fangji huangqi tang ( fht ) is a classical formula of traditional chinese medicine ( tcm ) .
it was firstly recorded in  jing gui yao lue  by zhongjing zhang in the han dynasty . the formula consisted of four herbs , including stephania tetrandra s. moore , astragalus membranaceus ( fisch . ) bge . , atractylodes macrocephala koidz . , and glycyrrhiza uralensis fisch .
lots of doctors recommended fht for the treatment of chronic glomerulonephritis , cardiac edema , and rheumatic arthritis in chinese clinical application based on their actual experience .
also , experts began to do their research work to reveal the potential effective compounds in fht .
for safe and effective use of fht in the clinic , quality control and evaluation of this formula with multiple compounds ' identification and determination became essential and important .
it has already been found out that fht contains different kinds of components , such as flavonoids ( liquiritigenin , isoliquiritigenin , liquiritin , formononetin , and calycosin ) , alkaloids ( fangchinoline and tetrandrine ) , lactones ( atractylenolide i , atractylenolide ii , atractylenolide iii , and atractylon ) , and saponins ( astragaloside i , astragaloside ii , astragaloside iii , and astragaloside iv ) . among these compositions ,
fangchinoline , tetrandrine , and liquiritigenin , as well as calycosin , all showed good effects on anti - inflammatory and improving immunity [ 47 ] .
the critical strategy for quality control and evaluation of fht should be applied to study as much of these effective components as possible .
several common analytical techniques were usually used for multiple chemical compounds ' determination in tcm , including hplc - dad , uv , hplc - elsd , and lc - icpms [ 814 ] .
however , these methods showed lots of drawbacks and technical shortcomings , such as time - consuming sample running , poor sensitivity , peak interferences , and limitation of detectors on components identification .
as it is widely accepted , the uhplc - qqq - ms technique has a special advantage of high resolution and acute sensitivity . and it is now a very powerful tool for the determination of complex chemical compounds in tcm prescriptions , by which the identification of each analyte is also accomplished at the same time .
for this reason , a simple , rapid , and sensitive method for simultaneous determination of 10 components in fht was developed based on uhplc - qqq - ms technique in this paper , and the feasibility of this technique on tcm quality control and evaluation was strongly demonstrated .
stephania tetrandra s. moore and atractylodes macrocephala koidz . were obtained from hebei chinese medicine group corporation ( hebei , china ) .
fangchinoline , tetrandrine , calycosin-7-o--d - glucoside , atractylenolide i , atractylenolide iii , liquiritin , isoliquiritin , liquiritigenin , isoliquiritigenin , and glycyrrhizin with purity of 99% or higher were purchased from the sichuan victor biological technology co. ( chengdu , china ) .
hplc - grade methanol and acetonitrile were purchased from e. merck ( merck , darmstadt , germany ) .
purified water was from a milli - q system ( millipore corporation , bedford , ma , usa ) .
formic acid was of analytical grade and was obtained from nanjing chemical reagent company ( nanjing , china ) .
the uhplc system ( shimadzu , kyoto , japan ) consisted of lc-30ad binary pump , an autosampler ( model sil-30sd ) , an online degasser ( dgu-20a5r ) , and a column temperature controller compartment ( cto-30a ) .
an extend - c18 column ( agilent , usa , 2.1  mm  100  mm , 1.8  m ) at a temperature of 30c was used for separation .
mass spectrometric detection was performed using triple quadrupole 5500 instrument ( ab sciex , concord , ontario , canada ) equipped with an electrospray ionization ( esi ) source .
the separation of these ten compounds was achieved by linear gradient elution using a mobile phase consisting of 0.1% ( v / v ) aqueous formic acid ( a ) and acetonitrile ( b ) .
the gradient program was as follows : 0.10.8  min , 5% b50% b ; 0.83  min , 50% b90% b ; 33.1  min , 90% b5% b. the flow rate was set at 0.4  ml / min .
the samples were placed in an autosampler at 4c prior to injection and the injection volume was 10  l . both positive and negative ionization mass spectrometric analyses were conducted due to the physicochemical properties of the ten analytes .
the scan mode was set as multiple reactions monitoring ( mrm ) and the selected monitor ions were m / z 623.2/174.1 for tetrandrine , m / z 609.3/367.1 for fangchinoline , m / z 233.1/187.2 for atractylenolide i , m / z 249.1/231.0 for atractylenolide iii , m / z 447.2/285.1 for calycosin-7-o--d - glucoside , m / z 821.3/351.2 for glycyrrhizin , m / z 417.1/255.1 for liquiritin , m / z 417.1/255.1 for isoliquiritin , m / z 256.2/135.0 for liquiritigenin , and m / z 256.2/135.0 for isoliquiritigenin .
also , another selected monitor ion pair was selected for the identification purpose for each analyte .
they were m / z 623.2/181.3 for tetrandrine , m / z 609.3/192.2 for fangchinoline , m / z 233.1/215.1 for atractylenolide i , m / z 249.1/163.0 for atractylenolide iii , m / z 447.2/270.0 for calycosin-7-o--d - glucoside , m / z 821.3/193.1 for glycyrrhizin , m / z 417.1/135.0 for liquiritin , m / z 417.1/135.0 for isoliquiritin , m / z 256.2/119.1 for liquiritigenin , and m / z 256.2/119.1 for isoliquiritigenin .
source condition parameters were as follows : curtain gas set at 35 , ion source temperature adjusted to 550c , and ion source gas 1 ( gs 1 ) and ion source gas 2 ( gs 2 ) both at 55 . according to the original composition and preparation process of fht recorded in  jin - gui - yao - lue ,  fht was obtained in the following procedure : pieces of stephania tetrandra s. moore ( 12  g ) , astragalus membranaceus ( fisch . ) bge .
( 6  g ) were mixed together in a ratio ( 4  :  5  :  3  :  2 , w / w / w / w ) and macerated in deionized water for 30  min before being decocted twice with boiling water ( 1  :  10 , w / v and 1  :  8 , w / v , resp . ) , 20  min for each time .
the solution was then filtered through a four - layer mesh and was diluted with water to 1000  ml .
2  ml of the above solution was added to 50  ml vessel , and 15  ml of 60% methanol solution was added for ultrasonic extraction .
then , moderate volume of 60% methanol solution was added to the scale of the 50  ml vessel .
the solution was centrifuged at 12,000  rpm for 5  min and was filtered through 0.22  m membrane .
stock solutions of the 10 analytes were separately prepared by diluting the reference standards with methanol .
all the standards were soluble in methanol and completely dissolved in the mixed working solution . and the mixed working solution containing 10 compounds was diluted to appropriate concentration ranges for the construction of calibration curves .
the calibration curve of each compound was performed with at least seven appropriate concentrations levels .
the concentrations of the 10 components in fangji huangqi tang should be included in the range of the standard curve .
the limit of detection ( lod ) and limit of quantification ( loq ) under the present chromatographic conditions were determined at a signal - to - noise ( s / n ) ratio of 3 and 10 , respectively .
intraday variations within one day were all taken into consideration to determine the precision of the developed method . the relative standard deviation ( rsd )
certain amounts of 10 standards were accurately added to a fht sample and then processed and analyzed as described in sections 2.2 and 2.3 .
the total amount of each analyte was calculated from the corresponding calibration curve , and the recovery of each analyte was calculated by the following equation : recovery ( % ) = ( amountdetermined  amountoriginal)/amountspiked  100% , where amountdetermined is the determined total amount of each analyte , amountoriginal is the original amount of each analyte in samples determined , and amountspiked is the spiked amount of each analyte with 80% , 100% , and 120% ratio of the original amount . in order to investigate the stability of the processed sample solution ,
the same sample solution was stored at 4c and analyzed every 12  h for 1 day .
in addition , the 10 stock solutions were all determined every week to investigate whether obvious degradation had happened under their preservation condition .
according to the published papers , many different kinds of components were identified in fht including alkaloids , flavonoid , and lactones .
also , these chemical compounds were proved to be the main effective components in fht .
the ten selected compounds for simultaneous determination belonged to different chemical classes and covered a wide polarity range .
so , it is of great significance to screen out an appropriate sample preparation method based on the effective extraction for various effective compounds .
therefore , a novel sample method has been developed to collect different polar components on a solid phase extraction column .
this simple and convenient technique was suitable for traditional chinese medicine prescriptions detection by enhancing the sample polarity scope , as well as reducing the pollution of the instrument .
it was difficult for researchers to perform good separation for the chemical constituents in fht because of the complexity of the composition and the existence of isomers . in this paper , the uhplc conditions were well optimized , including the type of column , column temperature , mobile phase system , and flow rate .
after a comparison of different brands of columns including agilent c18 , waters c18 , and thermo c18 , agilent column finally gave out sound separation for the ten compounds to be determined .
in addition , different kinds of mobile phase systems such as acetonitrile and methanol with a variety of modifiers ( including formic acid and acetic acid ) were tested . as a result , a solution of 0.1% ( v / v ) aqueous formic acid and acetonitrile at 0.4  ml / min was used as the optimal chromatographic elution system .
typical source conditions were used as follows : curtain gas was set at 35 ; ion source temperature was adjusted to 500c ; ion source gas 1 ( gs 1 ) and ion source gas 2 ( gs 2 ) were both set at 55 .
independent parameters for the detection of each compound were optimized by injecting standard solution into ms system under mrm mode , including the declustering potential ( dp ) , the collision energy ( ce ) , the entrance potential ( ep ) , and the collision cell exit potential ( cxp ) .
chromatographic peaks of fht were identified by comparing the retention time with that of each reference compound .
the representative extract ions chromatograms of the standard mixture solution were shown in figure 2 . ( a ) stands for fangchinoline ( 0.98  min ) ; ( b ) stands for atractylenolide i ( 2.28  min ) ; ( c ) stands for tetrandrine ( 1.01  min ) ; ( d ) stands for atractylenolide iii ( 1.83  min ) ; ( e ) stands for calycosin-7-o--d - glucoside ( 1.04  min ) ; ( f ) stands for liquiritigenin ( 1.47  min ) and isoliquiritigenin ( 1.72  min ) ; ( g ) stands for liquiritin ( 1.17  min ) and isoliquiritin ( 1.31  min ) ; ( h ) stands for glycyrrhizin ( 1.60  min ) . according to the mass - to - electric charge ratio of each compound ,
table 2 shows the regression equation for each reference compound , as well as the lod and loq values and the mass spectrometry information .
all the components showed good linearity ( r > 0.9991 ) in a relatively wide concentration range .
tables 3 and 4 provide precision and recovery results of the 10 components , respectively .
the intraday variations of the compounds were < 2.53% , the relative standard deviation ( rsd ) of repeatability test was below 2.68% for all the analytes of interest , and the extraction recoveries of the components ranged from 98.51% to 101.10% .
stability was evaluated by analyzing all the analytes in samples kept at 4c for 24  h. they were all found to be stable within 24  h ( rsd < 3% ) . as a result ,
the developed method was proved to be precise , accurate , and sensitive for the simultaneous quantification of the major compounds of fht .
the developed uhplc - ms / ms method was subsequently applied to analysis and quality evaluation of fht .
a rapid uhplc - ms / ms method was established for the comprehensive analysis of fht .
furthermore , ms / ms proposed high sensitivity which is helpful for the quantification of the low - content compounds .
the method was successfully applied to the simultaneous determination of 10 bioactive compounds in fht .
this available , rapid , and reliable method is fit for routine analysis and effective quality control of this chinese herbal prescription .
 the aim of this study is to develop an ultrahigh performance liquid chromatography method coupled with triple quadrupole mass spectrometry for simultaneous determination of tetrandrine , fangchinoline , atractylenolide i , atractylenolide iii , calycosin-7-o--d - glucoside , glycyrrhizin , liquiritin , isoliquiritin , liquiritigenin , and isoliquiritigenin in fangji huangqi tang ( fht ) . 
 the chromatographic separation was performed on a reversed - c18 column , eluted with a mixture of 0.1% acetic acid and acetonitrile at 0.4  ml / min . 
 the separation of these ten compounds was achieved by linear gradient elution . 
 the method was strictly validated with respect to specificity , precision , accuracy , and repeatability . 
 all the compounds showed good linearities ( r  0.999 ) . 
 the loqs of the ten components were 0.36 , 0.18 , 0.09 , 0.43 , 0.02 , 1.89 , 0.26 , 0.18 , 0.61 , and 0.48  ng / ml for tetrandrine , fangchinoline , atractylenolide i , atractylenolide iii , calycosin-7-o--d - glucoside , glycyrrhizin , liquiritin , isoliquiritin , liquiritigenin , and isoliquiritigenin , respectively . 
 the lods of the ten components were 0.11 , 0.05 , 0.03 , 0.13 , 0.01 , 0.57 , 0.08 , 0.05 , 0.18 , and 0.14  ng / ml for tetrandrine , fangchinoline , atractylenolide i , atractylenolide iii , calycosin-7-o--d - glucoside , glycyrrhizin , liquiritin , isoliquiritin , liquiritigenin , and isoliquiritigenin , respectively . 
 the method was proven to be specific and reliable , which would provide a meaningful basis for the quality control and evaluation of fht during its clinical application . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusions