Anal. Chem. 1995, 67, 1968-1 975
Determination of Acidic Pesticides in Water by a Benchtop Electrospray Liquid Chromatography Spectrometer Carlo Crescenzi, Antonio Di Corcia,* Stefan0 Marchese, and Roberto Samperi Dipartimento di Chimica, Universita “La Sapienza’; Piazza Aldo Mor0 5, 00185 Roma, Italy
A very sensitive and specific analytical procedure for determining 20 acidic pesticides in aqueous environmental samples using pneumatically assisted eledrospray (ES) LC/MS is presented. This procedure involves passing 1and 4-L river water and drinking water samples, respectively, through a 1-g graphitized carbon black (GCB) extraction cartridge. By exploiting the presence of positively charged active centers on the GCB surface, isolation of the acidic pesticides from baseheutral species was made possible by differential elution. Recoveries of the analytes were higher than 85%,irrespective of the aqueous matrix in which they were dissolved. Adoption of the ion pair technique by addition of 0.1 mmoVL KzHP04 and 0.2 mmol/L tetrabutylammonium fluoride to the mobile phase allowed analytes to be analyzed as preformed ions. A conventional 4.6-mm4.d. reversed-phase LC C-18column operating with a 1 a m i n flow of the mobile phase was used for chromatographing the analytes. A flow of 30 pWmin of the column etlouent was diverted to the ES source, while the rest of the mobile phase was delivered to a W detector set at 220-nm wavelength. The effects of the nature of the ion pair forming agent and its counterion as well as its concentration in the mobile phase on the response of the ES/MS detector were investigated. The effects of varying the skimmer cone voltage on the production of diagnostic fragments and the response of the MS detector were also evaluated. For the analytes considered, the response of the mass detector was linearly related to the amount of the analytes injected between 2.5 and 200 ng. A certain variation of the ion signal for the analytes considered occurred after several hours of continuous use of the LC/ ES/MS instrumentation. For routine use, then, analyte quantitation could be better performed by the UV trace method while entrusting unambiguous identification of the analytes to the MS detector. The limits of sensitivity (signal-to-noiseratio = 3)of the method for the pesticides considered in drinking water and surface water samples were estimated to be about 2-6 and 8-25 ng/L, respectively. A number of liquid chromatographic methods have been proposed for determining those analytes of environmental interest that are not amenable to analysis by the gas chromatography/ mass spectrometry technique. However, because of the legal implications of many environmental data, coupling LC with MS is a key element for the future of LC procedures. 1968 Analytical Chemistty, Vol. 67, No. 73,July 7, 1995
Electrospray (ES) ionization has rapidly emerged as a very promising technique for interfacing LC to MS. Similarly to thermospray, ES ionization produces ions at atmospheric pressure, but without the need for high temperatures that could decompose labile compounds. Briefly, the sequence of events in the electrospray process leading to generation of gas phase ions involves formation of charged small droplets by an electrical field. The droplets are gradually reduced in size by heat transfer and repeated Coulombic explosion until the radius of curvature of the daughter droplets becomes so small that field-assisted ion “evaporation”competes with further droplet disintegration. The LC/ES/MS system has proven to be a sensitive technique for analyzing compounds of environmental interest that exist as ions in solution.’,* Notably, although the electrospray process generates gas phase ions in a gentle way, structural information on analytes can also be easily achieved by collision-induced fragmentation with a suitable adjustment of the electrical field existing in the intermediate-pressure desolvation chamber located between the ionization source and the mass analyzer region. By suitably exploiting this possibility, Voyksner and Pack3 showed that a single mass analyzer can provide structural information very similar to that obtained by the much more costly tandem MS technique. Recent legislation enacted in many european countries (members of the European Community, EC) states that pesticides must not exceed the 100 ng/L level in waters intended for human consumption. In order to judge with sufficientconfidence whether a water sample is in compliance with this EC Directive, analytical methods able to detect pesticides at 20-30 ng/L levels are needed. In the recent past, we elaborated a very sensitive and rapid LC/ W method able to analyze more than 120 pesticides in aqueous environmental samples at the ng/L le~e1.~-6 This method involves the use of a 1-g graphitized carbon black (GCB) reversible cartridge for extracting pesticides from water samples. Passing sequentially through the cartridge two suitable solvent systems, isolation of acidic pesticides from nonacidic ones was successfully achieved by differential elution. Class fractionation was made possible because the GCB surface framework is contaminated by (1) Conboy, J. J.; Henion, J. D.; Martin, M. W,; Zweigeinbaum, J. A. Anal. Chem. 1990,62,800-807. (2) Popenoe, D. D.; Moms, S. J.: Hom, P. S.; Norwood, IC T.Anal. Chem. 1994, 66, 1620-1629.
(3)Voykner. R. D.; Pack, T. Rapid Commun. Mass Spectrom. 1991,5,263268. (4) Di Corcia, A,; Marchetti, M. Anal. Chem. 1991,63, 580-585. (5) Di Corcia, A; Marchetti, M. Environ. Sci. Technol. 1992,26, 66-74. (6)Di Corcia, A,; Marcomini, A; Samperi, R.; Stelluto, S. Anal. Chem. 1993, 65, 907-912. 0003-2700/95/0367-1968$9.00/0 0 1995 American Chemical Society
some positively charged adsorption sites that enable this material to behave as both a nonspecific sorbent and an anion e x ~ h a n g e r . ~ , ~ The purpose of this work has been to evaluate the potentiality of coupling our sample preparation procedure with the high confirmational power of the LC/ES/MS system for monitoring unambiguously acidic pesticides in drinking water and surface water samples. EXPERIMENTAL SECTION Reagents and Chemicals. Authentic acidic pesticides were purchased in part fom Eurobase (Milano, Italy) and in part from Riedel-de Haen (Seelze, Germany). The compounds considered in this work are listed below, together with their common names and pK, values (in parentheses): 3,&dichloro-2-methoxybenzoic acid (dicamba, 1.9); 2-isopropyl-(lL!)-bem2,1,3-thiadi&4 (3H)one 2,Y-dioxide (bentazone, 3.2); 2,4dinitrophenol (2,4DNPh, 4.5); khloro-2-oxo-3(2H)-benzothiazoleaceticacid (benazolin, 3.6); 3-(a-acetonylfurfuryl)-4hydroxycoumarin(coumafuryl, 4.9); 3 , s dibromo-4-hydroxybenzonitrile (bromoxynil, 4 4 ; d,&dinitro-& cresol @NOC, 4.7); 4hydroxy-3-(3-oxo-l-phenylbutyl)coumarin (warfarin, 4.9); 2-methyl-4chlorophenoxyaceticacid (MCPA, 3.1); 2,4dichlorophenoxyaceticacid (2,4D, 2.6); 3,5diiodo-2-hydroxybenzonitrile (ioxynil, 3.96); 2-(%methyl-4chlorophenoxy)propionic acid (mecoprop,3.8); 2-(2,4dkhlorophenoxy)propionic acid (dichlorprop, 3.0); 2,4,5trichlorophenoxyaceticacid (2,4513 2.1); Zmethyl4chlorophenoxybutyric acid (MCPB, 4.8) ; 2,4dichlorophenoxybutyric acid (2,4DB, 4.8); 2-(2,4,5trichlorophenoxy)propionic acid (2,4,5TP, 3.3); 2-sec-butyl-4,&dinitrophenol(dinoseb, 4.6); 2-tert-butyl-4,6dinitrophenol(dinoterb, 4.6); and pentachlorophenol (PCP, 4.7). Although 2,4DNPh is not used in agriculture as a pesticide, it was included in this study as it is a metabolite of pesticides largely used in agriculture. Linear octyl benzenesulfonate (C-8 LAS) sodium salt was from Fluka AG (Buchs, Switzerland) and was used as internal standard (IS). h e a r alkyl benzenesulfonate (L&)compounds are important surfactantsthat can be present in environmental waters. In spite of this, C-8 LAS was chosen as IS as commercial formulations invariably contain (2-10 through C-14 LAS mixtures. Individual standard solutions were prepared by dissolving 50 mg of each pesticide in 50 mL of acetonitrile. A composite working standard solution was prepared by mixing 0.5 mL of each pesticide standard solution and diluting to 50 mL with acetonitrile. For LC, distilled water was further purified by the Milli-Q RG apparatus (Millipore, Bedford, MA?). Methanol "plus" and acetonitrile "plus" of LC grade were from Carlo Erba (Milano, Italy). kHP04, trinuoroacetic acid ('FA), and various quaternary ammonium salts were from Fluka AG. All other solvents were of reagent grade and were used as supplied. GCB, commercially referred to as Carbograph 1or Carbopack B, was kindly supplied by Alltech (Deerfield, IL), while the other materials for preparing extraction cartridges were from Supelco (Bellefonte, PA). The preparation and pretreatment of the 1-g reversible extraction cartridges were carried out as previously r e p ~ r t e d .With ~ respect to this work, the design of the cartridge was sligthly modified in that the Teflon piston had a Luer tip and (7) Campanella, L.; Di Corcia, A; Samperi, R; Gambacorta, A Mater. Chem. 1982, 7, 429-438. (8) Di Corcia, A; Marchese, S.; Samperi, R j. Ch~omatogr.1993, 642, 163174. (9) Di Corcia, A.; Marchese, S.; Samperi R j. Assoc. OfiAnal. Chem. 1994, 77, 446-453.
was inserted into the syringetype cartridge after water extraction and washings before the baseheutral fraction was eluted. The trap was fitted into a sidearm filtering flask, and liquids were forced to pass through the cartridge by vacuum from a water Pump. Grab samples of river water (from the Tiber River) were collected in empty solvent bottles and stored at 4 "C until used. Procedure. The extraction of the analytes from aqueous samples was accomplished in a manner similar to that previously d e ~ c r i b e d .After ~ , ~ water sample extraction, the cartridge was sequentially washed with 6 mL of distilled water and 1 mL of methanol. Thereafter, a Teflon piston was forced to enter the cartridge that was turned upside down. The cartridge was firstly back-flushed with another 1mL of methanol, followed by 6 mL of a methylene chloride/methanol(80/20 v/v) solution to wash out coextracted baseheutral subtances, and then with 6 mL of methylene chloride/methanol(80/20 v/v) acidified with 0.2%(v/ v) TFA to elute acidic species. Before solvent removal, the acidcontaining extract was partially neutralized by addition of 50 pL of a water/methanol solution of ammonia obtained by diluting 2 mL of concentrated ammonia to 10 mL with methanol. When the acidic extract was dried without the addition of ammonia, severe losses of those pesticides that are more readily esterified by methanol in an acidic ambient, especially coumafuryl and warfarin, were noted. Furthermore, 100 pL of a 15 ng/pL C-8 LAS methanolic solution was added as internal standard. After exhaustively drying the extract in a water bath at 40 "C under a stream of nitrogen, the residue was reconstituted with 200 pL of a water/methanol (70/30 v/v) solution containing 4 mmol/L &HPOl and 0.2 mmol/L tetrabutyhmonium fluoride 0'BA.F'). A 50-pL portion of the final extract was injected into the LC column. Lc/uV/Es/MS Analysis. Liquid chromatography was carried out with a Varian (Walnut Creek, CA) Model 9010 instrument equipped with a Rheodyne Model 7125 injector having a 50-pL loop. The analytes were chromatographed on an Alltima 25cm x 4.hI"mi.d. column Wed with 5mp C18 reversed-phase packing (Alltech). Phase A was a methanol/acetonitrile mixture (85/15 v/v), and phase B was water. Both phases contained 0.1 mmol/L ~ H P O and I 0.2 mmol/L TBAF. The initial composition of the mobile phase was 30%A, increased linearly to 75%A over 40 min. The flow rate of the mobile phase was 1mL/min. A 30-pL portion of the column effluent was diverted to the ES source, while the rest of the mobile phase was delivered to a Model 2550 W detector (Varian) set at 22@nmwavelength. A Fisons VG Platform benchtop mass spectrometer (Fisons Instruments/VG BioTech, Milano, Italy) equipped with a pneumatically assisted electrospray LC/MS interface and a single quadrupole was used for analyzing target compounds in the column effluent. This was introduced into the ES interface through a 4oCm length of 75pm-diameter poly(ether ether ketone) (PEEK) capillary tube. The mass spectrometer was operated in the negative ion mode by applying to the capillary a voltage of 2.9 kV, while the skimmer cone voltage was set at 20 V. Mass spectra collected in full-scan mode were obtained by scanning over the range 139 5 m/z 5 380 in 2 s. The source temperaturewas maintained at 60 "C. Ions were generated using highly pure air as drying and nebulizing gas. The optimum flow rates of the drying and nebulizing gas were found to be 300 L/h and 13 mL/min, respectively. On a weekly basis, the counter electrode and the sampling cone were cleaned with a concentrated nitric acid/water (1/1 v/v) Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
1969
mixture and 10 min of sonication. Thereafter, they were extensively washed with distilled water, followed by acetone and methanol. The entire cleaning procedure required no more than 25 midweek. For recovery studies, the concentrations of pesticides in water were calculated by measuring the peak area for each analyte relative to that of the IS and comparing those results with those obtained from standard solutions. The standards were prepared by dissolving known and appropriate volumes of the working standard solution in the eluant phase used for desorbing acidic pesticides from the Carbograph surface and then following the procedure reported above. For those pesticides producing overlapping peaks, recoveries were calculated by measuring peak areas obtained after extracting chromatograms for selected m/z values. The mass spectrometry data handling system used was the Mass Lynkx s o h a r e from Fisons Instruments. RESULTS AND DISCUSSION In our previous work? LC fractionation of acidic pesticides was performed by using the ion suppression technique with addition of a suitable amount of TFA to the mobile phase. Under this chromatographic condition and with the ES/MS detector operating in the positive mode, however, very low signals for the analytes were obtained because of the scarce tendency that un-ionized acids have to form adducts with positive ions. Thus, the ion suppression technique was replaced with the ion pair (IP) technique by adding suitable amounts of HP0d2- and a quaternary ammonium salt. In this way, the analytes were delivered to the ES source operating in the negative mode as ‘‘preformed”ions. Typical IP-LC methods discussed in the literature involve concentrations of buffers and quaternary ammonium salts in the mobile phase between 5 and 20 mmol/L. It is known that a severe limitation of the ES interface is that it cannot afford mobile phases containiig such relativelyhigh salt concentrations, as they provoke signal instability and, ultimately, electrical breakage.1° Also, mobile phases rich in salts can rapidly occlude the sampling cone orifice, thus precluding ions from reaching the mass analyzer region. Therefore, our efforts were devoted to minimizing the concentrations of both the buffer and the quaternary ammonium salts dissolved in the mobile phase while still obtaining sharp and well-retained peaks for the target compounds. The addition of only 0.1 mmol/L &HP04 to the mobile phase sufficed to give a pH of 7.1, thus ensuring dissociation of the acidic species considered. With the view of optimizing the chromatographic performance of the analytical column and achieving well-defined ion signals for the analytes, the effects of the nature of the ion pair forming agent and its counterion as well as its concentration in the mobile phase on the two parameters discussed above were investigated. The effect of varying the alkyl chain length of the quaternary ammonium salt, Le., tetramethyl, tetraethyl, tetrapropyl, tetrabutyl, and tetrapentylammonium chloride, dissolved in the column effluent on the ion signals is shown in Figure 1. This experiment was conducted by dissolving 0.2 mmol/L of the various quaternary ammonium salts considered in 0.1 mmol/L HP042- buffered water/methanol (50/50 v/v) mixtures and measuring the ion signal intensities relative to known amounts of three selected (10) Hopfgartner. G.; Wachs, T.; Bean, IC; Henion, J. Anal. Chem. 1993,65, 439-446.
1970 Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
600 -
Methyl
I
I
I
Ethyl
Propyl
Butyl
Pentyl
Figure 1. Ion signal for three selected analytes obtained by varying the alkyl chain length of the quaternary ammonium ion dissolved in the mobile phase. (m) Bromoxynil, (A)2,4-D,and (0)dicamba.
pesticides. These compounds were separately and directly injected into the ES source by the flow injection analysis PIA) technique. Measurements were made in duplicate. As can be seen, a steady decrease in the signals was observed as the alkyl chain length was increased. This result may be explained on the basis of two considerations. One is that the extent of solvation of the quaternary ammonium ion decreases as the alkyl chain length increases, with the result that more and more stable ion pairs with analyte anions are formed. The other is that the electrospray process produces only a partial charge separation, so that the electrosprayed solution still maintains, during the sequence of events leading ultimately to gas phase ion formation, a certain amount of quaternary ammonium ions. Thus, anions leave the charged liquid droplets in part as such and in part recombined with the quaternary ammonium ions. From these two considerations, it follows that the recombination process will decrease the ion signal intensity to the extent that quaternary ammonium ions are able to form stable ion pairs with analyte anions. It is worth noting that the ion signal intensity was also dependent on the nature of the analyte. This effect could be accounted for by considering that, as found by Hiraoka et al.,” the less volatile the organic ion, the more is its tendency to recombine with quaternary ammonium ions. Thus, less volatile ions will be detected with lower sensitivity by the ES/MS system. From a chromatographic point of view, it was observed that mobile phases containing quaternary ammonium ions with short alkyl chains were unfortunately unable to elute the most polar analytes with sufficiently high retention times or to produce untailed peaks for the earlyeluting phenol derivative analytes, such as 2,4DNPh, DNOC, bromoxynil, and, to a lesser extent, ioxynil. These two adverse effects could be in part lessened by both decreasing the organic modifier percentage and increasing the ion pair forming agent concentration in the mobile phase. Under these conditions, however, a significantly weaker and less stable ion signal for the analytes was observed. Definitively, the best compromise was reached by adding to the buffered mobile phase a suitable amount of tetrabutylammonium (TBA) ions. (11) Hiraoka, IC;Murata, K ; Kudaka, I. Rapid Commun. Mus Spectrom 1993, 7, 363-373.
I
Table I.Effect of the Nature of the Counterion of the Ion Pair Agent on the ion Signals of Some Selected Anaiytes
600
ion signals compound 2,4D
F68
mecoprop bromoxynil
65 139 86
PCP
C138 32 108
Br-
I-
25 20 113
60
64
20 15 105 55
Arbitrary units.
400
-
2m
3
200
The effect of the nature of the counterion of the quaternary ammonium salt on the response of the ES/MS detector for the analytes considered was evaluated. These experiments were conducted under FIA conditions by introducing separately into the ES source equal amounts of some selected analytes, namely, 2,4D, mecoprop, bromoxynil, and PCP. The liquid phases were 0.1 mmol/L HP0d2- buffered water/methanol (50/50 v/v) mixtures containing equal concentrations (0.2 mmol/L) of various TBA salts having respectively F-,C1-, Br-, and I- as counterions. Measurements were made in duplicate. Results are reported in Table 1. As can be seen, for the conjugated bases of the two phenoxyacids, 2,4D and mecoprop, a remarkable decrease in the signal was observed on changing from F- to C1- as counterion. Although to a lesser extent, this trend was maintained by further increasing the counterion size. The behaviors exhibited by bromoxynil and PCP were counterintuitive. On steadily increasing the size of the counterion, their signals first decreased and then increased. These findings could be explained by making some assumptions. One is that competition effects occurring between halide ions and analyte ions in forming stable ion pairs with residual tetrabutylammonium ions present in the electrosprayed aerosol can affect the transition efficiency of organic anions to the gas phase. Among the counterions considered, the fluoride ion has the highest charge density, and thus a second reasonable assumption is that it forms the most stable ion pair with a quaternary ammonium ion. When these assumptions are accepted, the highest analyte ion signals obtained with F- as counterion can be accounted for by a decrease in the actual concentration of residual TBA- ions in the electrosprayed solution. With respect to 2,4D and mecoprop, differences in the behaviors of the ionized forms of bromoxynil and PCP could be explained assuming that the latter two species are more volatile, and so their transition to the gas phase is less affected by recombination processes. Studies performed by Raffaelli and Bruins12with the view of assessing whether the nature of the counterion could affect the transition efficiency of quaternary ammonium ions from an extremely diluted electrosprayed methanol solution to the gas phase led these authors to the conclusion that the small differences observed could not be considered significant. Nevertheless, their experimental results appear to partially substantiatethe fkst two assumptions reported above, as they obtained a definite decrease in the ion current for the TBA- ion on replacing C1with F- as counterion. The dependence of the ion signal for some selected analytes on the concentration of TBAF present in the mobile phase was (12) Raffaelli, A.; Bruins, A. P.Rapid Commun. Mass Spectrom. 1991,5,269275.
0
I 0
0.1
0.2
0.3
0.4
mmollL TBAF
Figure 2. Ion signal for three selected analytes vs the concentration of tetrabutylammonium fluoride in the mobile phase. (W) loxynil, (A) mecoprop, and (0)MCPA.
I
300
200
{
j
0
50
100
150
200
amount injected, ng
Figure 3. Ion signal vs amounts of two selected analytes injected into the LC column. ( 0 )Bromoxynil and (W) 2,4-D.
assessed. For this experiment, increasing concentrations of TBAF were added to buffered water/methanol (50/50 v/v) mixtures. Measurements were made in duplicate. Results reported in Fgure 2 show that the ion signal intensities were severely affected even by the presence of a small concentrationof TBAF in the buffered solution. This indicated once more that significant recombination effects taking place between the ion pair forming agent and the analyte ions in the electrosprayed droplets deeply influence the ion transition yield to the gas phase. From a chromatographic point of view, the addition of 0.2 mmol/L TBAF to the mobile phase sufficed to satisfy the requirement of well-retained and sharp peaks for the analytes considered. Under the chromatographic conditions adopted, the linear dynamic range and the absolute sensitivity of the ES/MS detector were estimated. These measurements were performed by injecting in triplicate into the LC column known and variable amounts of bromoxynil and 2,4D and measuring their peak areas by extracting chromatograms relative to m/z 274-279 (2,gdibromo4cyanophenoxy ion) and 161-166 (2,4dichlorophenoxyion)
+
Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
1971
Table 2. Variation of the Ion Signal Intensity and the Relative Abundance of Diagnostic Fragments for Some Selected Analytes on Varylng the Voltage Applled to the Skimmer Cone
signalhoise [m/z (relative abundance)1 20 v 30 V
10 v
compound dicamba
125
270
bentazone
145
360
[220 (lo), 176 (loo)]
[22@(go), 176b(loo)]
[239 (100) I
[239 (loo)]
40 V
124 [220 (6), 176 (loo)]
45 [220(lo), 176 (loo)]
560
530
[239 (loo)]
[239 (100)
ioxynil
420
970
1080
900
2,4-D
110
283
530
715
dich 1orprop
100
360
584
650
2,4 DB
40
280
320
dinoseb
200
650
PCP
190
[370 (loo)]
[370 (loo)]
[220 (loo), 161 (go)]
[234 (60), 161 (loo)]
[234 (loo), 161 (511 [248 (loo), 161(50)1
[248 (lo),
161(100)]
[239 (loo)]
[239 (loo)]
(234 (lo),
[220 (lo), 161 (loo)]
161 (loo)]
[234 (5), 161 (loo)]
380
[248 (3), 161 (loo)]
[248 (5), 161(100)1
940
1040
548
420
E239 (100)1
400
[265 (100)I
(370 (loo)]
[370 (100)1
[220 (loo), 161 (90)l
[220 (loo), 161d(5)l
[239 (loo), 193e(20)l
[265 (loo), 2301 (lo)]
1265 (100)I
196c(20)l
[265 (loo), 230 (20)]
4 The value for the molecular ion is indicated in italic type. m/z relative to 3,6-dichloro-2-methoxrphenoxy ion. m/z relative to 1H-benzo2,1,3-thiadiazin-4-one2,Zdioxide ion. m/z relative to 2,4dichlorophenoxy ion. e m/z relative to 2-sec-butylnitrophenoxyion. f m/z relative to tetrachlorophenoxy ion.
250
First Day
, ,
I
/
Second Day
I
MS
c 200
-0 150 c
0)
5
100
-Sample Cone Cleaning
uv
50
0
I
1Oam
3pm
8pm
loam
3pm
8Pm
time Figure 4. Variation of the MS and UV signals for three selected analytes over 2 working days. (A)loxynil, (W) dichlorprop, and (O),2,4-D.
219-225 (2,4dichlorophenoxyacetate ion). The column was operated isocratically with 50%of the organic modfier. As can be seen from the plot in Figure 3, the response of the ES/MS detector was linearly related to injected amounts of the two model compounds from 2.5 to 200 ng. By measuring the signal-to-noise ratio (S/N) from extracted chromatograms relative to the lowest injected amounts of both bromoxynil and 2,4D, the absolute limits of sensitivity (S/N = 3) of the ES/MS were set at about 0.3 and 1 ng, respectively. As the response of the ES/MS detector is related to the analyte concentration in the mobile phase, the absolute limits of detection reported above could be significantly decreased by adopting narrower LC columns. However, in our experience and as already extensively discussed by Hopfgartner et al.,I3the use of small-bore LC columns does not offer practical advantages, since lower extract volumes of actual samples must 1972 Analytical Chemistry, Vol. 67,No. 73,July 7, 7995
be injected into these columns to avoid overloading effects and retention time variations of the early-eluting analytes. For unambiguous confirmation of the presence of a certain target compound in complex matrices, a spectrum having at least one diagnostic daughter ion in addition to the corresponding molecular ion is desirable. As mentioned above, structural information on analytes can be simply obtained by suitably adjusting the potential difference set between the extraction and skimmer cones to produce collision-induced dissociation (CID) of ions in the region of intermediate pressure. In addition, an increase of the skimmer cone voltage usually enhances the S/N for an analyte by reducing the background ion current and (13) Hopfgartner, G.; Bean, IC;Henion, J.; Henry, R. J. Chromatogr. 1993,647, 51-61.
Table 3. Percentage Recovery ( n = 6) of 20 Acidic Pesticides Added to 1 4 River Water and 4 4 Drinking Water Samples’
compound
dicamba bentazone 2,4-DNPh benazolin coumafuryl bromoxynil DNOC warfarin MCPA 2,4D ioxynil
mecoprop dichlorprop 2,4,5T MCPB 2,4-DB
2,4,5TF’ dinoseb dinoterb PCP
recovery f RSD,” % drinking water river water 99 f 3 96 f 3 97 f 4 93 f 4 86 f 6 98 f 3 102 f 4 86 f 7 97 f 3 94 f 4 96 f 3 95 f 4 96 f 3 97 f 4 96 f 3 97 f 4 97 f 4 94 f 4 95 f 3 99 f 3
t
94 f 2 101 f 3 98 k 4 96 f 3 94 f 4 99 f 2 97 f 5 93 f 3 98 f 4 100 f 4 97 f 6 95 f 5 97 f 4 94 f 5 93 f 4 94 f 5 96 f 3 95 f 3 94 f 4 97 f 3
5
10
a Spike levels: 1 pg/L for river water and 0.1 pg/L for drinking water. RSD = relabve standard deviation.
improving the ion focusing effect.14 The effects of increasing the cone voltage on both the response of the MS detector and the production of diagnostic ions were investigated. This experiment was conducted by introducing into the source known amounts of eight selected analytes via FIA using a water/methanol (50/50 v/v) mixture containing 0.1 mmol/L &HP04 and 0.2 mmol/L TBFA and calculating S/N values at an extraction cone voltage increasing from 10 to 40 V. Measurements were made in triplicate. Results are reported in Table 2. Except for dicamba, s i g n i h n t increases of the S/N values for the other species considered in this experiment were obtained on increasing the skimmer cone voltage from 10 to 30 V. As expected, this increase was due to both lower background noise and higher peak heights. By further increasing the cone voltage from 30 to 40 V, the S/N for some of the analytes decreased, presumably due to the fact that high-energy collisions generated small daughter ions having m/z out of the mass scan range selected (see the Experimental Section). For dicamba, the CID process generated C1- ions whose abundance increased as the cone voltage was increased from 20 to 40 V, as observed by decreasing the lower limit of the m/z scan range down to 30. Large differences were noted in the stability of the analyte ions. Within the cone voltage range reported in the Table 2, no daughter ion was obtained for ioxynil. Only by setting the cone voltage at 50 V was it possible to detect a fragment produced by the loss of a iodine atom. At 40 V, the fragmentation of the dinoseb and bentazone parent ions by the CID process generated two diagnostic ions, resulting from the respective losses of a nitro group and an isopropyl group. From the fragmentation of the ionized forms of the three phenoxyacids included in this experiment, i.e., 2,4D, 2,4dichlorprop, and (2,4 DB), the same daughter ion, Le., 2,rlrdichlorophenoxyion, was generated by CID. The relative abundance of this charged fragment was dependent upon the nature of the parent ion and (14) Duffin, IC L.; Wachs, T.; Henion, J. D.Anal. Chem. 1992, 64, 61-68.
I
’
fi
5.00
10.00
1500
Z0.W
25.00
30.00
35.00
Figure 5. TIC chromatogram obtained by injecting 50/200 of a final extract relative to 1 L of a river water sample (from the Tiber River) spiked with the acidic pesticides at the level of 0.5 pg/L each: (1) dicamba; (2) bentazone, (3), 2,4-DNPh, (4) benazolin, (5) coumafuryl, (6) bromoxynil, (7) DNOC, (8) warfarin, (9) MCPA, (10) 2,4-D, (1 1) ioxynil, (12) mecoprop, (13) dichlorprop, (14) 2,4,5-T, (15) MCPB, (16) 2,4-DB, (17) dinoseb, (18) 2,4,5-TP, (19) dinoterb, and (20) PCP; IS,C-8 LAS.
the voltage applied to the skimmer cone. Analogous behavior was exhibited by the three ionized forms of three phenolcyacids (MCPA, mecoprop, and MCPB), differing from those mentioned above in that one chlorine is replaced by a methyl group. In this case, the CID process produced a daughter ion at m/z 141, corresponding to the 2-methyl-4-chlorophenoxyion. To obtain a good response of the MS detector for all of the analytes considered, we chose to set the skimmer cone voltage at 20 V. To obtain diagnostic ions even from the most stable analytes considered, the only way could be to appropriately modify the cone voltage during the chromatographic run. The robustness of this method was tested over two uninterrupted working days. This investigation was conducted by injecting at 1-h intervals 25 ,uL of a solution containing 2,4D, ioxynil, and dichlorprop at the level of 4 ng/L each into the LC column and measuring the peak areas produced by both the MS and UV detectors for the three model compounds. The column was operated isocratically with 50% of the organic modifier. Between injections, the colum effluent was continuously delivered to the ES source. Signals were plotted vs time, and the resulting graph is shown in Figure 4. Over the initial 10 h of the first Analytical Chemisiy, Vol. 67, No. 13, July 1, 7995
1973
XM ES20 263-267 /I 3%S &*a
100-
'I I
Scan ES.
12
100-
i
l41_144 6.17*5 .-
I
I
I l ~
A
0 1600
1800
2000
2200
,I
2400
&*I
15
1 1
2600
2800
n 30W
32W
3400
Figure 6. Selected ion current chromatograms for selected m/z values extracted from the chromatogram in Figure 5. Table 4. Time-Scheduled SlM Conditions for Monitoring 20 Acidic Pesticides in Drinking Water Samples.
compound
selected m/z
start-end times (min)
dicamba bentazone 2,4DNPh benazolin coumafouryl bromoxynil DNOC warfarin MCPA
176 239 183 170,198 297 275 197 307 142,200 162,220 370 142,213 162,234 255 142 162 196 239 239 265
14.0-19.0
2,4-D
ioxynil mecoprop dichlorprop 2,4,5-T MCPB 2,4-DB 2,4,5TP
dinoseb dinoterb PCP a
19.0-26.6
26.6-31.5
31.5-36.0
Dwell time, 0.1 s; span, 1.
working day, ion signals for 2 , 4 D and dichlorprop did not show signi6cant variations. Conversely, a steady signal loss for ioxynil was observed after the instrumentation was operated uninterrupted for 8 h. After 10 working hours, a 50%signal loss for ioxynil 1974 Analytical Chemistry, Vol. 67, No. 73,July 7, 7995
I W
1000
1500
2000
2500
3000
3500
It 4000
Figure 7. TIC chromatogram obtained by injecting 50/200of a final extract relative to 4 L of a drinking water sample spiked with the acidic pesticides at the level of 100 ng/L each. Peak numbering is the same as in Figure 5.
was observed. This difference in behaviors between the two phenoxyacids and ioxynil was unclear to us. A check of the sample cone revealed the presence of a very thin salt layer. With the system beyond the cone still under vacuum, cleaning the sample cone was easily addressed with use of a methanol-imbibed paper, taking care to pass it repeatedly over the cone orifice to dissolve salt particles that could partially obstruct the opening. As a result of this cleaning procedure, the ion signal for ioxynil was restored. A similar behavior was observed on the second day, with the difference that, after 9 working hours, a signifcant signal decrease occurred even for 2 , 4 D and dichlorprop. In addition, this signal loss was accompanied by a largely unstable background current. Again, the initial condition in terms of S/N was restored after the extraction cone cleaning procedure mentioned above. As expected, the W signals for the three model compounds did not show any significant variation over the two working days. For a routine application of the method under evaluation, the results obtained by the test described above indicate that the best solution could be to exploit the W signals for analyte quantitation while entrusting unambiguous analyte identitication to the ES/MS detector. The present analytical procedure was applied to the determination of acidic pesticides in drinking water and river water samples.
rt
Figure 8. U$-and SIM chromatograms obtained by injecting 50/ 200 of a final extract relative to 4 L of a drinking water sample spiked with the acidic pesticides at the level of 10 ng/L each. Peak numbering
is the same as in Figure 5. The spike recoveries of the overall isolation and chromatographic procedure are reported in Table 3. Pesticides were added at the indicated concentrations to six individual samples of each of the two matrices considered and analyzed. When recovery experiments were performed in hypochloritecontaining tap water samples, 0.5 g of Na&O3.5HzO/L of water was added before addition of the analytes, to prevent their oxidation and other unwelcome effects previously di~cussed.~
Figure 5 shows a TIC mass chromatogram obtained by spiking 1 L of a pesticidefree river water sample fortifled with the 20 analytes considered at the level of 0.5 pg/L and analyzing this sample by the procedure described above. As can be seen, distinct peaks were obtained for all of the analytes included in this study. By extracting chromatograms for selected m/z values corresponding to some selected analytes, Figure 6 shows that the speciiic determination of acidic pesticides in surface water samples can be achieved at concentrations far below 0.5 pg/L. A 4-L drinking water sample was spiked with the pesticides under evaluation at the level of 100 ng/L each and analyzed by the procedure described above. The TIC mass chromatogram shown in Figure 7 demonstrates that this method can satisfy the stringent requirements imposed by the EC Directive mentioned above. In terms of sensitivity and specificity, the potentiality of the ES/MS instrumentationused was fully exploited by extracting a 4-L drinking water sample fortified with the analytes at the level of 10 ng/L each and analyzing the final extract by a timescheduled, selected ion monitoring (SIM) scheme, the results of which are reported in Table 4. The SIM mass chromatogram obtained by following this procedure (Figure 8) shows that unambiguous identification of acidic pesticides in water can be performed even at concentrationsbelow 1ng/L. The corresponding W trace reported in the same figure shows that distinct peaks can be obtained for few a nanograms of any injected pesticide under study. For the acidic pesticides under evaluation, considering the analysis of 4 L of drinking water, reconstituting the residue with 200 pL, and injecting 50 p L of this, the quantification limits estimated from the W signals (5 times the limits of detection) ranged between 8 and 30 ng/L. For pesticides in surface water samples, the &res reported above should be increased of a factor 4,as only 1L of surface water can be extracted by this procedure without loss of the most polar pesticides. ACKNOWLEDGMENT We wish to thank the Consiglio Nazionale delle Ricerche for financially supporting this work. Received for review November 30, 1994. Accepted March 1, 1995.@ AC9411637 Abstract published in Advance ACS Abstracts, May 1, 1995
Analytical Chemistty, Vol. 67,No. 13, July 1, 1995
1975
