any countries have now passe rislation to ensure that pes,ides are used safely. This activity has brought a greater role for analytical chemistry in protecting the public from unsafe levels of pesticide residues. Traditionally, pesticides at levels of concern have been identi6ed by GC with e l e ment-selective detectors. These methods often required a check analysis by another scientist and lacked the ability to positively identify the analytes. With the development of MS, scientists were able to provide structural confirmation so that FDA could initiate consumer protection. In this article we will focus on the forensic role of MS in providing unambiguous proof of trace levels of pesticide residues.
L
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How do they get in our food?
Pesticidescan be introduced into the food chain in several ways. ?he legal and intelli-
Thomas Caims Richard A. Baldwin U S . Food and Drug Administration
Pesticide residue analysis The 1980s brought concerns about environmental issues such a s dioxins and polychlorinated biphenyls (1,Z).As a result, there was a quantum leap in monitoring capabilities, and regulatoty scientists could routinely measure contaminants at the part-per-trillion level. This advance was possible because of the reliability of MS and the strengths it offered in gent use of insecticides,fungicides, and mi. terms of reproducibility, repeatability, specificity, and limits of detection. ticides to curb infestations and increase Confirmationof trace levels of pesticrop yield generally produces pesticide re& cides detected by various elementdues at or below the legal tolerance level. sensitive (e.g., P, s,N, CI) GC detectors When pesticides are applied to crops for which they are not yet registered, residues using a multiresidue screening procedure has led to reliance on MS because of its at any level constitute a violation of law. ability to help define structure (3).In addiOnce pesticides are applied, they and their metabolites often persist for long perids in tion, regulatory scientists must be able to rigorously support their findings so that the environment, which can be viewed as an indirect route to transpofl in the ecosys proof can be provided in court. In a criminal case, scientific proof must meet the tem. Fiially, both nondeliberate contamitest of “beyond a reasonable doubt”; in a nation of the food supply via chemicalsa p proved for industrial applications only and civil matter, the standard is “the preponderance of evidence.” Because MS condeliberate criminal contamination to re firmation methods are often developed on ceive media attention or cause public reacan ad hoc basis to deal with an acute situtinn sometimes o c m .
MS allows detection
of pesticide residues in food at the part-per-billion level
552 A Analytical Chemistry, September 1, 1995
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ation, they cannot always be validated by interlaboratorytesting. What has emerged over the past decade is a set of evidentiary criteria generally recognized as scientifically sound. The highest level of confirmationthat can be provided by MS is the exact correlation b e tween the full mass spectral scans of a reference standard and the sample performed within the same analytical conditions. Usually electron ionization (EI) spectra contain sufficient structurally r e lated fragment ions to permit absolute identification. Under such conditions, the relative abundance ratios should experimentally fall within 5%quite often, however, the presence of background ions may severely interfere with exact comparisons. This practice of direct comparison represents the highest level of specificity obtainable by MS. In tracelevel analyses,however, using full mass spectral scans for confirmation is often impractical. To fully enhance sensitivity, multiple ion detection (MID) offers a conveniently efficient method of ignoring potential intetferencesand concentratingon ions that belong to the compound under im vestigation. It is in this area that the evolving criteria for continnation have received the most attention. Scrutiny has focused on the exact number of ions to be monitored to provide proof of presence. Setting this criterion has been complicated because of the many MS techniques available for analyzing food samples. More than a decade ago, Sphon argued that a minimum of three structurally related ions would he necessiuy to provide proof of presence (4). This assumption was based on a statistical approach using an extensive MS database as a model of a universal repositoly containing all possible organic compounds. Without paying attention to relative abundance ratios, three ions were r e quired to eliminate compounds with similarfragment ion selections from consideration. To improve the criteria for confirmation, the relative abundance ratios were required to he within 5%when compared with a reference standard recorded under
similar conditions. Evolution of new techniques, particularly soft ionization methods, has prompted a reinvestigation of supporting evidence for confirmation b e cause little or no fragmentation is observed. Over the past two decades, several key reviews of trace analysis by MS (5-8)have reported specific case histories illustrating the ability of various techniques and their hybrids to provide identificationand
confirmation.The evolution of the criteria for confirmation of trace residue levels in food and drugs has been discussed in detail using arguments derived from experimental case histories of what constitutes proof of presence (9),and these lend sup port for the three-ion concept. Supporting evidence provided by the GC or U:r e tention time, as well as appropriate sample preparation and cleanup, has been recognized as fundamentally important.
m/r 193 P-Mevinphos
m h 152, 181 Tetrahydrophthalimide 1I
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Figure I.Analysis of a tomato extract spiked with methamldophos. pmevinphos, tetrahydrophthallmlde,dlmethoate, dlchlofluanid, chlorpyrilos, folpet, and qpDDE. (a) Total ion chromatogram and (b) single ion monitoring detection.
Analytical Chemisfry, September 1, 1995 553 A
Chemical ionization of pesticides Chemical ionization (CI) techniques to favor the production of a protonated molecular ion for characterization have been the cornerstoneof resolving both the identi6cation and confirmation of pesticide residues over the past decade (10).The power behind this strategy is based on the fact that under CI only a few fragment ions, in addition to the protonated molecule ion, appear in the mass spectrum. In the case of EI, many more fragment ions are produced, and often the molecular ion is a b sent. The advantage of CI over E1 as the ionization mode for pesticides is the concurrent reduction in the number of background ions belonging to the ma&. The appearanceof a protonated molecular ion for the target pesticide is a key structural piece of evidence indicating the molecular weight of the compound. Unlike element-selective detectors, the mass spectrometer detects all eluting co pounds resulting from the GC analysis o a fruit or vegetable extract. Such extracts contain many matrix compounds representing flavor compounds or pyrolysis products from various thermally labile macromolecules characteristic of the crop being analyzed. The resulting chromatcgram under CI thus contains a large number of interfering compounds among the actual pesticide residues. Figure 1 illustrates the total ion chromatogram obtained from a spiked tomato extract (8different pesticides) at the 0.05 ppm level. Although CI has the ability to simplifythe chromatogram by producing mainly pre tonated molecular ions, the number of interfering matrix compounds is still relatively b e . Interrogation of this chromatogram for the various pesticides can be conducted by specifyingone of the major ions (usually the protonated molecular ion) for each pesticide and having the data system detect each of the pesticides at the correct re tention time. This process of data reduction is referred to as singleion monitoring (SIM). Detection, however, is contingent upon lolowing which ion should he used to detect each pesticide. In practical residue analysis, the necessary level of sensitivity (50 ppt) can be reached only by mass scans for the major ion (i.e., a peak at a specific amu) belonging to the pesticide
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Figure 2. Effect of interfering substances on confirmation of identity of aflatoxin B,. (a) NCI mass spectrum of aflatoxin6, standard, (b) NCI mass spectrum of sample in which the presence of aflatoxin B, is confirmed,and (c) NCI mass spectrum of sample in which aflatoxin 8, is detected but not confirmed.(Adaptedwith permission from Reference 11.)
and not full mass scans covering a wide range of mass (60-500 amu). In MID, three structurally related ions belonging to a target pesticide are scanned as unit mass ranges one after the other, culminating in three chromatograms providing concurrent confirmation of presence. Case histories Full-mass scans. In the case of fullmass spectral scans derived under the various ionization techniques (e.g., EI, CI, NCI, FAB), the reference standard and the sample recorded under similar conditions on the same instrument should have no less than a 5%disagreement in relative abundance ratios. This logic, although obvious, is often dficult to practice experimentally. Whereas reference standards are pure compounds. the sample extract can introduce interfering ions into the mass spectrum, complicating the confirmation process. Using chromatographic separation prior to MS can often guarantee that interfering ions are re-
554 A Ana/ytica/Chemistry, September 1, 1995
duced to a minimum, allowing direct spectral matching to take place. However, when probe samples are used for coniirmation. gross interference can occur. In the case of c o n h a t i o n of ailatoxin B, in peanuts under NCI. Park and co-workers argued that although the three ions rep resenting the compound ( m l r 297,311, and 312) were present in the correct relative abundance ratios (Figure 2), they could be fragment ions from higher molecular weight compounds ( 1 1 ) . They stated that when the interfering ions constitute more than 20% of the total intensity of the mass spectrum, confirmation cannot be deduced in spite of the pres ence of the three ions at the correct relative abundance ratios. This lower limit of 20%represents a responsible judgment by the authors based on practical experience (i.e., the expert opinion factor). MID. Confirmation of trace levels is generally carried out using MID to lower the detection limit of the mass spectrometer to match the residue problem. This
Figure 3. Continnationd dimethoate in a mango extract. (a) Methane CI m a s spectrum of dimethoate and (b) multiple ion detection chromatograms obtained from the sample extract showing the confirmation of dimethoate at scan 175. (Adapted with permission from Reference 12.)
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Figum 4. Mass spectra for etrimphor. (a) EI mass spectrum, (b) CI m a s spectrum. and (c) product ion SpeCtNm from m/zzg3 as the precursor ion. (Adapted with permission from Reference 13.)
method mandates that chromatographic separation of the sample be performed prior to MS to concentrate the compound of interest into an appropriate elution profile for analysis as well as potential quantification,if desired. The literature contains a large number of case histories in which more than three or four ions were adopted for the confirmationprocess. For example, in the case of trace levels of dimethoate in mangoes (12).four ions produced using GC/MS with methane CI were selected for confirmation of the eluting peak believed to represent the pesticide in the extract (Figure 3). It would seem that the expert consensus prefers to adopt no less than four ions for confirmation, whereas a minimum of three shllcturaUy related ions would generally be recognized as scientificallysound for screening and some confirmation work. In the case of quantification, however, a single ion is often used to further reduce the limit of detection, optimize dwell times, and improve precision and accuracy. This method of approaching quantification is generally accepted only after confirmation has been performed or a deliberate study is underway using spiked samples in a recovery study. Product ion chemistry. Soft iOniZation methods have become popular techniques for the analysis of trace-level residues because they provide two distinct advantages over EI. First, the ionization process favors the production of solitary protonated molecular ions or adduct ions, depending on the reagent gas employed. Second, soft ionization methods tend to suppress the background interference ions because of a lack of fragmentation. Observation of protonated molecular ions can be considered the most important criterion for identification,but the burden of proof of presence placed on a single ion species cannot be regarded as sufficient for confirmation, Most residue samples anaiyzed by LC/MS suffer from this same disadvantage: CI provides only protonated molecular ions for confirmation. Because molecules anaiyzed by K / M S are u s d y thermally labile or nonvolatile, the opportunity to use E1 to obtain sufkient fragment ions is not an experimentaloption. Only two LC interfaces permit El studies the moving belt int e h c e and the particle beam device. As a
Ana/flica/ Chemistry, September 1, 1995 555 A
result, the specficity of the analysis has been increased by using tandem MS (MS/ MS). In the case of etrimphos, both the El and CI spectra did not contain sufficient fragment ions for the confirmation process (Figure 4). However, the product ion spectrum derived from the protonated m o lecular ion (m/z 293) provided five ions to meet the confirmation criteria (13).When the protonated molecular ion is used as the precursor ion, the product ion spectra normally contain sufficient ions to meet the criteria of a minium of three structuraUy r e lated product ions. Therefore, the recent shii in emphasis to the use of LC/MS in. terfaces has created a reliance on product ion spectra to satisfy the criteria for confirmation. The limitations imposed by reducing the mass range scanned (Le., a few atomic mass units) to achieve the necessary level of detection of a pesticide do not permit a concurrent confirmation of presence through structural analysis of related fragment ions. In the CI mode, the base peak or strongest ion is predominantly the protonated molecule ion. Therefore, a collision study of product ions resulting from isolation and interaction of this ion with argon or some other inert collision gas can produce a product ion spectrum to meet the criteria of three ions for confir. mation of presence. In Figure 5, the methane CI spectrum produced the major fragment ion for the insecticide carbaryl at vn/z 145, corresponding to protonated a-naphthol. The product ion spectrum yielded a large number of product ions that confirmed, without ambiguity, the aromatic character of this pesticide by illustrating atom-byatom fragmentation of the overall structure (m/z 127 for naphthalene with subse quent losses defining the benzoid nucleus, m/z 91,77, and 65). The product ion spectrum represents a much higher level of structural confirmation than that obtained by matching full-scan data acquired under CI or El. This technique is ideally suited to CI for two reasons. First, the predominant ion produced under CI is the protonated molecule ion, which reduces the contribution of interfering compounds in the sample extract and simplifies the resulting spectrum. Second, the collision experiment under MS/MS can use this proto556 A
nated molecule ion to produce a product ion spectrum characteristic of the whole molecule. With the advent of the ion trap and its recent ability to perform such product ion chemistry at residue levels, the marriage between CI and MS/MS is well suited to pesticide confirmations.
residue spectrum for confirmation;and the calibration curves upon which to perform quanIiiication of the analyte(s) versus one of the internal standards. Additionally, we conducted a comparison study using a single selected ion versus the total scan that revealed no loss in precision and accuracy. However, six compounds (mostly sulfurcontaining pesticides) were insensiEmerging ion-trap technology tive to this procedure. The sensitivity Evidence that the ion trap could detect and data generated for the selected 245 target quantify 245 target pesticides extracted compounds revealed that full-scan data ria Pesticide Analytical Manual methods can be collected for most compounds (3) while concurrently providing full-scan down to the 0.5-0.25 ppm level. data at the 0.25-1 ppm level has recently The main accomplishment of this r e been reported by our lab (14-E). The p r e search, however, is the clear d e m o n s b cision and accuracy data indicated no tion that the ion trap approach provides an acceptable qualitative and, in many ingreater than a 15%RSD with a correlation coefficient of 0.995 for a calibration stances, quantitative detector for multiresicurve between 0.25 and 1ppm. This exper- due analysis. We believe that this emergimental database (11 calibration mixing technology has strong potential as a tures containing the 245 target pesticides unique screening tool for pesticide analyrun 3 times at 3 concentrations) provided sis. However, the issue of acceptable quanthree important pieces of information tification for regulatory purposes remains to be proven. Recent commercial into establish criteria for the proposed method the ion($ selected from the spec- troduction of a new generation of ion trum to detect the target compound in traps capable of performing MS/MS exthe extract; the reference spectrum, which periments with increased sensitivity to the would allow a direct comparison with the femtogram level will open up a new ave
I
200
Figure 5. Mass spectra for carbaryl. (a) Methane CI mass spectrum and (b) argon collision-activated dissociation of the precursor ion at mir 145.
Analyticai Chemistry, September 1, 1995
nue of structural confirmation where sample interferences might otherwise prevent detection. References (1) Cairns, T.; Fisbein, L.; Mitchum, R. K. Biomed. Mass Spectrom. 1980, 7,484. (2) Waid, J. S. PCBs and the Environment; CRC Press: Boca Raton, FL, 1986. (3) U S . Department of Health and Human Services, Food and Drug Administration.
Pesticide Analytical Manual; US.Government Printing Office: Washington, DC, 1994. (4) Sphon, J. A. J. Assoc. Ox Anal. Chem. 1978,61,1247. (5) Self, R Biomed. Mass Spectrom. 1979, 6, 361. (6) Gilbert, J. Applications of Mass Spectrome-
try in Food Science;Elsevier: New York, 1987. (7) Gilbert, J.; Startin,J. R; Crews, C. Pestic. Sci. 1987, 18,273. (8) Tosine, H. M.; Clement, R. E. Mass Spectrom. Rev. 1988,8, 593. (9) Cairns, T.; Siegmund, E. G.; Stamp, J. J. Mass Spectrom. Rev. 1989,8,93. (10) Cairns, T.; Siegmund, E. G.; Stamp, J. J. Mass Spectrom. Rev. 1989,8, 127. (11) Park, D. L.; Diprossimo, V.; Abdel-Malek,
E.; Trucksess, M.; Nesheim, S.; Brumley, W. C.; Sphon, J. A,; Barry, T. L.; Petzinger, G. J. Assoc. Ox Anal. Chem. 1985, 68,636. (12) Cairns, T.; Siegmund, E. G.; Doose, G. M. Bull. Environ. Contamin. Toxicol. 1984, 32,645. (13) Cairns, T.; Siegmund, E. G. J. Assoc. Ox Anal. Chem. 1987, 70,858. (14) Cairns, T.; Chiu, K. S.; Siegmund, E. G. Rapid Commun. Mass Spectrom. 1992, 6, 331. (15) Cairns, T.; Chiu, K.S.; Siegmund, E. G. Rapid Commun. Mass Spectrom. 1992, 6, 449. (16) Cairns, T.; Chiu, K. S.;Navarro, D.; Sieg-
J
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mund, E. G. Rapid Commun. Mass Spec-
trom. 1993, 7,971. Thomas Cairns is vice president of technology, research, and development at Psychemedics Corp., afinn that analyzes hairfor drugs of abuse. Before joining Psychemedics in July 1995, he was a senior research scientist assigned to the FDA’s Ofice of Regulatory Affairs located at the Mass Spectrometry Center in Los Angeles. His research primarily involved the application of M S in support of a regulatory program to provide consumer protection. Richard A. Baldwin is director of the Division of Field Science in FDA’s Ofice of Regulatory Affairs in Rockville, MD, where he has played a pivotal role in bringing to the 18 FDA field laboratories emerging technologies, such as the ion trap, to improve and refine regulatory methods of analysis. Address correspondence about this article to Cairns at Psychemedics Corp., 5832 Uplander Way, Culver City, CA 90230.
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Analytical Chemistry, September 1, 1995 557 A
