aconite rootstocks of aconitum carmichaelii ( chuanwu ) and a. kusnezoffii ( caowu ) have been used in traditional chinese medicine for over two millennia . they are commonly used as a key ingredient in many herbal prescriptions in the orient for rheumatalgia , heart failure , contracture of limbs , and pain in joints . the principal active ingredients in chuanwu and caowu are alkaloids with c19-diterpenoid skeleton , including aconitine ( a ) , mesaconitine ( ma ) , and hypaconitine ( ha ) [ 1 , 2 ] . however , these alkaloids are also toxic and have a very narrow safety range , because they could easily induce ventricular tachycardia and fibrillation even at therapeutic dose levels . down the ages , various processing methods , including prolonged steaming and boiling , were developed to reduce their toxicity [ 2 , 3 ] . during the course of steaming and boiling , aconitines would be hydrolyzed to benzoylaconitines and aconitines , which still retain analgesic properties while their toxicity is reduced about a hundred - fold . from 1989 to 2006 , over 45 aconite poisoning cases were reported in hong kong , among which three cases were fatal [ 46 ] . aconite poisoning cases often occur in other asian countries , as well as , for example , india   and japan . in order to ensure the safety and effectiveness of clinical diagnosis as well as forensic investigation of aconite poisoning , it is necessary to develop convenient , selective , and accurate methods to identify and determine these alkaloids in herbal medicine . various methods have been published , including ir , hplc [ 10 , 11 ] , hpce , hplc / ms [ 1315 ] , and gc / ms . however , there have been few reports about the analysis of aconite alkaloids in herbal medicine using hplc in combination with an automated analytical system and esi / ms / ms . in the present study , an hplc method was established and validated to determine a , ma , and ha in raw and processed caowu and chuanwu . then an automated analytical system and esi / ms / ms were applied to the qualitative analysis of these alkaloids and their semihydrolyzed products . the results indicated that ha was the most stable compound among a , ma , and ha , after undergoing the prolonged heating treatment , which suggested that ha plays a major role in the toxicity of aconite alkaloids in processed chuanwu and caowu . hence , ha could be used as an indicator when one alkaloid is required as a reference to monitor the quality of aconite alkaloids . in addition , raw and processed chuanwu and caowu can be distinguished by monitoring the ratio of a and ma to ha . the reference standard of a was purchased from sigma chemical co. ( st . louis , u.s.a ) . the reference standards of ma and ha were purchased from the institute for the control of pharmaceutical and biological products , china . raw and processed herbs ( chuanwu , caowu ) were purchased from herb market in hong kong . benzoylaconitine , benzoylmesaconitine , and benzoylhypaconitine were prepared from a , ma , and ha , respectively , in our laboratory . lc - grade acetonitrile ( acn ) and tetrahydrofuran and analytical - grade phosphoric acid and triethylamine ( tea ) were purchased from riedel - de haen co. ( germany ) . three standard solutions were prepared by accurately dissolving the a , ma , and ha in acn - tea ( 75  :  25 , v / v ) buffer , respectively . raw and processed herbs ( chuanwu and caowu ) were pulverized into powder . after passing through a 0.45  mm sieve , the powder was dried in an oven at 55c for 6 - 7  h , then three samples with 1.0  g weight were accurately weighed . each weighed sample was extracted with 1  ml aqueous ammonia solution ( 30% ) for 20  min at room temperature and then was extracted with ethyl ether ( 20  ml ) in an ultrasonic bath for 10  min . after the sample was placed at room temperature for 16  h , the liquid phase was filtered . the residue was further extracted for 3 additional times in the same manner ( and the final extraction was assured complete ) . the filtrate was pooled together and was extracted with 2% hydrochloric acid for 4 times ( 25  ml each time ) . the aqueous solution was adjusted with ammonia solution to ph 10 and further extracted with ethyl ether for 3 times ( 25  ml , each time ) . after being washed with water ( 10  ml ) , the combined ether solution was dried with anhydrous sodium sulphate and then was evaporated to dryness at 40c in an evaporator . the residue was further dissolved with 1  ml acn - tea ( 75  :  25 , v / v ) . all final solutions were filtered through a 0.45  m filter membrane , and 20  l filtrate was injected into the hplc system for analysis . the agilent / hp 1090 series hplc system ( hewlett packard , ca , usa ) consisted of quaternary pump , guard column ( econosphere c18 , 5  m , alltech , usa ) , analysis column ( 25  cm  4.6  mm i.d . , microsorb c18 , 5  m , usa ) , and a diode - array detector . the optimal hplc conditions were as follows : a mixture of acn - tea ( 75  :  25 , v / v ) was selected as the solvent of standards and samples , while a mixture of acn , tea buffer ( ph 3.0 ; 25  mm ) , and thf was used as the mobile phase in the gradient program , and the flow rate was maintained at 1  ml / min . detection wavelength was set at 238  nm and the column temperature was maintained at 45c . the automated analytical system mainly included an autosampler , four tandem columns ( concentration column , extraction column , separation column i , and separation column ii ) , a uv detector , the intelligence software , and a spectral library . automated analytical system has several distinct advantages , including automated pretreatment and sample analyses , short analysis time , and high specificity . the system was used for the direct qualitative analysis of a , ha , and ma , and sample pretreatment was not necessary in this system . the esi / ms / ms system consisted of an esi source and a model mat tsq 700 tandem mass spectrometer ( finnigan , ca , usa ) . the esi source was operated in the positive ion mode , and the high voltage for the cylindrical electrode was set at 4510  v. in the above experiments , extracted sample dissolved in methanol - water - acetic acid ( 50  :  50  :  0.005 , v / v / v ) was directly infused into the esi source at a rate of 5  l / min . the mass spectrometer was scanned from 100 to 700  amu per second . it was found that the resolutions of aconitine , mesaconitine , and hypaconitine were markedly affected by the concentration of triethylamine phosphate in buffer solution . since the best separation resolution for the alkaloids was achieved at the concentration of less than 30  mm and the background signal noise increased at less than 10  mm , therefore , 25  mm buffer solution was finally chosen as the hplc mobile phase for subsequent analysis . different gradient modes of mobile phases were chosen according to the following gradient programs ( table 1 ) and the subsequent chromatograms were shown in figure 1 . the best result in terms of peak shape , resolution , and run time was obtained with a mixture of acn , tea buffer ( ph 3.0 ; 25  mm ) , and thf , in a gradient mode of mode d of table 1 . so the optimal hplc conditions were as follows : a mixture of acn - tea ( 75  :  25 , v / v ) was selected as the solvent of standard and samples , and a mixture of acn - tea buffer ( ph 3.0 ; 25  mm ) and thf was used as the mobile phase in a gradient mode of 0  :  90  :  10 ( 0  min ) , 6  :  84  :  10 ( 20  min ) , and 26  :  64  :  10 ( 40  min ) . the regression equation , linear range , correlation coefficient ( r ) , and limit of detection obtained from the established hplc method for the assay of a , ma , and ha are listed in table 2 . the intraday relative standard deviations ( 3 run for each concentration ) were less than 1.61% , and interday relative standard deviations ( 3 run per day for each concentration within 3 consecutive days ) were less than 7.3% . the average recovery of a , ma , and ha was found to be 91% , 89% , and 87% , respectively . a , ma , and ha in raw and processed aconite samples were determined by the established hplc method . it could be seen that the contents of the three alkaloids in processed chuanwu samples were much lower than those in raw chuanwu samples ( figures 1(d ) and 2 ) , the apparent reason of which was that these aconite alkaloids in raw chuanwu were hydrolyzed to their corresponding semi - hydrolyzed products during the treating process . in addition , we have also conducted preliminary study to identify the benzoylaconitines hydrolysed from the three alkaloids in raw and processed chuanwu and caowu , by heating a , ma , and ha in dioxin / h2o ( 7.5/2.5 , v / v ) at 120c for 50  min . benzoylaconitine , benzoylmesaconitine and benzoylhypaconitine were obtained from a , ma , and ha , which lost the acetal groups during the hydrolysis process . after heating treatment , most of a and ma transformed to benzoylaconitine analogues , while , ha had little change , which makes its content the highest among the three alkaloids ( a , ma , and ha ) in the processed herbs . moreover , the results , obtained from boiling raw herbs in water for 15 , 30 , 60 , 90 and 150  min , revealed that contents of a and ma dropped rapidly with an increase in boiling time , and a and ma disappeared completely at 150  min . however , the reduction of ha was small after the same treatment , and its amount still remained significant even at 150  min . it is interesting that ha could survive the heating process , which suggested that ha might play a major role in the toxicity of aconitum alkaloids in processed chuanwu and caowu . hence , it could be used as an indicator when one alkaloid is required as a reference to monitor the quality of aconite alkaloids . in addition , the raw and processed chuanwu and caowu can be distinguished by monitoring the ratio of a and ma to ha . in the qualitative analysis of these alkaloids , the standard solution of a was injected into the automated analytical system . the spectral fingerprint parameters of the unknown sample are shown in table 4 and are compared with those of the candidates suggested by the intelligence software . the searching results for match - fingerprint parameters in the spectral library proved that the unknown sample was a. the identification of ma and ha was achieved by the same procedure . the automated analytical system chromatogram of a mixed standard ( a , ma , ha ) is shown in figure 3 . to confirm the results obtained by automated analytical system , aconite alkaloids the mass spectra showed the fragments of protonated molecules ( [ m+h ] ) and the characteristic losses of side chain ch3cooh ( 60 ) , c6h5cooh ( 122 ) , m / z 152 , and 279 ( table 5 ) . the esi / ms spectra of a mixture of a , ma , and ha standards at 1  g / ml level and a decocted chuanwu ( the time of decoction was 0.5  h ) were shown in figure 4 . the mass spectra showed [ m+h ] ion at m / z 646 for a , at m / z 632 for ma , at m / z 616 for ha , at m / z 603 for benzoyl - aconitine , at m / z 589 for benzoyl - mesaconitine , and at m / z 573 for benzoyl - hypaconitine . the protonated molecular ion has minimal fragmentation because of the soft ionization of the esi . as shown in figure 4(a ) , the [ m+h ] peaks of the aconite alkaloids were the characteristic peaks , which could be used to simultaneously identify these alkaloids , though the sample was directly introduced into the esi / ms . figure 4(b ) shows the relative amounts of the three aconite alkaloids and semi - hydrolyzed products in a decocted chuanwu . the identity of each alkaloid could be further confirmed by esi / ms / ms . after undergoing cid ( collision - induced decomposition ) by argon gas , the protonated molecular ion ( the parent ion , selected by the first mass analyzer ) was transformed into fragments , which could facilitate structural identification of the parent ion . it can be seen from figures  5(a ) , 5(b ) , and 5(c ) that the protonated molecule ions ( [ m+h ] ) of a , ma , ha are at m / z 646 , 632 , and 616 , respectively , and the major fragment ions ( the neutral loss 60 , 122 , 152 , 279 ) are at m / z 524 , 494 , 367 for a , m / z 510 , 480 , 353 for ma , and m / z 494 , 464 , 337 for ha , respectively . figure  5(d ) shows that the esi / ms / ms spectrum of ha in decocted chuanwu is the same as that of the standard ha . hplc in combination with automated analytical system and esi / ms / ms was employed to analyze a , ma , ha , and their benzoyl analogs in our study . the analytical results obtained from of automated analytical system are identical to those from esi / ms / ms . therefore , the automated analytical system would be a good complementary method for the quality control of the herbal preparations containing aconite alkaloids . the most important finding in this research is that the analytical results indicated that ha was the most stable compound among a , ma , and ha , even after prolonged heating treatment , and thus suggest that ha might play a major role in the toxicity of processed chuanwu and caowu . hence , ha could be used as an indicator when one alkaloid is required as a reference to monitor the quality of aconitum alkaloids , and the raw and processed chuanwu and caowu can be distinguished by monitoring the ratio of a and ma to ha .
hplc in combination with automated analytical system and esi / ms / ms was used to analyze aconitine ( a ) , mesaconitine ( ma ) , hypaconitine ( ha ) , and their benzoyl analogs in the chinese herbs caowu and chuanwu . 
 first , an hplc method was developed and validated to determine a , ma , and ha in raw and processed caowu and chuanwu . 
 then an automated analytical system and esi / ms / ms were applied to analyze these alkaloids and their semihydrolyzed products . 
 the results obtained from automated analytical system are identical to those from esi / ms / ms , which indicated that the method is a convenient and rapid tool for the qualitative analysis of herbal preparations . 
 furthermore , ha was little hydrolyzed by heating processes and thus it might account more for the toxicity of processed aconites . 
 hence , ha could be used as an indicator when one alkaloid is required as a reference to monitor the quality of raw and processed chuanwu and caowu . 
 in addition , the raw and processed chuanwu and caowu can be distinguished by monitoring the ratio of a and ma to ha .