paclobutrazol ( chemical structure as in figure 1 ) was a plant growth regulator registered for the reduction of terminal growth and pruning volume , the inhibition of gibberellins and sterol biosynthesis , and hence the rate of cell division [ 1 , 2 ] . due to its toxicity , the agreed adi ( accepted daily intake ) and arfd ( acute reference dose ) were all 0.1  mg / kg bw / day . according to reg . number 396/2005 ( ec ) annex i , the mrl for paclobutrazol in fruit was 0.5  mg / kg , and the national food safety quality standard ( gb 2763 - 2014 , china ) was 0.5  mg / kg in wheat and rice . however , no information was available concerning the residual status and pattern of paclobutrazol in potato . growth inhibition characteristics had been reported for paclobutrazol with soil drenches , soil sprays , and foliar sprays . cross - comparisons among these studies were difficult since they were conducted under different growth conditions ( field , greenhouse , and growth chamber ) . two other factors that influenced the paclobutrazol residues were absorption rate and longevity or persistence of the compound in treated plant tissue and soil . traditionally , gas chromatography ( gc ) and gas chromatography - mass spectrometry ( gc / ms ) was once popular for analyses of paclobutrazol , but gc / ms required prederivatization and target compounds may be decomposed by injection at high temperatures . more recently , hplc - based methods for analyses of paclobutrazol had been published in plant [ 69 ] and pear pulps   using uv detection and different cleanup steps in order to get recoveries of 70% at the 0.01  mg / kg level . thus , using these conventional techniques , it was necessary to perform one or more cleanup steps to decrease interferences and preconcentration steps in order to obtain adequate detection levels . nowadays , lc coupled to tandem ms ( ms - ms ) [ 1113 ] had also been applied to plant growth regulator analysis in fruits as a powerful confirmation tool , improving the sensitivity and reducing the sample pretreatment steps , such as solid phase extraction ( spe ) techniques . recently , the liquid - liquid extraction with low temperature partitioning ( lle - ltp ) was promising for multiresidues analysis since this technique presented some advantages in relation to other extraction techniques , such as practicability , reduced number of steps , and low consumption of organic solvents , as well as being reliable and selective [ 11 , 14 ] . in this study , it was to develop and validate a uhplc - ms / ms method for determination of paclobutrazol in potato and soil , taking advantage of liquid - liquid extraction at low temperature . in addition , the paclobutrazol residue in potato and soil and the dynamics dissipation in soil were investigated through field trials . the purpose of this study was to evaluate the influence of paclobutrazol residue in potato , and a mrl of paclobutrazol in potato was recommended . a high - performance liquid chromatography- ( hplc- ) grade acetonitrile and methanol were obtained from merck co. ltd . sodium chloride and sodium acetate of analytical grade were obtained from the chemical reagent company ( shanghai , china ) . highly purified water ( milli - q , millipore , bedford , ma ) was used throughout the preparation of the mobile phase . stock solution was prepared at 1000  mg / l in methanol and stored in dark vials at 20c . for the calibration curve , matrix - matched calibration was used with five series concentrations of extraction solution ( 0.001 , 0.01 , 0.05 , 0.1 , and 0.5  mg ultimate residue experiments were conducted in experimental fields located in songming agriculture demonstration garden , yunnan province , china , from april 18 to june 29 , 2013 . the soil was sandy clay loam , with content of sand 46% , clay 28% , organic matter 2.02% , and ph 6.94 . the experiments were designed according to ny / t 7882004 ( guideline on pesticide residue trials ) issued by the ministry of agriculture , china . according to the usage guide , paclobutrazol formulation spray application was recommended one time during the early flowering stage of potato ( in 45 days ) . plants were treated with 3 dosage levels , 40  g ( available ingredient)/ha ( the low dosage , recommended dosage ) , 60   g ( available ingredient)/ha ( the middle dosage , 1.5 times the recommended dosage ) , and 100  g ( available ingredient)/ha ( the high dosage , 2.5 times the recommended dosage ) . treatments were consisted of foliar sprayed ( with or without thick polypropylene soil cover ) , and the tuber on the paclobutrazol residual soil with 60  g ( available ingredient)/ha , and the control plants were treated with distilled water . foliar sprays were applied with and without a ground cover to evaluate the impact of foliar adsorption and indirect soil absorption from foliar runoff on soil residue . to check the possibility of paclobutrazol contamination from surface and peel of tuber , twenty tubers ( 4.8  kg ) were sprayed with paclobutrazol ( 1.0  mg / l ) , five of which were rinsed with 500  ml distilled water and 100  ml acetonitrile ; then the rinsing acetonitrile was determined by uhplc - ms / ms after 30  min . another five samples were peeled ; then the peel and pulp were analyzed for comparison with potato harvest . as reported paper , potato was harvested about one month after flowering stage , so the tuber and soil were collected to determine final residue at ten days before harvest ( 22 days ) , harvest time ( 32 days ) , and eight days after harvest ( 40 days ) after foliar spraying . to determine dissipation rate of paclobutrazol in soil , soil samples of approximately 500  g from depth of 010  cm were collected randomly from five points in each plot at 1  h , 1 , 3 , 7 , 14 , 21 , 32 , 40 , and 50 days after soil spray . the soil samples were air - dried , clod broken , mixed , and sieved through a 2  mm sieve as described by sharma . soil : 10  g soil sample was weighed into a 100  ml centrifuge tube and then 50  ml acetonitrile was added . the centrifuge tube was extracted in an ultrasonic bath for 10  min and centrifuged at 3000  g for 5  min . afterwards , the tube was stored at low temperature ( 20c ) in refrigerator ( haier , qingdao , china ) for 10  min to easily separate organic layers . two - milliliter portions of the organic layer were filtered through 0.2  m membrane filters prior to analysis by uhplc - ms / ms . potato : 25  g sample was weighed into a 100  ml centrifuge tube and extracted with 50  ml acetonitrile . the same procedure was followed as that described for soil up to uhplc - ms / ms . sample analyses were performed on tandem mass spectrometry ab 4000 ( ab sciex , ontario , canada ) which consisted of a 1290 ultrahigh performance liquid chromatography ( agilent technology , usa ) . a zorbax eclipse plus c18 column ( 50  mm  2.1  mm i.d . , 1.8  m particle size ) was employed for the separation of the analyte and was maintained at 30c . the mobile phase was comprised of acetonitrile / water containing 0.1 percent formic acid ( 78/22 , v / v ) and was delivered at a constant flow of 0.3  ml / min . the injection volume was 1  l . the spectral acquisition was operated in positive electron spray ionization mode , and multiple reactions monitoring ( mrm ) was utilized . the gas temperature was set at 350c with a flow rate of 8.0  l / min . the precursor ion was m / z 294.2 , and production ion was m / z 129.0 , 70.0 for paclobutrazol , with relative intensity of 4.5%/68% , in which the most intense production ion m / z 70.0 was the quantitative ion , and collision energy and fragmentor were 110 and 50  ev , respectively . solid - phase extraction ( spe ) was a preferred way to prevent matrix interference , but it was time - consuming with high cost . the advantage of liquid - liquid extraction with low temperature was that the sample components remained in the ice phase , whereas paclobutrazol was extracted by the completely transparent liquid , which was easily obtained and directly analyzed by uhplc - ms / ms . as it had been previously indicated , cho et al . the result showed that the sample would be frozen and separated easily under 20c to 10  min . so it can be operated under facile condition by home refrigerator . in order to gain a favorable extraction yield , the extraction yield of the various solvents for paclobutrazol was listed in the following sequence : acetonitrile ( recovery 95% ) > acetone ( recovery 86% ) > dichloromethane ( recovery 65% ) > n - hexane ( recovery 10% ) . furthermore , the ratio of acetonitrile to the sample showed that the best results were with 2  :  1 ( potato ) and 5  :  1 ( soil ) , resulting in 85% recovery greater than other ratios with 1  :  1 or less . the tuning solution was introduced into the electrospray ionization ( esi ) source by direct infusion . the main ions produced in ms and ms / ms were identified in positive ionization modes . the obtained precursor ions indicated a clear relation between the structure of paclobutrazol and its ability for positive ionization . the precursor ion was m / z 294.2 , and production ion was m / z 294.2129.0 and 294.270.0 ( quantitative ion ) . the composition of mobile phase can influence the performance of the ionization process in esi mode . so , different mobile phases ( acid , base , and neutral ) were tested . the result showed that the response of paclobutrazol can be increased in acid mobile phase ( 0.1% formic acid ) . the optimized mobile phase was acetonitrile / water containing 0.1% formic acid ( 78/22 , v / v ) , as shown in figure 2 . for this reason , residues of paclobutrazol in samples were quantified with matrix - matched five - series standard calibration ( 0.001  mg / l , 0.5  mg the calibration curves showed good linearity with the following equations and relative coefficients : y = 1.95693  10 + 964964x , correlation coefficient r = 0.9976 . the instrument detection limit ( lod ) and limit of quantification ( loq ) were estimated through ten repetitive injections of standard solution , which can detect at a signal - to - noise ratio ( s / n ) of three multiples and ten multiples , respectively . the lod value was 0.5  g / kg , and the loq values were always 2  g / kg in the potato and 5  g / kg in soil . repeatability and reproducibility were expressed as relative standard deviations ( rsd ) of retention time ( rt ) and peak area ( ar ) . reproducibility of rt and peak area was good for 1 month ; all values were 3.4% ( rt ) and 5.2% ( peak area ) or less . furthermore , repeatability of rt and peak area , which was evaluated on 1 day , proved more satisfactory , with values of 1.5% ( rt and peak area ) or less . the accuracy of the whole method was evaluated by the development of a recovery study carried out at three concentration levels ( 5 , 50 , and 100  g / kg ) . all experiments were carried out in quintuplicate at each level ( results are shown in table 1 ) . as it can be seen in table 1 , recovery values were satisfactory , ranging between 83 and 106% with rsd lower than 10% . as it can be seen from the rsd values , the method was reproducible and applicable to the analysis of paclobutrazol in potato and soil . foliar spray without cover treatment showed that amounts of paclobutrazol were absorbed by foliage and foliar runoff was absorbed by the soil . .   showed result that paclobutrazol residues in the soil increase due to falling leaves from nearby sprayed trees . apparently , the amount of paclobutrazol absorbed by stolon , then transported to tuber , was larger than by foliar absorptive capacity . to check the possible contamination of paclobutrazol from surface and peel of tuber the content in organic solvent was very lower than the expected concentrations in whole potato . so the paclobutrazol residue in potato harvest was mainly from absorption and transport from soil , which was not removed by peeling . the result showed that the initial deposits of paclobutrazol in soil were 1.14  mg / kg . except for a slight increase on 1st day , the residue value showed a steady decrease and was below 0.22  mg / kg on 50th day . the dissipation equation of paclobutrazol was y = 1.0784e , r = 0.9403 . to establish a recommended maximum residue limit ( mrl ) of paclobutrazol in potato , two guideline values , with need maximum daily intake ( nedi ) and acceptable daily intake ( adi ) , the value of adi with paclobutrazol was 0.1  mg / kg bw / day . neda was derived on the supervised trials median residue ( stmr ) or processing factor ( stmr - p ) and food intake rate ( f ) . safe  level of exposure ( mrl ) does not exceed the estimated level of actual exposure ( adi ) . the equation was ( 1)nedi=stmristmr - pifi . according to the investigation of nutrition and health by chinese health ministry in 2002 , the f of potato was 0.0496  kg , stmr in potato harvest ( recommended dosage : high dosage level ) was 00.521  mg / kg , and then the high value nedi ( 0.0258  mg ) was not beyond 5% the value adi  63 ( adult weight ) of 6.3  mg . liquid - liquid extraction with low temperature partitioning was developed and validated for analysis of paclobutrazol in potato and soil using uhplc - ms / ms . the optimal chromatographic separation and sensitivity were successfully applied to the analysis of paclobutrazol residue in potato and soil . according to risk assessment with nedi and adi ,
a simple , accurate , and highly sensitive analytical method was developed for determining the paclobutrazol residue in potato and soil , the dynamics dissipation in soil . 
 extraction was carried out by low temperature partitioning and analyzed by ultrahigh performance liquid chromatography tandem mass spectrometry ( uhplc - ms / ms ) . for a favor extraction yield 
 , the parameters such as temperature and solvent were optimized . 
 the result showed that sample would be easily frozen and separated using acetonitrile under 20c for 10  min . 
 the limit of detection ( lod ) was 0.5  g / kg , and the limit of quantification ( loq ) was 2 and 5  g / kg for potato and soil , respectively . 
 the influence of paclobutrazol residue in potato was evaluated . 
 the possible contamination of paclobutrazol from surface can be rinsed by distilled water or peeled off , but the paclobutrazol in potato harvest comes mainly from absorption and transport , which could not be removed by peeling . 
 the half - life of paclobutrazol in soil was 20.64 days , and the residue was below 0.22  mg / kg on 50th day after spraying . according to the risk assessment with need maximum daily intake ( nedi ) and acceptable daily intake ( adi ) , a maximum residue limit ( mrl ) of paclobutrazol in potato was recommended as 1.0  mg / kg .