Report
Ρesticide
Analysis
in Food by MS
any countries have now passed legislation to ensure that pes ticides are used safely. This ac tivity has brought a greater role for ana lytical chemistry in protecting the public from unsafe levels of pesticide residues. Traditionally, pesticides at levels of con cern have been identified by GC with ele ment-selective detectors. These methods often required a check analysis by an other 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.
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How do they get in our food?
Pesticides can be introduced into the food chain in several ways. The legal and intelli-
T h o m a s Cairns Richard A. Baldwin U.S. Food and Drug Administration 552 A
MS allows detection ofpesticide residues in food at the part-per-billion level
Pesticide residue analysis
The 1980s brought concerns about envi ronmental issues such as dioxins and polychlorinated biphenyls (2, 2). As a re sult, there was a quantum leap in moni toring capabilities, and regulatory scien tists could routinely measure contami nants at the part-per-trillion level. This advance was possible because of the reli ability 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 crop yield generally produces pesticide resi Confirmation of trace levels of pestidues at or below the legal tolerance level. cides detected by various elementWhen pesticides are applied to crops for sensitive (e.g., P, S, N, Cl) GC detectors which they are not yet registered, residues using a multiresidue screening procedure at any level constitute a violation of law. has led to reliance on MS because of its Once pesticides are applied, they and their ability to help define structure (3). In addi metabolites often persist for long periods in tion, regulatory scientists must be able to the environment, which can be viewed as rigorously support their findings so that an indirect route to transport in the ecosys proof can be provided in court. In a crimi tem. Finally, both nondeliberate contami nal case, scientific proof must meet the nation of the food supply via chemicals ap test of "beyond a reasonable doubt"; in a proved for industrial applications only and civil matter, the standard is "the prepon deliberate criminal contamination to re derance of evidence." Because MS con ceive media attention or cause public reac firmation methods are often developed on tion sometimes occur. an ad hoc basis to deal with an acute situ-
Analytical Chemistry, September 1, 1995
This article not subject to U.S. copyright. Published 1995 American Chemical Society.
ation, they cannot always be validated by interlaboratory testing. What has emerged over the past de cade is a set of evidentiary criteria gener ally recognized as scientifically sound. The highest level of confirmation that can be provided by MS is the exact correlation be tween the full mass spectral scans of a ref erence standard and the sample per formed within the same analytical condi tions. Usually electron ionization (EI) spectra contain sufficient structurally re lated fragment ions to permit absolute identification. Under such conditions, the relative abundance ratios should ex perimentally fall within 5%; quite often, however, the presence of background ions may severely interfere with exact com parisons. This practice of direct compari son represents the highest level of speci ficity obtainable by MS. In trace-level analyses, however, using full mass spectral scans for confirmation is often impractical. To fully enhance sensitiv ity, multiple ion detection (MID) offers a conveniently efficient method of ignoring potential interferences and concentrating on ions that belong to the compound under in vestigation. It is in this area that the evolv ing criteria for confirmation have received the most attention. Scrutiny has focused on the exact num ber of ions to be monitored to provide proof of presence. Setting this criterion has been complicated because of the many MS techniques available for analyz ing food samples. More than a decade ago, Sphon argued that a minimum of three structurally related ions would be necessary to provide proof of presence (4). This assumption was based on a statisti cal approach using an extensive MS data base as a model of a universal repository containing all possible organic com pounds. Without paying attention to rela tive abundance ratios, three ions were re quired to eliminate compounds with simi lar fragment ion selections from consider ation. To improve the criteria for confirma tion, the relative abundance ratios were required to be within 5% when compared with a reference standard recorded under
similar conditions. Evolution of new tech niques, particularly soft ionization meth ods, has prompted a reinvestigation of supporting evidence for confirmation be cause little or no fragmentation is ob served. Over the past two decades, several key reviews of trace analysis by MS (5-8) have reported specific case histories illustrat ing the ability of various techniques and their hybrids to provide identification and
confirmation. The evolution of the criteria for confirmation of trace residue levels in food and drugs has been discussed in de tail using arguments derived from experi mental case histories of what constitutes proof of presence (9), and these lend sup port for the three-ion concept. Support ing evidence provided by the GC or LC re tention time, as well as appropriate sam ple preparation and cleanup, has been recognized as fundamentally important.
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Figure 1 . Analysis of a tomato extract spiked with methamidophos, -mevinphos, tetrahydrophthalimide, dimethoate, dichlofluanid, chlorpyrifos, folpet, and ο,ρ-DDE. (a) Total ion chromatogram and (b) single ion monitoring detection. Analytical Chemistry, September 1, 1995 553 A
Report Chemical ionization of pesticides
Chemical ionization (CI) techniques to fa vor the production of a protonated molecu lar ion for characterization have been the cornerstone of resolving both the identifi cation and confirmation of pesticide resi dues 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 pro duced, and often the molecular ion is ab sent. The advantage of CI over EI as the ionization mode for pesticides is the con current reduction in the number of back ground ions belonging to the matrix. The appearance of 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 com pounds resulting from the GC analysis of a fruit or vegetable extract. Such extracts contain many matrix compounds repre senting flavor compounds or pyrolysis products from various thermally labile macromolecules characteristic of the crop being analyzed. The resulting chromatogram under CI thus contains a large num ber of interfering compounds among the actual pesticide residues. Figure 1 illus trates the total ion chromatogram ob tained from a spiked tomato extract (8 dif ferent pesticides) at the 0.05 ppm level. Although CI has the ability to simplify the chromatogram by producing mainly pro tonated molecular ions, the number of in terfering matrix compounds is still rela tively large. Interrogation of this chromatogram for the various pesticides can be conducted by specifying one of the major ions (usually the protonated molecular ion) for each pesticide and having the data system de tect each of the pesticides at the correct re tention time. This process of data reduc tion is referred to as single-ion monitoring (SIM). Detection, however, is contingent upon knowing which ion should be used to detect each pesticide. In practical resi due analysis, the necessary level of sensi tivity (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 554 A
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Figure 2. Effect of interfering substances on confirmation of identity of aflatoxin B 1 . (a) NCI mass spectrum of aflatoxin B, 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 B., is detected but not confirmed. (Adapted with 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, culminat ing in three chromatograms providing concurrent confirmation of presence.
duced to a minimum, allowing direct spec tral matching to take place. However, when probe samples are used for confir mation, gross interference can occur. In the case of confirmation of aflatoxin B1 in peanuts under NCI, Park and co-workers argued that although the three ions rep resenting the compound (m/z 297, 311, Case histories and 312) were present in the correct rela Full-mass scans. In the case of fulltive abundance ratios (Figure 2), they mass spectral scans derived under the could be fragment ions from higher mo various ionization techniques (e.g., EI, CI, lecular weight compounds (11). They NCI, FAB), the reference standard and stated that when the interfering ions con the sample recorded under similar condi stitute more than 20% of the total inten tions on the same instrument should have sity of the mass spectrum, confirmation no less than a 5% disagreement in rela cannot be deduced in spite of the pres tive abundance ratios. This logic, although ence of the three ions at the correct rela obvious, is often difficult to practice ex tive abundance ratios. This lower limit of perimentally. Whereas reference stan 20% represents a responsible judgment by dards are pure compounds, the sample ex the authors based on practical experi tract can introduce interfering ions into ence (i.e., the expert opinion factor). the mass spectrum, complicating the con MID. Confirmation of trace levels is firmation process. Using chromato generally carried out using MID to lower graphic separation prior to MS can often the detection limit of the mass spectrome guarantee that interfering ions are re ter to match the residue problem. This
Analytical Chemistry, September 1, 1995
method mandates that chromatographic separation of the sample be performed prior to MS to concentrate the com pound 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 confirmation process. For example, in the case of trace levels of di methoate in mangoes (12), four ions pro duced using GC/MS with methane CI were selected for confirmation of the eluting peak believed to represent the pes ticide in the extract (Figure 3). It would seem that the expert consensus prefers to adopt no less than four ions for confirma tion, whereas a minimum of three structur ally related ions would generally be rec ognized as scientifically sound for screen ing 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 re covery study.
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Product ion chemistry. Soft ioniza tion methods have become popular tech niques for the analysis of trace-level resi dues 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 suffi cient for confirmation. Most residue samples analyzed by LC/MS suffer from this same disadvantage: CI provides only protonated molecular ions for confirmation. Because molecules analyzed by LC/MS are usually thermally labile or nonvolatile, the opportunity to use EI to obtain sufficient fragment ions is not an experimental option. Only two LC inter faces permit EI studies: the moving belt in terface and the particle beam device. As a
Analytical Chemistry, September 1, 1995 555 A
Report result, the specificity of the analysis has been increased by using tandem MS (MS/ MS). In the case of etrimphos, both the EI and CI spectra did not contain sufficient fragment ions for the confirmation pro cess (Figure 4). However, the product ion spectrum derived from the protonated mo lecular ion (ml ζ 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 nor mally contain sufficient ions to meet the cri teria of a minimum of three structurally re lated product ions. Therefore, the recent shift in emphasis to the use of LC/MS in terfaces has created a reliance on product ion spectra to satisfy the criteria for confir mation. The limitations imposed by reducing the mass range scanned (i.e., a few atomic mass units) to achieve the necessary level of detection of a pesticide do not per mit a concurrent confirmation of pres ence 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 ml ζ 145, corre sponding to protonated α-naphthol. The product ion spectrum yielded a large num ber of product ions that confirmed, with out ambiguity, the aromatic character of this pesticide by illustrating atom-byatom fragmentation of the overall struc ture (mlζ 127 for naphthalene with subse quent losses defining the benzoid nucleus, ml ζ 91, 77, and 65). The product ion spectrum represents a much higher level of structural confir mation than that obtained by matching full-scan data acquired under CI or EI. This technique is ideally suited to CI for two reasons. First, the predominant ion produced under CI is the protonated mol ecule ion, which reduces the contribu tion of interfering compounds in the sam ple extract and simplifies the resulting spectrum. Second, the collision experi ment under MS/MS can use this proto 556 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 confir mations. Emerging ion-trap technology Evidence that the ion trap could detect and quantify 245 target pesticides extracted via Pesticide Analytical Manual methods (3) while concurrently providing full-scan data at the 0.25-1 ppm level has recently been reported by our lab (14-16). The pre cision and accuracy data indicated no greater than a 15% RSD with a correlation coefficient of 0.995 for a calibration curve between 0.25 and 1 ppm. This exper imental database (11 calibration mix tures containing the 245 target pesticides run 3 times at 3 concentrations) provided three important pieces of information to establish criteria for the proposed method: the ion(s) selected from the spec trum to detect the target compound in the extract; the reference spectrum, which would allow a direct comparison with the
(a)
residue spectrum for confirmation; and the calibration curves upon which to perform quantification 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 accu racy. However, six compounds (mostly sulfur-containing pesticides) were insensi tive to this procedure. The sensitivity data generated for the selected 245 target compounds revealed that full-scan data can be collected for most compounds down to the 0.5-0.25 ppm level. The main accomplishment of this re search, however, is the clear demonstra tion that the ion trap approach provides an acceptable qualitative and, in many in stances, quantitative detector for multiresidue analysis. We believe that this emerg ing technology has strong potential as a unique screening tool for pesticide analy sis. However, the issue of acceptable quan tification for regulatory purposes re mains to be proven. Recent commercial in troduction of a new generation of ion traps capable of performing MS/MS ex periments with increased sensitivity to the femtogram level will open up a new ave-
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Analytical Chemistry, September 1, 1995
nue of structural confirmation where sam ple interferences might otherwise pre vent detection.
STAY AHEAD OF
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. Must Reading for Fast Track Professionals Pesticide Analytical Manual; U.S. Govern ment Printing Office: Washington, DC, Who reads Industrial & Engineering Chemistry 1994. (4) Sphon, J. A. J. Assoc. Off. Anal. Chem. Research? Leading chemical engineers and industrial 1978, 61,1247. chemists worldwide. (5) Self, R. Biomed. Mass Spectrom. 1979, 6, 361. (6) Gilbert, J. Applications ofMass Spectrome Each month Industrial & Engineering Chemistry Research delivers try in Food Science; Elsevier: New York, up-to-the-minute original studies and reports in the area of kinetics 1987. (7) Gilbert, J.; Startin, J. R; Crews, C. Pestic. and catalysis, materials and interfaces, process engineering and Set. 1987,18,273. design and separations. Peer-reviewed, quality information on the (8) Tosine, H. M.; Clement, R. E. Mass Spec trom. Rev. 1988,8, 593. fundamental and theoretical aspects of chemical engineering, (9) Cairns, T.; Siegmund, E. G.: Stamp, J. J. process design and development, and product R&D. And I & EC Mass Spectrom. Rev. 1989,8, 93. (10) Cairns, T.; Siegmund, E. G.; Stamp, J. J. Research satisfies your need for in-depth knowledge with special Mass Spectrom. Rev. 1989,8,127. sections or entire issues devoted to selected topics, including (11) Park, D. L; Diprossimo, V.; Abdel-Malek, E.: Trucksess, M.; Nesheim, S.; Brumley, current research notes, correspondence, and reviews. W. C; Sphon, J. Α.; Barry, T. L.; Petzinger, G.J. Assoc. Off. Anal. Chem. 1985, Look to I S. EC Research for vital information you need to stay 68, 636. (12) Cairns, T.: Siegmund, E. G.; Doose, G. M. ahead of the curve, and push into new frontiers. Bull. Environ. Contamin. Toxicol. 1984, 32,645. Timely. Practical. Reliable. Comprehensive. "Aggressive... (13) Cairns, T.; Siegmund, E. G.J. Assoc. Off. Anal. Chem. 1987, 70, 858. innovative... indispensible" are how leading industrial and chemical (14) Cairns, T.; Chiu, K. S.; Siegmund, E. G. engineers around the globe describe I & EC Research. Don't miss Rapid Commun. Mass Spectrom. 1992, 6, 331. a single issue. Call now! (15) Cairns, T.; Chiu, K.S.; Siegmund, E. G. Rapid Commun. Mass Spectrom. 1992, 6, 449. Call Toll-Free 1-800-333-3511 (16) Cairns, T.; Chiu, K. S.; Navarro, D.; Sieg mund, E. G. Rapid Commun. Mass Spec in the U.S. trom. 1993, 7,971. Outside the U.S.: 614-447-3776. Thomas Cairns is vice president of technol Fax: 614-447-3671. ogy, research, and development at PsycheOr Write: medics Corp., a firm that analyzes hair for American Chemical Society drugs of abuse. Before joining Psychemedics Member and Subscriber Services in July 1995, he was a senior research sci P.O. Box 3337 · Columbus, OH 43210 entist assigned to the FDA's Office ofRegula 1995 Subscription Rates tory Affairs located at the Mass Spectrome Canada fi All Other U.S. Mexico Europe* Countries * try Center in Los Angeles. His research pri ACS Members marily involved the application of MS in $117 $131 One Year $ 65 $ 89 $221 $248 support of a regulatory program to provide Two Years $117 $165 Nonmembers consumer protection. Richard A. Baldwin is $747 $761 One Year $695 $719 director of the Division of Field Science in * Air Service Included. Published by the American FDA's Office of Regulatory Affairs in RockChemical Society under the American Chemical Society ville, MD, where he has played a pivotal role editorial leadership of 1 1 5 5 Sixteenth Street, N.W. Donald R. Paul, University in bringing to the 18 FDAfieldlaborato Washington. D.C. 2 0 0 3 6 of Texas, Austin. ries emerging technologies, such as the ion ACS III PUBLICATIONS Environmental Research for the Chemical Science trap, to improve and refine regulatory meth ods of analysis. Address correspondence Member subscription rates are for personal use only. Subscriptions are based on a calendar year. Foreign pay ment must be made in U.S. currency by international money order, UNESCO coupons, or U.S. bank draft, or about this article to Cairns at Psychemedics order through your subscription agency. For nonmember rates in Japan, contact Maruzen Co., Ltd. This publi Corp., 5832 Uplander Way, Culver City, cation is available on microfilm and microfiche. The full text is available online on STN International. CA 90230.
THE CURVE
Analytical Chemistry, September 1, 1995 5 5 7 A
