The Mass Spectrometer as a Gas Chromatograph Detector

margin of difference—most particu- larly to address the novel ways in which the ..... call these a spectrum, and throw out all the ions whose intens...
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Instrumentation

Catherine Fenselau Department of Pharmacology and Experimental Therapeutics The Johns Hopkins University School of Medicine 725 North Wolfe Street Baltimore, Md. 21205

The Mass Spectrometer as a Gas Chromatograph Detector T h e enormous analytical potential of the combined gas chromatograph/ mass spectrometer was first realized in 1957 when Holmes and Morrell reported their rudimentary coupling of the two instruments with a stream splitter as an interface (1 ). T h e combination is a natural one since samples are analyzed in the gas phase in both instruments, sample levels are comparable, temperature range is comparable, and neither instrument needs to be modified excessively in the t a n d e m arrangement. Most importantly, the two instruments perform highly complementary analytical functions. Early problems in coupling the two included the fast scan rate required of the mass spectrometer to analyze the contents of a single gas chromatographic peak. Initially, this was best met by time of flight analyzers. Now, however, magnetic instruments can be scanned through broad mass ranges and reset in 4- or 5-s intervals, and quadrupole analyzers also offer appropriate scan rates. T h e early major incompatibility, and one which is still a problem, is the difference in pressure required for the operation of the two instruments. Thus, while the gas chromatograph operates at positive pressures, the mass spectrometer is designed to run under high vacuum. An associated problem is the presence of much carrier gas and little sample in the eluent from the gas chromatograph. In the first instrument referred to above, both of these problems were addressed with a simple splitter. This is not a particularly desirable interface since most of the sample is lost. In recent years improved pumping speeds for the mass spectrometer have permitted the direct introduction of gas at low flow rates, such as those associated with the use of capillary columns in the gas chromatograph. Another solution has been proposed in the use of atmospheric pressure sources on the mass spectrometer. However, the in-

terfaces most commonly used between a gas chromatograph and a mass spectrometer are those called separators or enrichers, which are designed on the basis of flow dynamics and molecular effusion to eliminate most of the carrier gas from the system, while transmitting as much of the sample into the mass spectrometer as possible (2, 3). Both gas chromatography and mass spectrometry have improved greatly since the first combined instrument was reported. In addition, unique innovations have been introduced into the combined gas chromatograph/ mass spectrometer (GC/MS) and in our understanding of how to use it, so t h a t the analytical potential of the combined GC/MS is now clearly greater than the sum of its parts. It is the purpose of this article to address this margin of difference—most particularly to address the novel ways in which the mass spectrometer can be used as a detector for the gas chromatograph. First, several ways will be described in which the mass spectrometer can be used as a "real t i m e " detector for the gas chromatograph; subsequently, the reconstruction of chromatograms by computerized analysis of previously acquired scans will be considered. Real Time Detection

Total Ion Current. Total ion current is measured in different ways in different kinds of mass spectrometers (4), but basically it is a measure of the total number of ions formed from material eluting from the gas chromatograph, recorded as a function of time. Figure 1 provides a comparison between a gas chromatogram detected by flame ionization and a total ion current chromatogram detected by the mass spectrometer. There is no difference in the retention times (no lag time) if the system is properly constructed. There are some differences here and there in response to different

Time (min) Figure 1. Gas chromatograms of urine extract recorded simultaneously by a flame ionization detector (FID) and total ion current (TIC) monitor in a mass spectrometer

compounds. Of particular interest is the fact t h a t the flame ionization detector does not respond as efficiently to compounds with very long retention times as the mass spectrometer. This difference may merely reflect plumbing problems, but it is frequently observed. Selected Ion Monitoring. In selected ion monitoring the intensities of preselected ions are recorded as a function of time. Although some commercial companies have devised proprietary names for this technique, the concept is quite straightforward, and it is difficult to justify special names for minor variations in hardware (5). Single ion monitoring and multiple ion monitoring will be discussed separately here. Single ion monitoring was used by early workers to analyze gas chroma-

ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977 • 563 A

Figure 2. Single ion monitoring at m/e 436.319, the molecular ion of a set of iso­ meric 5«-androstanediol standards (a) and that ion formed by components of pros­ tate extract (b) (7). Column: 3 % OV-17, temperature: 245 ° C Reproduced by permission of Heyden & Son Ltd.

tographic eluents before magnetic an­ alyzers could be rapidly scanned (6). An ion characteristic of a class or of a particular compound was monitored throughout the elution of the gas chromatogram, for example, mass 43 ions characteristic of hydrocarbons. In re­ cent years, single ion monitoring has been favored for analyses requiring the highest sensitivity. Particularly in environmental and biological work, single ion monitoring is often carried out at higher resolution to provide in­ creased selectivity. For example, high resolution single ion monitoring of the a b u n d a n t molecular ions of stereoisomeric 5«-androstanediols has been shown to provide sufficient selectivity and sensitivity to identify and assay Βα,Πβ-; 30,17α-; and 3/3,17/3-androstanediol in prostatic tissue extracts (7). Single ion records of a trimethylsilylated standard mixture and a trimethylsilylated tissue extract are shown in Figure 2. Ions of mass 436.319 were monitored with mass resolution of 1 p a r t in 10 000. As little as 20 femtograms (2 X 10~ 14 g) of standard ste­ roid could be detected by this tech­ nique. Reagent ion monitoring is an inter­ esting variation of single ion monitor­ ing, wherein the intensity of reagent ions used in a chemical ionization source is monitored as a function of time (8). T h e intensities of reagent ions decrease when they react with material eluted from the gas chromatogram. In Figure 3 such an ion rec­ ord is compared with a flame ioniza­ tion detection chromatogram for the same mixture. T h e responses of the two detection systems differ consider­ ably, reflecting the fact t h a t the tbutyl ion transfers a proton only to

compounds t h a t are more basic and that other ion molecule reactions can occur with some compounds. In multiple ion monitoring the in­ tensities of two or more preselected ions are recorded as a function of time. To achieve this, the analyzer of the mass spectrometer cycles through the group of ions being monitored, switch­ ing each into the detector in turn. Thus, the intensity of each of the ions is recorded several times per second, resulting in an essentially continuous analog ion record. This kind of contin­ uous peak stepping was used as early as 1948 in process analysis of the puri­ fication of uranium isotopes (9). T h e utility of multiple ion monitoring for deconvoluting overlapping compo­ nents in a gas chromatogram and for

0

1 2 Time (min) Figure 4. Abundance as a function of time of molecular ions of carbamaze­ pine (m/e 236) and dihydrocarbamazepine (m/e 238) coinjected into GCIUS(11) Reproduced by permission of C. V. Mosby Co.

assaying stable isotope incorporation was demonstrated in conjunction with a gas chromatograph in 1967 (10). In Figure 4 the molecular ion of carbam­ azepine is monitored concurrently with the molecular ion of dihydrocarbamazepine (11). In this case, the dihydro compound has been added as an internal standard for assaying blood level of carbamazepine. T h e ions re-

Figure 3. Abundance as a function of time of C 4 Hg + reactant ions in a chemical ionization source (trace inverted) compared with a flame ionization gas chromato­ gram of a mixture of hydrocarbons and alcohols (8)

564 A • ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977

Figure 6. Spectra scanned repetitively under computer control of a trimethylsilylated mixture containing glucuronide of benzyl alcohol Total ion chromatogram is shown underneath at several attenuations. Obtained by electron impact on a DuPont 491 GC/MS interfaced to Incos computer system by use of a 2 ft X % in. glass column of 3 % OV-17 on Supelcoport

Figure 5. Abundance as a function of time of molecular ions of phencyclidine and d 5 -phencyclidine and their major fragment ions formed in a chemical ion­ ization source ( 12) Reproduced by permission of Heyden & Son Ltd.

corded are the Μ · + ions for each com­ pound. Of particular interest in this figure is the fact t h a t although the two compounds are not completely re­ solved by the gas chromatograph, their molecular ions can be resolved by the mass spectrometer. Multiple ion monitoring can be used to record the masses and relative in­ tensities of several ions formed from a single compound to add specificity to an assay. T h e technique can be ap­ plied to quantitative studies in which stable isotope analogs are used as in­ ternal standards. Since the sample and its stable isotope-labeled analog are usually poorly resolved by gas chromatography, the mass spectrome­ ter is the only chromatographic detec­ tor which can be used to distinguish them. Figure 5 shows the molecular ions (m/e 243 and 248) and major fragment ions (m/e 159 and 164) of phencyclidine and ds-phencyclidine extracted from 1 m L of blood (12). Methane chemical ionization was more effective than electron impact because it minimized interference by fragment ions from other components of the extract. Quantitative determi­ nations could be made as low as 1 ng/mL blood. T o use selected ion monitoring ef-

Figure 7. Total ion current chromatogram (a) and mass chromatograms [m/e 375 (b) and 217 (c)] of trimethylsilylated urine extract Obtained by electron impact on Dimaspec GC/MS computer system by use of a 5 ft X 1/4 in. glass col­ umn of 3 % OV-101 on Supelcoport

566 A • ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977

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fectively, the investigator must be familiar with the spectra of the compounds or class of compounds sought. In rigorous reporting the scanned spectrum on which the selected ion monitoring is based should be made available. Although it varies with the sample, the sensitivity of selected ion monitoring is reported by many laboratories to fall in the range 1 0 - 1 2 to 1 0 - 9 g, between 1 pg and 1 ng. Often, sensitivity is limited by interference or by absorption on the GC column. Computer-Reconstructed Chromatograms

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Reconstructed Total Ion Chromatograms. A computer system can provide automated acquisition, normalization, and plotting of spectra; this kind of support is especially attractive when one is analyzing the compounds in a multicomponent mixture in a gas chromatograph/mass spectrometer. It is not necessary to wait until a peak elutes to scan the mass spectrometer. With computer control, spectra can be scanned and acquired repetitively every few seconds throughout elution of the entire gas chromatogram. This approach generates three-dimensional ion signals, each characterized by mass, intensity, and also by time (scan number) as shown in Figure 6. With this approach one can easily end up with several hundred spectra stored in the computer, and considerable ingenuity is required on the part of the investigator to retrieve useful information in an efficient manner. Figure 7 (top panel) shows a total ion chromatogram reconstructed by summing the ion intensities in each repetitive scan. The scan number is indicated on the axis. This kind of chromatogram looks very much like a total ion current chromatogram or flame ionization detector chromatogram, although peak shapes can be distorted if scans are not made frequently enough. Mass Chromatograms. With all of the repetitive scans stored in the computer, the intensities of any ion may be retrieved from each scan and plotted as a function of time. In Figure 7 in addition to the reconstructed total ion chromatogram, two mass chromatograms are shown, also reconstructed from about 250 repetitive scans made while a trimethylsilylated urine extract was eluted from the gas chromatograph. T h e mass chromatograms were reconstructed of ions prominent in the scanned spectra of trimethylsilylated glucuronides. These mass chromatograms resemble selected ion records, although they are less sensitive by as much as 10 4 , because of the shorter sampling times used in fast repetitive scanning. T h e limited mass range chromato-

c o n s t r u c t e d s p e c t r u m r e s u l t i n g from t h i s k i n d of d e c o n v o l u t i o n of c o m p o n e n t s of a u r i n e e x t r a c t . B y a n d large, such resolved spectra are reported to p r o v i d e v e r y good m a t c h e s w h e n lib r a r y s e a r c h e s a r e u s e d for t h e i r i d e n t i f i c a t i o n . O n c e t h e s p e c t r u m in e a c h r e p e t i t i v e s c a n h a s b e e n r e s o l v e d in t h i s m a n n e r , t o t a l ion i n t e n s i t i e s c a n be f o u n d a n d c o n v e r t e d b a c k i n t o r e solved or d e c o n v o l u t e d t o t a l ion c h r o m a t o g r a m s . T h e s u c c e s s of t h i s a p p r o a c h is i l l u s t r a t e d in F i g u r e 9 in w h i c h a r e s o l v e d c h r o m a t o g r a m maybe c o m p a r e d with t h e original total ion c h r o m a t o g r a m of a n e x t r a c t from g a s t r i c lavage. Conclusions Figure 8. Spectrum of indole acetic acid 3-methyl ester from a GC/MS analysis of human urine (top) and same spectrum resolved by automated data processing (bottom) ( 14)

Figure 9. Total ion current chromatogram of an extract from gastric lavage, reconstructed from repetitive scans (top) and from resolved or deconvoluted repetitive scans (bottom) ( 15) Reproduced by permission of Marcel Dekker

g r a m is a v a r i a t i o n of m a s s c h r o m a t o g r a p h y t h a t has proven especially useful in a n a l y z i n g p o l y c h l o r i n a t e d hyd r o c a r b o n s . In t h i s t e c h n i q u e t h e c o m p u t e r s u m s ion i n t e n s i t i e s t h r o u g h a l i m i t e d m a s s r a n g e as a f u n c t i o n of s c a n n u m b e r or t i m e . T h e s p e c t r u m of a c o m p o u n d s u c h as m i r e x , w i t h 12 c h l o r i n e a t o m s , will c o n t a i n m o l e c u l a r i o n s a t 13 m/e v a l u e s (13). T h e e n t i r e m o l e c u l a r ion p e a k g r o u p c a n be

s u m m e d to provide increased sensitivity w i t h s o m e sacrifice in specificity. P e a k D e c o n v o l u t i o n to P r o v i d e Reconstructed Spectra and Deconv o l u t e d C h r o m a t o g r a m s . Classically, t h e d e c o n v o l u t i o n of o v e r l a p p i n g gas c h r o m a t o g r a p h i c p e a k s is a p p r o a c h e d by e m p h a s i z i n g p e a k s h a p e s a n d s l o p e s . H o w e v e r , if o n e u s e s t h e m a s s s p e c t r o m e t e r as a d e t e c t o r , o n e c a n m o n i t o r t h e rise a n d fall of v a r i o u s ion i n t e n s i t i e s a n d u s e t h i s as a b a s i s for p e a k d e c o n v o l u t i o n . As w a s p o i n t e d o u t a b o v e , t h e profiles of t h e i n t e n s i t i e s of t h e t w o m o l e c u l a r ions in Pagure 2 are resolved even t h o u g h the two c o m p o u n d s are not separated comp l e t e l y on t h e gas c h r o m a t o g r a p h . S i m i l a r l y in F i g u r e 7, profiles of s p e cific ions c a n b e s e e n t o b e r e s o l v e d from o v e r l a p p i n g c o m p o n e n t s in t h e r e c o n s t r u c t e d t o t a l ion c h r o m a t o g r a m . T o a first a p p r o x i m a t i o n , o n e c a n d e t e r m i n e w h i c h ion i n t e n s i t i e s a r e m a x imized t o g e t h e r a t e a c h p o i n t in t i m e , call t h e s e a s p e c t r u m , a n d t h r o w o u t all t h e i o n s w h o s e i n t e n s i t i e s a r e n o t maximal at t h a t same time. This a p p r o a c h was used m a n u a l l y more t h a n 15 y e a r s a g o , p r e c i s e l y t o d e c o n v o l u t e o v e r l a p p i n g c o m p o n e n t s in t h e G C e l u e n t , a l t h o u g h a s it is p r a c t i c e d by p r e s e n t day c o m p u t e r p r o g r a m m e r s , it is c o n s i d e r a b l y r e f i n e d (14, 15). F i g u r e 8 p r e s e n t s a r e s o l v e d or r e -

Table 1. Sensitivity of GC Detectors Total ion current monitoring Mass chromatogram Flame ionization Electron capture Selected ion monitoring

570 A • ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977

10-8 g 10-8g 10-9g 10~ 1 2 g 10-12g

T h e m a s s s p e c t r o m e t e r is a u n i v e r sal d e t e c t o r for gas c h r o m a t o g r a p h s , since a n y c o m p o u n d t h a t can p a s s t h r o u g h a G C will be c o n v e r t e d i n t o ions in t h e M S . A t t h e s a m e t i m e t h e h i g h l y specific n a t u r e of m a s s s p e c t r a p e r m i t s t h e m a s s s p e c t r o m e t e r t o be u s e d as a highly specific G C d e t e c t o r . S u i t a b i l i t y for q u a n t i t a t i v e m e a s u r e m e n t s has been d e m o n s t r a t e d , and s e n s i t i v i t y is c o m p e t i t i v e w i t h t h a t of t h e e l e c t r o n c a p t u r e d e t e c t o r as s h o w n in T a b l e I. Perhaps the most intriguing aspect of t h e m a s s s p e c t r o m e t e r as a G C d e t e c t o r is its g r e a t v e r s a t i l i t y , s i n c e it can provide scanned spectra, selected ion r e c o r d s , m a s s c h r o m a t o g r a m s , r e solved t o t a l ion c u r r e n t c h r o m a t o grams, and more, at variable mass resolving p o w e r , w i t h a n e v e r - g r o w i n g s e l e c t i o n of i o n i z a t i o n t e c h n i q u e s . References (1) J. C. Holmes and F. A. Morrell, Appl. Spectrosc, 11,86(1957). (2) C. F. Simpson, CRC Crit. Rev. Anal. Chem. (September 1, 1972). (3) G. A. Junk, Int. J. Mass Spectrum. Ion Phys., 8,1 (1972). (4) C. Fenselau, Appl. Spectrosc, 28, 305 (1974). (5) F. C. Falkner, B. J. Sweetman, and J. T. Watson, Appl. Spectrosc. Rev., 10, 51 (1975). (6) C. Merritt, ibid., 3, 263 (1970). (7) D. S. Millington, M. E. Buoy, G. Brooks, M. E. Harper, and K. Griffiths, Riomed. Mass Spectrom., 2, 219 (1975). (8) F. Hatch and B. Munson, Anal. Chem., 49,731 (1977). (9) F. Falkner, Riomed. Mass Spectrom., 4,67 (1977). (10) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Rvhage, Anal. Chem., 38, 1549 (1966). (11) I.. Palmer, L. Bertilsson, P. Collste, and M. Rawlins, Clin. Pharmacol. Ther., 14,827 (1973). (12) D.C.K. Lin, A. F. Fentiman, Jr., R. L. Foltz, R. D. Forney, Jr., and I. Sunshine, Riomed. Mass Spectrom., 2, 206 (1975). (13) D. A. Carlson, K. I). Konyha, W. B. Wheeler, G. P. Marshall, and R. G. Zaylskie, Science, 194, 939 (1976). (14) R. G. Dromey, M. J. Stefik, T. C. Rindfleisch, and A. M. Duffield, Anal. Chem., 48, 1368(1976). (15) J. E. Biller and K. Biemann, Anal. Lett., 7,515(1974).