the slope of a calihration curve (see box). Sensitivity may he a useful figure of merit for “pure” analytes, but it can become meaningless for the determination of analytes in increasingly complex mixtures. In the analysis of complex mixtures, interferences can arise from other chemical constituents of the mixture or background and can be referred to as “chemical noise” (12). The effectiveness ofa trace analysis technique, therefore, may he determined not so much hy its sensitivity, but rather by its ability to discern the signal of the analyte from the chemical noise. This is the second S, selectivity. Thus, a more descriptive figure of merit for a trace analytical technique is the limit of detection, defined as the amount of analyte that results in a signal-to-noise ratio ade-
Tandem mass spectrometry (MS/ MS) has gained rapid acceptance in the analytical community since its introduction in the 1970s. Although it has been applied successfully to structure elucidation of unknowns (1). its acceptance has been due largely to its ability to provide sensitive and selective analysis of complex mixtures rapidly, often with minimal, if any, sample cleanup (2-7). A recent book ( 8 ) and several recent reviews ( M 2 ) contain extended explanations of the theory, instrumentation, and applications of MS/MS. This REPORT will deal with the application of MSlMS to the determination of trace organic compounds in complex mixtures. The fundamentals and difficulties of trace organic analyses and the basic MS/MS operating modes will be discussed. Examples will illustrate how MS/MS can meet the needs and overcome some of the difficulties associated with trace analyses. Requirementsfor trace analysis To perform trace analyses successfully, it is necessary to consider carefully what McLafferty has referred to as the “4 Ss of trace analysis” (13): sensitivity, selectivity, speed, and $. To obtain a detectable signal for a trace amount ofanalyte, a technique must have high sensitivity, defined by 758A
ANALYTICAL CHEMISTRY, VOL.
quate to provide the desired confidence (typically S/N = 3). The equation below shows that the limit of detection takes into account both the sensitivity and the selectivity (inversely related to the level of chemical noise) of an analytical technique. To improve the limits of detection in many trace analyses, the selectivity of an instrumental technique is often increased by the use of extensive cleanup, chromatographic separation, and derivatization procedures. Such sample manipulations, however, increase the possibilities for sample contamination and sample loss through adsorption onto glassware, oxidation, etc. In addition, if the methods necessary to increase the selectivity become too time-consuming and expensive, the analytical method may become
Sensitivity = I slope of the calibration cur\
57.
NO. 7, JUNE
1985
0003-2700/85/0357-758ASO1 50/0 @ 1985 American Chemical Society
Report Jcdie V. Johnson Richard A. Yosl Depanment of Chemistry University of Florida Gainesville. Fla. 32611
impractical for routine work. Thus, the final two Ss, speed of analysis and cost effectiveness, are also important. Tandem instrumental methods An alternative approach to increase selectivity is the use of two or more analytical techniques in tandem. Cooks and Busch have shown that, as the number of stages of analysis is increased, the absolute levels of the signal and noise decrease; however, if the additional stages increase the selectivity of the technique, the chemical noise level will decrease more rapidly than the signal (Figure 1) (14). If the chemical noise dominates over the electrical noise (as is almost always the case in MS)an overall improvement in the S/N ratio will result. As long as there is a detectable signal above the electrical noise level, therefore, an increase in the number of tandem analytical stages will result in improved limits of detection. There has been intense interest in tandem analytical techniques (also called "hyphenated" techniques) in recent years (15).Although much of this interest has resulted from the availability of complementary information from tandem techniques ( I @ , there has been increasing interest in the use of tandem techniques for enhanced selectivity and improved lim-
I
its of detection in trace analysis (15). These techniques include spectrofluorometry and gas chromatography/ mass spectrometry GC/MS), as well as MSiMS. It is informative to consider each of these techniques in light of the discussion above. Spectrofluorometry involves the use of two monoFigure 1. Variation in the signal, chemical noise, and SIN chromators in "ratio with increasing stages of analysis used in tandem ries-the first to select the wavelength duces the spectral interference or for absorption and the second to select the emission wavelength. Because of chemical noise relative to the signal, often resulting in lower limits of dethe nonunity quantum efficiency, the tection than are possible with absorpselection of only a small portion of the emission wavelength range, and the tion spectrophotometry. transmission losses in the second monIn GC/MS the selectivity is improved, compared to MS alone, by the ochromator, there is a loss in the absophysical separation of the components lute signal detected compared to absorption spectrophotometry and, of a mixture by chromatography prior to mass analysis. For a mixture comtherefore, a loss of sensitivity. Howevponent to interfere with the analyte, it er, for a mixture component to intermust elute from the chromatographic fere with the analyte, it must not only absorb photons at the same excitation column a t the same retention time waveleneth hut must also emit radiand be ionized to Droduce ions of the same m D . The sensitivity is again reation at the same emission waveduced, largely because of the dilution length. This increase in selectivity reI
ANALYTICAL CHEMISTRY. VOL. 57, hO. 7. ANE 1985
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of the analyte during the chromatographic separation. Furthermore, the chromatographic separation increases the analysis time considerably.
MS/MS In MS/MS, two mass analyzers function analogously to the two monochromators in spectrofluorometry (12). For a mixture component to interfere with the analyte, it must be ionized to form an ion (parent ion) of the same m/z value, which must then fragment between the two mass analyzers to form a fragment ion (daughter ion) of the same m/z value as that of the analyte. This increased selectivity is achieved at the expense of sensitivity, because the conversion of the parent ion into a specific daughter ion is not complete, and the transmission efficiency of the second mass analyzer is not unity. A tandem mass spectrometer consists of an ion source, two mass analyzers separated by a fragmentation region, and an ion detector. The principles of MS/MS are straightforward and can be compared to conventional GC/MS. A mixture is introduced into the ion source of the tandem mass spectrometer, where ionization of the mixture produces ions characteristic of the individual components. The separation of the analyte from the other mixture components (the chromatographic step of GC/MS) is then achieved by the mass selection of the characteristic ion of the analyte by the first mass analyzer. This parent ion undergoes collisionally activated dissociation (CAD) through collisions with neutral gas molecules in the fragmentation region to yield various daughter ions, analogous to the fragmentation occurring during the ionization step of GC/MS. Subsequent mass analysis of the daughter ions by the second mass analyzer permits identification of the separated components. The four most common MS/MS operating modes of tandem mass spectrometers are daughter scan, parent scan, neutral loss scan, and selected reaction monitoring. The specific operational mode chosen for a particular analysis will depend on the information desired. A daughter scan consists of selecting a parent ion characteristic of the analyte by the first mass analyzer, fragmenting it by CAD in the fragmentation region, and scanning the second mass analyzer to obtain a daughter mass spectrum. Analogous to a normal mass spectrum, the daughter mass spectrum can be used for identification of an analyte by standard mass spectral interpretation or by matching the spectrum to that of an authentic sample. In the parent scan mode, a specific daughter ion is 760A
selected with the second mass analyzer, and the first mass analyzer is scanned over a specific mass range, selecting parent ions of different m/z that fragment to yield the specific daughter ion. This mode can be used to screen for a class of compounds that fragments to yield a common substructure. Fragmentation of the (M HIf ions of most of the phthalates produced by positive chemical ionization (PCI),for instance, yields the characteristic daughter ion 149+ (17).Therefore, a parent scan of 149+ would be a method for screening mixtures for phthalates with few interferences. In a neutral loss scan, both mass analyzers are scanned with a constant difference in mass. The resulting neutral loss spectrum contains the daughter ions that arise from the loss of a specific neutral fragment from the parent ions. This mode is useful for screening for a class of compounds characterized by a common fragmentation pathway. The molecular ions of chlorinated organics often lose C1' or HC1 during CAD, and thus a neutral loss scan of 35 or 36 (and/or 37 and 38) would provide a screening procedure for chlorinated organics in a mixture (9).The parent and neutral loss scans are used for rapidly screening mixtures for classes of compounds. Confirmation could then be accomplished by comparing a complete daughter spectrum of each detected parent ion in the mixture with that obtained from an authentic standard. Although the three operational modes described are very selective, as in full-scan normal mass spectrometry, they may lack the sensitivity necessary for the determination of trace components. Thus, for trace analysis, selected reaction monitoring (SRM) is often used, wherein a limited number of parent ion-daughter ion pairs are monitored for each analyte. Thus, an enhancement in the sensitivity is obtained, albeit a t the expense of some selectivity. This selected reaction monitoring is analogous to selected ion monitoring (SIM) used for maximal sensitivity in conventional GUMS. The mass analyzers that have been used in tandem mass spectrometers include quadrupole, magnetic-sector, electric-sector,time-of-flight, and ion cyclotron resonance analyzers and combinations of these (Reference 9 and references therein). The two most common MS/MS instruments are the mass-analyzed ion kinetic energy spectrometer (MIKES) (18,19) and the triple-quadrupole mass spectrometer (5). A MIKES instrument consists of a double-focusing mass spectrometer in which the ions traverse the magnetic sector before the electric sector (re-
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* ANALYTICAL CHEMISTRY, VOL. 57, NO.
7, JUNE 1985
versed geometry). With this instrument, an ion is momentum-selected by the magnetic sector and allowed to undergo metastable (unimolecular) or collisionally activated dissociation in the following field-free region to produce various daughter ions and neutral fragments. The kinetic energy of the daughter ions is then analyzed with the electric sector. The resulting spectrum provides information about both the daughter ions' mass-tocharge ratios and the kinetic energy released upon fragmentation (20). A triple-quadrupole mass spectrometer consists of, in series, an ionization source (typically dual chemical ionization-electron impact [CI-EI]), a quadrupole mass filter (Ql),a radio-frequency-only quadrupole (Q2), a second quadrupole mass filter (Q3), and an electron multiplier. Whereas Q1 and Q3 are operated as mass filters in the MS/MS modes, Q2 acts as a collision chamber and focusing device. Applications of MS/MS for trace analysis
There have been two driving forces behind most trace analysis applications of MS/MS: the need for increased speed of analysis and decreased cost per sample and the need for improved limits of detection in complex samples. The increase in selectivity that results from the use of two mass analyzers in tandem, in conjunction with the excellent sensitivity of the electron multiplier for ion detection (21),has enabled the direct analysis of complex mixtures for components at the picogram and femtogram levels with little or no sample preparation. One of the earliest published applications of MS/MS for trace analysis of mixtures was the determination of cocaine in coca leaf samples and urine with a MIKES instrument (10, 22). Solids probe introduction and positive chemical ionization-selected reaction monitoring (PCI-SRM) analysis of cocaine standards resulted in a calibration curve extending over two or three orders of magnitude with a limit of detection of approximately 1ng and with an error in peak area estimated as 30%.This technique permitted the determination of 4 ng of cocaine in a 1-pg sample of coca leaf diluted in 1mg of chalk dust and of 1.7 ng of cocaine in a 1-pL urine sample. This work was extended to the simultaneous mapping of cocaine and cinnamonylcocaine in 1-mg samples of coca plant tissue. The only sample preparation used in these analyses was grinding of the plant samples in liquid nitrogen. The speed of analysis with MS/MS techniques can be increased both by minimizing the sample preparation
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The I21 is even better-easier to use and more sensitive, with superior signal-to-noise ratio. Because of our heavy workload, we need a detector that can be serviced easily In most detectors, the flow cells are
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Flgure 2. Screening for illicit drugs in 1 pL of an acid-neutral extract of spiked horse serum by
sollds probe methane PCI-SRM Negatives are observe5 fathe two basic canpounds (pocaine and internal standard t r i m y l amine). PaiUves are ob6Brved tw the five acldic or neUtrai compounds (theophylline. thecbromine. popyipraben, phenylbutazone. and internal standard.
.........
2-arnin~szhlaobenzopenone). Note thet thee of h s e drws (theophylline, theobromire. and propylparaben) have molecular w e i m of 1SO. Reprinted w n permission hom Reference 7
and by eliminating any chromatographic separation of the mixture components prior to mass spectrometric analysis. Commonly, mixtures are introduced directly into the ion source by a heatable solids probe. The rapidity of sample analysis possible with a solids probe is illustrated by the PCI-SRM determination of 20 ng of urea in 1-pL samples of diluted blood serum a t a rate of 15 samples per hour (6).The precision of the peak heights was f15% RSD. The increased speed of analysis enables replicate analyses of the sample to be performed, permitting more reliable determination of the amount of analyte and estimation of the Drecision of the analysis. The earlv research with MIKES instruments demonstrated that several trace components could be rapidly determined in complex mixtures with little or no sample preparation. However, MIKES instruments, and twosector MS/MS instruments in general, have limitations when used for mixture analysis. Besides having spectral artifacts and less-than-unit mass resolution in the MS/MS spectra, the scan laws for the parent and neutral loss scans are complex nonlinear functions, and the magnetic sector cannot be quickly and accurately jumped between ions, particularly if the ions have widely differing rn/z values (23). On the other hand, the triple-quadrupole MS/MS instruments have unit mass resolution for both the parent and daughter ions over the entire mass range and produce no spectral artifads. In addition, the quadrupoles, 702A
1
' . ~
having a linear scan function and lacking the hysteresis effects of a magnet, can be quickly and accurately jumped between different ions of widely differing r n h values. These same characteristics allow all the various MS/MS scan modes to be easily placed under computer control. With a center quadrupole as a collision cell and focusing device, very efficient CAD of parent ions and collection of daughter ions are possible. Triple-quadrupole MS/ MS instruments, however, are limited in mass range (to 2000 u) and mass resolution (unit) compared to that possible with multiple-sector MS/MS instruments. The application of triple-quadrupole MS/MS to trace mixture analysis is illustrated by the determination of illicit drugs in the urine and blood serum of racing animals by solids probe PCI-MS/MS techniques (7).Presently, screening for illicit drugs is most commonly performed by thin-layer chromatography (TLC), with confirmation performed on any positives by GC/MS. These methods entail extensive sample workup prior to analysis, limiting the number of animals that are tested. MS/MS, however, permits both rapid screening and confirmation with minimal sample workup. With the introduction of 1pL of blood serum via a heated solids probe, the detection limits for most of the illicit drugs studied were in the low partsper-million (ng/pL) range with PCISRM. With a simple solvent extraction of the blood serum, the detection limits were reduced to the low ppb (pg/pL) range. The selectivity of MS/
ANALYTICAL CHEMISTRY, VOL. 57. NO. 7, JUNE 1985
75
MS is illustrated by the fact that three isobaric (same nominal mass) drugs could be independently identified because of their unique daughter ions (Figure 2). Confirmation of the drugs a t the ppm (ng/pL) level in blood serum was possible by comparison of the complete daughter spectra of (M H)+ ions from the simple extract to those of authentic standards. With this procedure, it was possible to screen for as many as 50 drugs and metabolites in a single 1-pL sample in less than 5 min, at a cost estimated to be equivalent to that of current TLC screening (7). One area where MS/MS has made a major contribution is the analysis of nonvolatile and thermally labile compounds. These compounds are typically analyzed with a soft ionization technique, such as field desorption (FD), fast atom bombardment (FAB), or direct chemical ionization (DCI). These techniques generally produce protonated and cationized molecules of low internal energy and, therefore, very little fragmentation. In addition to the sample ions, FAB also generates a large background of ions due to the liquid matrix in which the sample is dissolved. This lack of structurally significant fragment ions and the large amount of matrix background (im FAB) make the identification of mixture components difficult without extensive cleanup. However, fragmentation of the sample's protonated molecules under CAD conditions yields structurally significant daughter ions and decreases the chemical noise. For example, FAB has been combined
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A
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Flgura 3. Chromatogram for GCltripiequadrupole MSlMS selected reaction monltwing for TCP (196-- 1607
with MS/MS to determine the endogenous levels of leucine enkephalin in a biological extract of canine caudate nucleus (24). With selected reaction monitoring of both the endogenous peptide and the '802-labeled internal standard, leucine enkephalin was determined to be present a t 250 ng/g of tissue. With FD/MS/MS, it was possible to d y e mixtures of cationic, anionic, and neutral surfactants without prior separation of the components (25).With simultaneous ion detection, complete FD-CAD daughter spectra were obtained with only 5 pg of sample. In another example, a confirmatory assay was developed for invermectin, a family of antiparasitic compounds, in animal tissues (26).These compounds have low volatility and low ionization efficiency and are thermally labile. FAB, FD, and LC/MS each lacked the sensitivity necessary for a limit of detection at the 10-25-ppb level required. Solids probe EI-MS did have the necessary sensitivity, but lacked the specificity; even with extensive sample cleanup and high-resolution MS (R = lO,ooO), there waa still too much chemical interference. With a necessary liquid chromatographic sample cleanup, DCVMS/MS permitted reliable determination of invermectin in the 5-10-ppb range. The application of MS/MS to trace analysis is further illustrated in a comparison of mass spectrometric approaches for screening for hexachloro764A
benzene (HCB) and 2,4,5-trichlorophenol (TCP) in human blood serum and urine (27). The traditional approach of capillary column GC/MS was compared with short-column GC combined with MS/MS (both triplequadrupole and MIKES) and highresolution MS (HRMS). The added selectivity of MS/MS and HRMS permitted the use of short (
