musty and earthy odors are troublesome in water samples ( such as tap water , reservoirs , and lakes ) because they dramatically influence the esthetic quality and consumer acceptability of the drinking water [ 1 , 2 ] . metabolites are produced in the process of degradation of green - blue algae , which are responsible for this malodor in water , especially during the period of algae blossom in summer [ 35 ] . the chemical by - products , geosmin ( trans-1,10-dimethyl - trans-9-decalol ) and 2-mib ( 2-methylisoborneol ) , are commonly found in lakes and reservoirs , where people can smell the odor of these compounds in the drinking water even at the determination of 10  ng  l or less , but it would be difficult to identify and quantify these two trace volatile organic compounds ( vocs ) [ 610 ] . therefore , how to extract and enrich them tends to be the key step for qualitative and quantitative analysis at this trace level . to date , a variety of techniques for extraction and enrichment have been established and applied for analyzing earthy and musty compounds . among these techniques , closed - loop stripping analysis ( clsa ) and some of its modified versions have been widely used for the extraction of trace amounts of malodor such as geosmin and 2-mib in water samples . the result showed that clsa was a good tool for the analysis of geosmin and 2-mib at a low - level threshold . some other methods , such as purge and trap coupled to gas chromatography with mass spectrometry [ 12 , 13 ] or to gc - fid , liquid - liquid microextraction ( llme ) , stir bar sorptive extraction ( sbse ) [ 1618 ] , and solid - phase extraction ( spe ) , can also be taken to detect the vocs in water at  ng  l levels . although these techniques greatly improve the limits and sensitivity of detection , some shortcomings such as being unsuitable for the analysis of low - boiling - point odors and time - consuming ( spe , sbse ) [ 20 , 21 ] and lacking the stability of droplet during extraction ( lpme ) restrict the extensive use of these methods . as technology advances , solid - phase microextraction ( spme ) was first developed , and it was reported that headspace spme ( hs - spme ) was effective for collecting volatile organic compounds from penicillium . hs - spme has become the most popular technique in pretreating and enriching a variety of water odors [ 2327 ] , because of requiring no solvents during extraction by hs - spme , which can not be achieved by llme ; being a simpler operation if compared with other methods as spe , clsa , and sbse ; and , the most important merit , being able to enrich the target selectively by suitable fiber , which can not be obtained by spe and llme . it had been reported that the efficiency of hs - spme was subject to several factors such as extraction temperature , ionic strength , and rotate - speed . the method of  one factor at a time  , the traditional method for optimizing experiment conditions of hs - spme or purge and trap ( p&t ) , was to change the level of one factor while keeping others constant [ 12 , 24 , 26 , 28 ] , which was almost the only one to optimize these factors in current reports for determining trace vocs in water samples . obviously , this method is time - consuming when these factors and their levels reach a certain number . in addition , it often overlooks the interactions among the factors , despite having no interactions in this study . in this paper , however , the orthogonal experiment design was applied to arrange and optimize experiment conditions , including extraction temperature , desorption temperature , extraction time , solution ph , ionic strength , and rotate - speed . besides , the process of mass spectrometry was optimized to improve the sensitivity and efficiency during the detection and analysis in this study . the good figures of merit for the analysis of the trace amount of odors in water samples have been obtained by combining them . finally , the analytical method has been validated and applied to test on water samples . two categories of compounds commonly observed in water , geosmin and 2-mib , and the internal standard 2-isobutyl-3-methoxypyrazine ( ibmp ) were obtained from sigma - aldrich ( usa ) at a concentration of 100  mg  l in methanol , 1  mg l mixed standard solutions of the two target compounds in methanol , and all of them were stored in the dark at 4c . deionized water was prepared on a water purification system ( gradient a10 ) supplied by millipore ( billerica , ma , usa ) . sodium chloride ( analytical grade , china ) , which was added to the samples before extraction , was conditioned by heating at 450c for 4  h before use . the spme apparatus was purchased from supelco ( usa ) , including fiber 30/50  m dvb / car / pdms ( no . 57348-u ) [ 26 , 29 ] , fiber holder , 60  ml specialized vials for spme , sampling stand , magnetic stirrer , and injection catheter ( no . the extraction conditions shown in table 3 were followed . after putting nacl and a stir bar in a 60  ml vial , 40  ml of mixed standard solutions ( 50  ng l ) for orthogonal experiment , or 40  ml of environmental water samples , was added , and 2  l ibmp ( 1  mg the vial was sealed with polytetrafluoroethylene ( ptfe ) septum cap and placed in a water bath . several minutes after the temperature was achieved in the vial , the outer needle of fiber was used to penetrate the septum , and the fiber was exposed to the headspace for extraction . after exposure , the fiber was immediately inserted into the gc injection port for desorption . a varian 300 gc / ms / ms ( varian , inc . , ca , usa ) with ion trap and mass spectrometer was obtained with a varian vf-5 ms capillarity column ( 30  m  0.25  mm  0.5  m ) . the temperature of the oven started at 40c and was held for 5  min . then , the temperature was 8c  min to achieve 160c ( total time : 20  min ) followed by 20c  min to achieve 260c ( total time : 25  min ) . the electron impact ( ei ) ms conditions were as follows : ion - source temperature : 230c ; ms transfer line temperature : 250c ; solvent delay time : 5  min ; and ionizing voltage : 70  ev . the mass spectrogram in full - scan mode was obtained at the m / z range of 80200  u. according to the ms scan function ( sim mode ) , the process was divided into three main segments as shown in table 2 . the method of internal standard was applied to construct the calibration curve and determine the concentrations of 2-mib and gsm in water . to obtain the qualitative and quantitative ions , the two target analytes and the internal standard compound were first identified simultaneously by hs - spme / gc - ms in the scan mode . the chromatogram ( full - scan mode ) is shown in figure 1 , and the full - scan mass spectra from 17.0 to 24.3  min were obtained with m / z range of 80200  u. in order to optimize the extraction of gsm and 2-mib by hs - spme , several parameters were investigated . the orthogonal design experiment is a valid approach by applying the orthogonal table ln ( m ) to arrange experiments . in the orthogonal table , n represents the number of experiments ; m is the number of factor levels ; and k is the number of factors . the multiple factors as well as the multiple levels can be dealt with by this design , and it also can obtain the optimal design with less experimental effort . and the experimental results can then be calculated by the analysis of variance , which can identify the main significant factors and levels . this method had been applied to optimize experimental conditions for determining the several phytohormones in natural coconut juice by wu and hu , and it has been used by huang et al .   for the determination of glycol ethers it had been also extraction of fat - soluble vitamins by sobhi et al .   and analysis of 17 organochlorine pesticides in water samples by qiu and cai . in this study , utilized orthogonal experiment design l32 ( 4 ) was applied to arrange and optimize experiment conditions . the concrete experiments are listed in table 3 , and , all experiments were repeated twice , and then , the average peak area was applied to weigh the efficiency of hs - spme under different conditions . according to the analytical results calculated by spss 19.0 in table 4 , for the maximum for 2-mib was k31 , k42 , k43 , k34 , k45 , k46 , and k47 , and that for gsm was k41 , k42 , k43 , k34 , k45 , k46 , and k27 . in other words , the optimum conditions for 2-mib were listed as follows : temperatures of extraction and desorption : 60c and 260c , respectively ; times of extraction and desorption : 40  min and 7  min , respectively ; ionic strength : 30% ( w / v ) ; rotate - speed : 600  rpm ; and solution ph : 8.0 . and those for gsm were listed as follows : temperatures of extraction and desorption : 70c and 260c , respectively ; times of extraction and desorption : 40  min and 7  min , respectively ; ionic strength : 25% ( w / v ) ; rotate - speed : 600  rpm ; and solution ph : 6.0 . table 5 shows the results of the analysis of variance ( anova ) for the significance of the main factors , which were based on the peak area of headspace in simulative water samples . as to 2-mib , extraction temperature , extraction time , desorption time , and ionic strength were significant factors ( p  0.01 ) , while other factors did not have a significant effect ( p  0.05 ) within the studied range . for the gsm , only extraction temperature and extraction time were significant factors ( p  0.01 ) , while others were not significant ( p  0.05 ) . according to the results of anova of the previous main factors , the significant factors were studied at 10  ng  l of mixed standard solutions by using the method of  one factor at a time . each of them was tested twice , and the results were obtained in figure 2 . responses were calculated upon the conditions of 40c , 50c , 60c , and 70c extraction temperatures ( time of extraction and desorption : 30  min and 5  min , resp . ; ionic strength : 25% ( w / v ) ; rotate - speed : 600  rpm ; desorption temperature : 260c ; the solution ph : 5.0 ) . as shown in figure 2(a ) , we studied the spme analyses run at the selected temperature , the extraction efficiency of the two analytes increased as the extraction temperature was , from 40c to 60c while a decrease was observed for 2-mib and a slow increase was observed for gsm between 60c and 70c as reported similarly by saito et al . . firstly , as the extraction temperature was growing , the increasing amount of water vapor would be assembled on the fiber , which would lower the extraction efficiency ; secondly , the different molecular weight of vocs was known to be inconsistently susceptive to the extraction fiber ; thirdly , this can be explained by the partition coefficient between the fiber and the analytes . in other words , kfs = k0exp[h / r(1/t  1/t0 ) ] ; that is to say , the partition coefficient ( kfs ) would be altered if the extraction temperature was changed from t0 to t , because the potential energy of the analyte on the coating material will be less than its potential energy in the sample if the kfs is more than one . therefore , the value of kfs will decrease as the extraction temperature is increasing , which can result in the decreased extraction efficiency as reported by chai and pawliszyn . when considering the extraction temperature , 65c was the optimal choice as obtained in figure 2(a ) . responses were calculated upon the conditions of 10 , 20 , 30 , and 40  min extraction temperature times ( desorption time : 5  min ; ionic strength , 25% ( w / v ) ; rotate - speed : 600  rpm ; extraction and desorption temperatures : 65c and 260c , resp . ; and solution ph : 5.0 ) . as shown in figure 2(b ) , we studied the spme analyses run at the selected time ; the extraction efficiency of the two analytes increased rapidly as extraction time was from 10  min to 30  min , while a slow increase was observed for both of them between 30 and 40  min . however , the equilibrium time for this fiber may be 40  min or more , but we desired shorter extraction time to maximize the sample . therefore , the extraction time 30  min was selected for experiments , and also this allowed the gc - ms analysis ( 25  min ) to be performed nearly in the approximate time as in the hs - spme procedure . the suitable salt addition could improve the transfer of analytes from the aqueous phase to the gaseous phase , so this can result in a higher concentration of the odors in the headspace . responses were calculated upon the conditions of 15% , 20% , 25% , and 30% ( w / v ) ionic strengths ( times of extraction and desorptions : 30  min and 5  min , resp . ; rotate - speeds : 600  rpm ; extraction , and desorption temperatures : 65c and 260c , resp . ; and solution ph : 5.0 ) . as shown in figure 2(c ) , it was fairly clear that 25% ( w / v ) was most suitable for the extraction process , and this concentration of salt was selected for the future experiments . as shown in figure 2(d ) , the desorption time ( 2 , 3 , 5 , and 7  min ) profile is studied . thus , 5  min was selected as the optimal time . according to the results of the analysis of variance , some factors such as rotate - speed , desorption temperature , and solution ph did not have significant effects ( p  0.05 ) within the given range . consequently , 600  rpm , 260c , and 5.0 were chosen , respectively , for rotate - speed , desorption temperature , and solution ph [ 24 , 26 ] . the 10  ng  l spiked mixed standard solutions were detected by two methods later . as shown in figure 3(b ) , the ms analysis progress in sim mode was separated into five segments . to be more specific , segment 1 started from 15.0  min , selected ion m / z = 124 , segment 2 from 18.4  min , m / z = 107 , segment 3 from 19.0  min , nothing , segment 4 from 22.0  min , m / z = 112 , and segment 5 from 22.4  min , nothing . in this case , the peaks can be separated effectively , and some disturbed peaks would be excluded . in contrast , as shown in figure 3(a ) , it would result in some unidentified peaks by not using the segments to analyze the compounds . the signal to noise ratio ( s / n ) was shown in table 6 , and it was fairly clear that the ms scan function was more effective for the determination of analytes . the proposed hs - spme / gc - ms method had been validated in terms of accuracy , linearity , limits of detection , relative standard deviation ( rsd ) , and recovery . linearity was studied by extracting the two odors in mixed standard solutions at five levels , ranging from 5 to 100  ng  l. calculated calibration curves gave high levels of linearity with a correlation coefficient value of 0.9999 for both of geosmin and 2-mib , and the rsds were 2.75%~3.80% and 4.35%~7.6% for gsm and 2-mib , respectively . under the optimized experimental conditions , limits of detection ( s / n = 3 ) and limits of quantitation ( s / n = 10 ) for geosmin were 0.04 and 0.14  ng tap water , deionized water , and lake water were used to verify the applicability of this method for the analysis of these two odors in water samples . as shown in table 9 , this fairly indicated that the proposed method could be used to analyze these musty and earthy odors in water samples . this study has demonstrated the application of the orthogonal experiment design for screening the significant factors of hs - spme for the analysis of volatile organic compounds in water samples . also , the ms scan function was an effective approach for the determination of analytes . the proposed method has been validated by excellent results ( i.e. , high sensitivity , low limits of detections , and high levels of linearity ) . therefore , this method would be most likely to be applied to optimize factors for future study for analysis of some other odors in water ( i.e. , 2-isopropyl-3-methoxy pyrazine and 2,4,6-trichloroanisole ) .
geosmin and 2-mib are responsible for the majority of earthy and musty events related to the drinking water . 
 these two odorants have extremely low odor threshold concentrations at ng  l1 level in the water , so a simple and sensitive method for the analysis of such trace levels was developed by headspace solid - phase microextraction coupled to gas chromatography / mass spectrometry . in this study , 
 the orthogonal experiment design l32 ( 49 ) was applied to arrange and optimize experimental conditions . 
 the optimum was the following : temperatures of extraction and desorption , 65c and 260c , respectively ; times of extraction and desorption , 30  min and 5  min , respectively ; ionic strength , 25% ( w / v ) ; rotate - speed , 600  rpm ; solution ph , 5.0 . under the optimized conditions , limits of detection ( s / 
 n = 3 ) 
 were 0.04 and 0.13  ng  l1 for geosmin and 2-mib , respectively . calculated calibration curves gave high levels of linearity with a correlation coefficient value of 0.9999 for them . 
 finally , the proposed method was applied to water samples , which were previously analyzed and confirmed to be free of target analytes . besides , the proposal method was applied to test environmental water samples . 
 the rsds were 2.75%~3.80% and 4.35%~7.6% for geosmin and 2-mib , respectively , and the recoveries were 91%~107% and 91%~104% for geosmin and 2-mib , respectively .