevertheless , it has been proved that vardenafil poses a serious health risk , such as headache , low blood pressure , flushing , dyspepsia , and nasal congestion or rhinitis .
thus , vardenafil could be legally obtained only with a doctor 's prescription . however , some illegal dealers add vardenafil into dietary supplement products for the sake of profit . for purposes of quality control and health safety , establishing sensitive and selective methods to detect illegal vardenafil addition in dietary supplements are necessary . as shown in the literatures ,
several methods have been reported for vardenafil determination in dietary supplements such as lc - ms and gc - ms method , lc - esi - ms method [ 1 , 4 ] , lc - esi - ms / ms method , mrm - esi - ms - ms method , and fticrms method .
ms provides sensitive and reliable results , but it also implies highly sophisticated apparatus , careful control of conditions and relatively high operation cost .
thus , simpler , automated and less expensive methods are still necessary . taking the advantages of perfect versatility and easy fluidic manipulation , flow injection ( fi )
the integration of fi and high - performance liquid chromatography ( hplc ) has also showed the power for the complex samples analysis .
cl - based technique as a means of detection for hplc aroused the analysts interest in recent years [ 12 , 13 ] , although cl detection is not as universal as fluorescence detection or ultraviolet visible detection , it is more selective for fewer interfering species observed in the chromatogram   and sensitive for a large cl signal - to - noise ratio ( s / n ) and wide linear working range .
the combination of hplc high resolution , cl sensitive detection and fi manipulation would be one of the plausible techniques for a sensitive , precise , and facile determination of trace levels of compounds in the complex mixture of substances .
recent studies witness rapid progress in hplc - cl method , which is widely used to perform diverse qualitative and quantitative analytical problems [ 1518 ] .
our experiments indicated that in alkaline medium the cl of luminal - k3fe(cn)6 could be greatly enhanced by the presence of trace vardenafil .
based on above phenomenon , an automated hplc - cl method for vardenafil determination in dietary supplements was developed .
the proposed method exhibited the advantages of automated operation , high analytical throughput , simple instrumentation , wide linear range , and reagent - saving and was applied to oral liquid , wine , and capsule samples analysis .
all the other reagents were of analytical reagent grade unless specified otherwise , and deionized and doubly distilled water was used throughout .
vardenafil hydrochloride trihydrate ( purity , 99.6% ) was purchased from qiyu biology co. , ltd .
other reagents including k3fe(cn)6 , h3po4 , ethylenediaminetetraacetic acid disodium salt ( na2edta ) , and naoh were supplied by xi'an chemical reagent factory ( xi'an , china ) .
mol / l ) was prepared by ethanol and stored at 20c in a refrigerator .
standard series from 8.0  10 ~ 1.0  10  mol / l were prepared daily by sequentially diluting stock solution with mobile phase solution .
a 1.0    10  mol / l k3fe(cn)6 stock solution was prepared by dissolving 164.6  mg of k3fe(cn)6 in 50  ml water , and it was protected against light and stored at 4c in a refrigerator . a 1.0    10  mol / l stock solution of luminol was prepared in 0.1  mol / l naoh solution .
the mobile phase consisting of ethanol and an aqueous solution containing 1.0  10  mol / l h3po4 and 2.0  10  mol / l na2edta ( 25  :  75 , v / v% ) was prepared .
mobile phase was filtered through 0.45  m millipore membrane of polytetrafluoroethylene ( ptfe ) and further degassed in an ultrasonic bath before use .
health food samples including one herbal oral liquid , one herbal medicated wine , and one herbal capsule claimed being capable of enhancing sexual performance for men that were under suspicion of adulteration , were purchased from local drug store and market .
a portion of oral liquid and wine sample were , respectively , 10-fold diluted with mobile phase solution , filtered through a 0.45  m ptfe membrane filter ( shanghai xinya purifier devices factory , shanghai , china ) and finally degassed prior to use . for capsule sample preparation ,
one capsule weight powder was transferred to a 50  ml beaker , and vortically extracted with 10  ml mobile phase solution for 1  h. a portion of the extraction solution was filtered through a 0.45  m ptfe membrane filter and diluted with mobile phase solution for further analysis . as illustrated in figure 2 , the hplc - cl experimental setup consisted of a cl analyzer ( xi'an remex analyse instrument co. , ltd , xi'an , china ) equipped with a hamamatsu r456 pmt ( tokyo , japan ) and a negative high voltage ( nhv ) generator , a planar coiled transparent ptfe flow cell , a 1.8  ml / min constant flow rate cl pump ( shanghai instrument electric motor factory , shanghai , china ) , a lc-10at liquid chromatography ( shimadzu , tokyo , japan ) equipped with a rheodyne 7725i syringe - loading sample valve ( 20  l - loop , cotati , ca , usa ) , a waters c18 column ( i.d . : 4.6  mm  150  mm , particle size : 5  m , usa ) and a dt-230a column oven ( tianjin do - chrom technology co. , ltd , tianjin , china ) .
stream a containing 1.0  10  mol / l luminol , 2.0  10  mol / l na2edta , and 1.0  mol / l naoh and stream b containing 1.0  10  mol / l k3fe(cn)6 delivered by cl pump merged in the t1 .
then , the reagent mixture merged with mobile phase in the t2 and generated a stable cl baseline . subsequently , when the sample valve was switched to the injection position , 20  l standard or sample was injected into the mobile phase .
outflow vardenafil reacted with the reagent mixture and generated the enhanced cl signal in the flow cell .
the cl signal was recorded as a function of time and cl intensity by the remex analyzer with the pmt operated at 400  v. a microcomputer equipped with remex software running under windows xp was employed for data treatment .
vardenafil peak identification was carried out by the standard addition method and the retention time ( tr ) of vardenafil .
the peak height , i ( i = is  i0 , where i0 was the cl of baseline , is was the peak cl of vardenafil ) was used to quantify vardenafil content by means of the related calibration equation .
for vardenafil cl determination , we considered several known cl reactions including alkaline luminol - h2o2 , alkaline luminol - k3fe(cn)6 , alkaline luminol - kio4 , acidic kmno4 , acidic kmno4-hcho , acidic kmno4-na2so4 , and naclo - naoh . to prevent the possible interference from metal ions , 2.0  10  mol / l na2edta was used in all luminol concerned reactions .
experimental results indicated that alkaline luminol - k3fe(cn)6 reaction contributed the strongest cl response to 1.0  10  mol / l vardenafil presence .
the kinetic curve of the alkaline luminol - k3fe(cn)6-vardenafil cl reaction was studied with a static injection method .
alkaline luminal and k3fe(cn)6 solution were mixed in the beaker placed in front of the pmt window . after a stable baseline
was obtained , 1.0  ml 1.0  10  mol / l vardenafil standard was injected into the beaker by a syringe . the kinetic curve recorded as a function of time and cl intensity , and it could be concluded that vardenafil greatly enhanced luminol - k3fe(cn)6 cl , which reached a maximum within 1.5  s. thus , the studied cl reaction was a fast reaction . as a result , the t2 was placed as near as possible to the flow cell inside the dark box as shown in figure 2 . to obtain the sensitive and fast vardenafil detection with the described hplc - cl system , a series of experiments were conducted to establish optimum analytical performance .
1.0  10  mol / l vardenafil standard was used in all optimization experiments . both mobile phase and
luminol and k3fe(cn)6 flow rates were controlled at 1.8  ml / min , respectively .
experimental optimum analytical performances were selected based on the biggest cl s / n values and on the average of three injections for each test when the relative standard deviation ( rsd ) of each test point was less than 5.0% , unless specified otherwise .
for hplc - cl analysis , the mobile phase should be , not only suitable for vardenafil separation in complex matrix but also compatible with cl detection . acetonitrile - acidic aqueous buffer [ 1 , 4 , 11 , 19 ] and methanol - acidic aqueous buffer [ 8 , 20 ] were the most commonly used mobile phases for vardenafil separations on c18 column .
when investigating the compatibility of acetonitrile with cl detection , it was found that acetonitrile brought about very high cl background .
similar experiments showed that when methanol and ethanol were used , relatively low cl backgrounds and smooth baselines could be obtained .
thus , methanol and ethanol were selected to constitute the mobile phases for the posterior study .
the effect of organic phase volume percentage ( v / v% , organic phase volume / organic + aqueous phase volume ) on tr , s / n value and column pressure ( p ) , were investigated in the range of 40%60% methanol and 20%30% ethanol , respectively . observing the experimental results listed in table 1 , it could be concluded that the higher the v / v% , the shorter the tr , and 30% ethanol contributed the biggest s / n value .
however , 30% ethanol also caused relatively high p. thereupon , as a compromise among the bigger s / n value , shorter tr and suitable p , 25% ethanol was chosen in subsequent experiments .
a number of organic or inorganic acids were employed to constitute mobile phase to improve vardenafil separation [ 1 , 4 , 8 , 11 , 19 , 20 ] .
considering the possible reaction between organic acid and cl reagents , inorganic acid was considered to be more suitable for cl detection .
experiment indicated that h3po4 could effectively suppress the peak - tailing , and a relatively narrow and symmetric chromatographic peak could be achieved ( figure 3 ) .
what is more , as expected , the tr was shortened . with respect to obtaining the biggest cl s / n value ,
the optimum h3po4 concentration was investigated by varying its concentration in the range of 1.0  10 ~ 1.0  10  mol / l .
experiments showed that s / n value increased with h3po4 concentration increasing . when h3po4 concentration was 1.0  10 
mol / l , the ph of mobile phase was about 2.0 , the biggest s / n value was obtained . according to the column supplier 's suggestion , h3po4 concentration higher than 1.0    10  mol /
1.0  10  mol / l h3po4 concentration was employed as the optimum . as could be seen the experimental results summarized in table 2 , by varying the flow rate from 0.3 to 0.9  ml / min , the effect of mobile phase flow rate upon cl detection was investigated .
it was found that the higher the mobile phase flow rate , the shorter the tr and the higher the p.
lastly , a flow rate of 0.8  ml / min was chosen as a compromise between the biggest s / n value and relatively shorter tr .
as was well known , the alkaline medium was necessary for luminal - k3fe(cn)6 reaction . in our experiments ,
the effect of naoh concentration on the cl detection was examined in the range of 0.2~1.3  mol / l .
experiments showed that the s / n value increased with the naoh concentration increasing , and a leveling off could be found when the naoh concentration was higher than 1.0 
the effect of luminol concentration on cl detection was evaluated in the range of 1.0  10 ~ 1.3  10  mol / l .
it was found that the s / n value increased with luminol concentration increasing and reached maximum at 1.0  10 
mol / l , whereas the s / n values decreased with further luminol concentration increasing .
consequently , 1.0  10  mol / l luminol concentration was selected . as an oxidant of cl reaction , k3fe(cn)6 affected the cl signal directly .
the effect of k3fe(cn)6 concentration on cl detection was assessed in the range of 1.0  10 ~ 3.0  10  mol / l .
it was found that the s / n value increased with k3fe(cn)6 concentration increasing in the range of 1.0  10 ~ 1.0  10  mol / l .
based on above results , 1.0  10  mol / l k3fe(cn)6 was selected .
a series of standard solutions ( 1.0  10 ~ 3.0  10  mol / l ) was used to determine the analytical figures of merit under the optimum conditions .
experiments showed that i to vardenafil concentration ( c ) was linear in the range of 8.0  10 ~ 1.0  10 mol / l with the regression equation of i = 3.17c + 9.91 ( c : 10  mol / l , n = 5 , r = 0.9980 ) and the detection limit was 5  10  mol / l ( 3 ) .
the reproducibility was investigated by injecting 2.0  10  mol / l vardenafil standard for 11 times , which was demonstrated by the rsd .
intraday and inter - day precisions of the described method were examined using the oral liquid sample spiked with 2.0  10  mol / l standard .
the intraday precision , defined as the rsd of 5 times determinations to the same sample within one day , five injections one time , was less than 3.5% .
the inter - day precision , defined as the rsd of five days determinations to the same sample , 5 injections one day , was less than 2.3% .
it was concluded that the described hplc - cl method presented relatively good precision .   following the procedures detailed in the experimental section ,
the proposed hplc - cl method was applied to determine vardenafil in oral liquid , wine , and capsule samples . the original and vardenafil standard spiked chromatograms of oral liquid ,
it could be seen from figure 4 that vardenafil was not detected in all three samples and there was no interfering from the endogenous substances at the tr of vardenafil .
it was noticed that the tr of vardenafil was 6.4  min , which was much shorter than the tr reported in relative literatures [ 1 , 4 , 8 , 9 , 11 , 19 ] .
this shorter tr also implied the time and mobile phase saving . to evaluate the applicability of the proposed hplc - cl method for the determination of vardenafil in oral liquid , wine , and capsule , recovery experiments were performed .
these experiments were conducted by spiking 3 levels of vardenafil content into oral liquid and wine and adding identical levels of vardenafil content into capsule as shown in table 3 .
the recovery percentages were estimated by comparing the concentrations detected from spiked samples with the nominal concentrations added . from experimental results summarized in table 3 , it was concluded that the proposed method afforded good precision and accuracy when applied to oral liquid , wine , and capsule samples .
we had carried out the automated hplc - cl method on vardenafil determination in oral liquid , wine , and capsule samples .
based on the obtained experimental results , the following conclusions could be drawn . a new mobile phase that is composed of 25% ethanol and 75% aqueous
was found to be nontoxic , environment friendly , and compatible with the alkaline luminol - k3fe(cn)6 cl detection .
furthermore , the cl detector was cheap , simple and no sophisticated technique required . in a word ,
our proposed method might be adopted as an alternative method for vardenafil determination of in dietary supplement .
 a flow method of high - performance liquid chromatography ( hplc ) seperation and chemiluminescence ( cl ) detection for sensitive vardenafil analysis in dietary supplements was developed . 
 the vardenafil separation was achieved on a c18 column at 30c using ethanol - h3po4 and ethylenediaminetetraacetic acid disodium salt ( na2edta ) aqueous solution ( 25  :  75 , v / v% ) as mobile phase . the followed continuous cl detection was conducted based on the strong cl enhancement by the presence of vardenafil to luminol - k3fe(cn)6 reaction in alkaline medium . at the flow rate of 0.8  ml / min , the vardenafil retention time ( tr ) was 6.4 min . 
 factors that affected the hplc resolution and cl detection were studied and optimized . 
 the calibration curve obtained for vardenafil standard was linear in concentration range of 8.0  107 ~ 
 1.0  104  mol / l . 
 the relative standard deviations ( rsd ) of intraday and interday precision were less than 3.5% . 
 the proposed method was applied to the vardenafil determination in oral liquid , wine , and capsule samples . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusions