Anal. Chem. 1989, 6 1 , 494-496
494
Nondiscriminatory Fast Atom Bombardment Matrix for Fatty Acid Mixture Analysis Sir: Fast atom bombardment FAB ( I ) is an established technique for producing gas-phase ions from nonvolatile and/or thermally sensitive compounds. The use of a liquid matrix provides for a long-lived ion current. Thus, time-demanding experiments can be conducted such as slow scanning of mass spectra and collisional activation of desorbed ions. The latter experiment utilizes FAB and tandem mass spectrometry. It is specially well-suited for sequencing peptides (2-4) and for d termining the structure of compounds that undergo charge-remote fragmentations such as fatty acids (5-7),lipids (8,9),surfactants (IO),and related compounds
1
(11).
It is also desirable that FAB mass spectrometry provide quantitative information on mixttues of like compounds. However, quantitative analysis of mixtures without the use of internal standards is hindered by discrimination effects, resulting in underrepresentation of some components. Several research groups have noted the discrimination of less surface-active and/or more hydrophobic mixture components in the full scan FAB mass spectrum and proposed various ways to circumvent the problem. Ligon and Dorn (12) observed that, in an equimolar mixture of alkyl quaternary amines desorbed from glycerol, the longest chain, most surface-active amine dominates the mass spectrum of desorbed ions. The authors showed that the underrepresentation of the shorter chain, less surface-active species can be reduced by adding an excess of a surfactant of opposite charge to dominate the surface and reduce the so-called surface activity discrimination. Lyon and Hunt (13) acquired spectra from a mixture of anionic surfactants desorbed from triethanolamine until there was no more analyte signal and summed the intensities for each component over all of the scans. This signal averaging technique gave better relative concentration information than was revealed in the first few scans alone because the early scans suffer from surface activity discrimination. As the more surface-active species are desorbed, the shorter chain surfactants become more fully represented. Williams et al. (14) used a hydrophobicity index created for the amino acids by Bull and Breese (15) to assign a relative hydrophobicity to the peptides in a tryptic digest of glucagon. It was found that the less hydrophobic, more hydrophilic peptides are discriminated against in the FAB mass spectrum of the peptide digest taken up in acidified glycerol, thioglycerol/diglycerol (l:l), and 1,2,64rihydroxyhexane. Using high-performance liquid chromatography fractionation, they were able to separate the hydrophilic and hydrophobic peptides and analyze the fractions separately. In another experiment, Williams et al. (14) esterified the polar head groups of peptides in a mixture to reduce the hydrophilicity of the peptides containing these groups. Caprioli (16) and co-workers showed that because of the dynamic nature of continuous flow FAB, the hydrophilic peptides from the tryptic digest of glucagon are more fully represented. The above discrimination problems are understood in terms of matrix-analyte interactions. Because the foremost requirement of a FAB matrix is that it be sufficiently nonvolatile to allow useful spectra to be obtained, H-bonding, polar matrices are typically used (e.g., glycerol). Ironically it is the strong H-bonding nature of the matrix that gives rise to many of the discrimination effects. For example, nonpolar, surface-activeand/or hydrophobic compounds such as fatty acids 0003-2700/89/0361-0494$01.50/0
(In,surfactants (12),and peptides with a low Bull and Breese derived hydrophobicity index (14) give good sensitivity in H-bonding, polar matrices. These species are surface-active because the free energy is lowered by self-assembly at the liquid surface (18). On the other hand, polar hydrophilic compounds such as peptides with a high Bull and Breese hydrophobicity index (14) and polyhydroxy compounds such as glycopeptides (19) show lower sensitivity by FAB. We present here preliminary results of a study of fatty acid mixtures desorbed from typical FAB matrices. One purpose is to determine the nature of the discrimination of mixture components in FAB at different total fatty acid concentrations. On the basis of previous work in this laboratory (20,21), it is apparent that analysis of fatty acid mixtures by FAB mass spectrometry suffers from discrimination against some components. The causes of these problems are more rigorously defined here by studying discrimination effects as a function of concentration. An understanding of the problem leads to the proposal of a nondiscriminatory FAB matrix for fatty acid mixture analysis.
EXPERIMENTAL SECTION Capric, palmitic, and stearic acids were purchased from Sigma (St. Louis, MO) or Aldrich (Milwaukee,WI) and used as received. 1-Dodecanol was purchased from Aldrich (Milwaukee, WI) and vacuum distilled once to remove higher series homologues. A stock solution of the three fatty acids in the relative ratio of C12C16C18 = 1.10:1.001.04 was prepared in carbon tetrachloride. Triethanolamine and glycerol, the latter of which had been made 0.5 F in KOH, were mixed with ethanol to form a 33% solution which could be more easily pipetted. The dodecanol, which melts just above room temperature, was heated and then pipetted. To obtain the data in Figure 1A,B, an appropriateamount of the fatty acid stock solution was mixed with the 33% solution of the matrices, and the volatile solvents were allowed to evaporate while standing at room temperature for several days. For the data in Figures 1C and 2, the dodecanol was mixed with an appropriate amount of the fatty acid stock solution. The concentrations were calculated after assuming complete evaporation of the volatile solvents. Just before analysis, the fatty acid/matrix solutions were heated to ca.35 "C in a Pierce hcti-Therm heating module. This caused the dodecanol to liquefy and the mixture to become a solution. The fatty acid mixtures at high fatty acid concentrations in the glycerol and triethanolamine matrices were milky at room temperature, but became homogeneous at 35 "C. A few microliters of the solutions were applied to the probe tip, and the spectra were obtained on a Kratos MS 50 triple analyzer (22) equipped with a Kratos FAE3 source operated at 8 kV and an Ion Tech FAE3 gun operated at 6 kV and 2 mA. The data in Figure 1 are from three scans averaged by hand. Because the relative concentrations were not l.O:l.Ol.O, the average ion abundances were divided by the relative concentration ratios and normalized to the C16 fatty acid so that all observed abundances should, in the absence of discrimination effects, be 1.0.
RESULTS AND DISCUSSION The analysis of fatty acid mixtures in the typical FAB matrices, glycerol and triethanolamine, is hindered by surface activity discrimination at total mixture concentrations below ca. F (see Figure lA,B). The relative abundance of the matrix ions decreases as the total concentration of fatty acids increases, underscoring the importance of the fatty acid surface activity. A t lower concentrations, the shorter chain, less surface-active fatty acids are underrepresented. This trend is consistent with the results that Ligon and Dorn (12) ob@ 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989 4
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At higher concentrations, the longest chain C18 fatty acid is inadequately represented. This is an example of reverse surface activity discrimination apparently due to the formation of micelles by the longest chain fatty acid (23). This conclusion is supported by the observation that underrepresentation of the C18 fatty acid occurs a t lower concentrations in the glycerol matrix containing 0.5 M KOH. There is no CH2CH20H moiety on glycerol as there is on triethanolamine to increase the solubility of the unassociated fatty acids. Furthermore, the high ionic strength of a 0.5 M KOH solution can be expected to lower the critical micelle concentration of all of the fatty acids, and the longest chain fatty acid is expected to have the lowest critical micelle concentration of the three (24). These results indicate that a FAB matrix is needed that shows little differential surface activity effects and minimizes micelle formation. The matrix should have the property of solubilizingthe fatty acids, and so it should resemble the fatty acids themselves. We chose 1-dodecanol, a nonpolar, hydrophobic matrix with a long alkyl chain, allowing more van der Waals interaction with the analyte, and an alcohol functionality allowing hydrogen bonding. Because the fatty acids are expected to be completely solvated, there should be no inherent surface activity discrimination or tendency to form micelles a t high total fatty acid concentrations. By taking advantage of the characteristics of the new dodecanol FAB matrix, one can obtain accurate quantitative information from a mixture of fatty acids. The discrimination effects that operate in typical FAB matrices have been reduced. Even at a relatively high total fatty acid concentration, the ions of the fatty acids do not dominate the mass spectrum as can be seen from their low abundances relative to the matrix ions (Figure 2). This indicates the lack of fatty acid surface activity in dodecanol. The formation of micelles leading to reverse surface activity discrimination is not apparent. Because the fatty acids are freely dissolved, the bulk relative ratios are represented in the FAB mass spectra. In fact, linear plots of nearly identical slope were obtained for each fatty acid component when relative abundance was plotted against the individual concentrations. However, the range of concentrations that can be investigated in dodecanol is reduced. These characteristics of the matrix have implications on the choice of a matrix for FAB MS/MS analysis. In the MS/MS experiment, an ion is selected and may be subjected to collisional activation, giving rise to a product ion spectrum free from matrix interferences. For this analysis, it is desirable to have a long-lived, intense ion signal. Dodecanol would not be as good as the typical FAB matrices because the fatty acids are not surface-active in dodecanol and because dodecanol is more volatile, giving a shorter lived ion beam. If qualitative or structural information from a mixture is desired, then the more typical FAB matrices are recommended because they
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F W o 1. Mscrknkratkn of a m i m e of three alkyl saturated fatty ackls as a function of concentration in (A) glycerol 0.5 M KOH, (6) triethanolamine, and (C) dodecanol. See text for details.
tained in the analysis of mixtures containing equimolar surfactants. 69
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Anal. Chem. 1989, 61, 496-499
provide for greater sensitivity and a longer lived ion current. CONCLUSIONS The two types of discrimination that occur in quantitative fatty acid mixture analysis by FAB mass spectrometry depend on the concentration regime of the analysis. By considering the origin of this discrimination, the requirements of an improved FAB matrix were determined. Dodecanol meek these requirements, a t least for mixtures of saturated fatty acid congeners. However, the lack of surface activity and the more volatile nature of this matrix make it less compatible with MS/MS than the typical FAB matrices. We are currently investigating the use of dodecanol in the analysis of mixtures containing more widely different fatty acids, as well as the possibility of quantification without the use of an internal standard. The application of the basic principles utilized in this study is being considered for other analyte systems.
Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Processes 1984. 6 1 , 3113-122. Lyon, P. A.; Hunt, S. Presented at the 36th ASMS Conference on Mass Spectrometry and Allied Topics, 1988. Naylor, S.; Findeis, F. A.; Gibson, B. A.; Williams, D. H. J . Am. Chem . SOC. 1988, 108, 6359-6362. Bull, H. 6.; Breese, K. Arch. Biochem. Bbphys. 1974, 161, 865. Caprioii, R. M.; Morre, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987, 1 , 15-18. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. Anal. Chem. 1985, 5 7 , 2018. Bull, H. B. An Introdwtlon to physical Bbchemisby; F. A. Davis Company: Philadelphia, PA, 1964; Chapter 9. Naylor, S.; Skelton. N. J.; Williams, D. H. J . Chem. Soc., Chem. Commun. 1986, 1619. Deterdlng, L. J.; Gross, M. L. Anal. Chlm. Acta 1987, 200, 431. Adams, J.; Gross, M. L. Org. Mass Spectrom. 1988, 2 3 , 307. Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge. H. Int. J. Mass Spectrom. Ion phys. 1982. 42, 243. Barber, M.; BordoH, R. S.; Ellott, 0. J.; Sedgwick. R. D.; Tyler, A. N. J . Chem. Soc., Faraday Trans. 11983. 79, 1249-1255. Mead, J. F.; Alfin-Slater, R. B.; Hewtan. D. R.; Popjak, G. LIPIDS Chemisby, Blochemlstry, and Nutrnotion; Plenum Press: New York, 1986; Chapter 4.
LITERATURE CITED Barber, M.; Bordoli, R. S.; Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 5 4 , 645A. Bleman, K.; Martin, S. A. Mass Specfrom. Rev. 1987. 6. 1. Bleman, K.; Scoble, H. A. ScEence 1987, 237, 992. Hunt. F. D.; Yates, J. R. III; Shabanowk, J.; Winston, s.; buer, C. R. Proc. Natl. Acad. Sei. USA 1986, 8 3 , 6233-6237. Tomer, K. 8.; Jensen, N. J.; Gross, M. L. Anal. Chem. 1988, 58, 2429. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. J. Am. Chem. Soc. 1985, 107. .- . , 1883. .- - -. Adams, J.; Gross, M. L. Anal. Chem. 1987, 5 9 , 1576. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. L@& 1987. 2 2 , 480. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. LIP& 1986, 2 1 , 580. p. A,; Crow, F. w.; Gross, M. L. Anal. che” 1985, 57, 2984. Tomer, K. 6.; Gross, M. L. Bbnmd. Environ. Mass. Spectrom. 1988, 15, 89.
Kenneth A. Caldwell Michael L. Gross* Midwest Center for Mass Spectrometry Department of Chemistry University of Nebraska-Lincoln Lincoln, Nebraska 68508
RECEIVED for review September 6,1988. Accepted December 2,1988. This research was supported by the National Science Foundation grant to the Midwest Center for M~~~ Spectrometry (Grant No. CHE-8620177).
Sorbent Isolation and Elution with an Immiscible Eluent in Flow Injection Analysis Sir: On-line preconcentration, e.g., of metal ions on an ion-exchange resin, followed by elution and detection has proved to be a versatile and sensitive technique for trace metal analysis, especially when coupled to atomic spectrometry (1-5). Various ion-exchange materials have been exploited for this purpose (5-9), and the utility of time-based, rather than valve-based, injection has been shown ( 2 , 3 , 5 ) . System configurations have been developed that prevent the sample matrix from entering the detector and allow elution to be carried out in a back-flushing mode (2, 7). The use of two preconcentration columns (one is in load mode while the other is in elution mode) has been introduced to increase sample throughput (2, 6, 7, 10). Although the advantages offered by such methods, namely, improvements in detectability and elimination of matrix effects, are desirable in many areas other than trace metal determinations, not many efforts have been made (11). While some ingenious membrane-based techniques for analyte isolation from the matrix and selective preconcentration have been developed (12-14), it is hard to equal the reliability of a packed-column-based device over extended periods of operation. Thus far, in all continuous analysis applications,the eluting solvents have been miscible with the sample matrix. The use of an immiscible solvent not only extends the scope of the general technique but also can provide unique selectivities and nearly ideal “plug elution”. In principle, the immiscible phase can be isolated with a membrane-based phase separator (see for example, ref 15 and citations therein). However, a potentially more robust approach is to actively sense the im0003-2700/89/0361-0496$01.50/0
miscible plug, isolate it, and accordingly direct it to the detector, using a miscible carrier and performing further chemistry en route, if desired. This paper demonstrates the above principle for preconcentration/sorption of mercaptans from a gasoline stream on an anion exchanger, elution by an aqueous alkali, and colorimetric determination in the aqueous eluate. A second example is shown for the preconcentration of aniline in a benzene stream on a cation exchanger, elution by dilute H2S04,and direct UV detection. EXPERIMENTAL SECTION Reagents. DTNB (5,5’-dithiobis(2-nitrobenzoicacid)) was obtained from Sigma Chemical Co. Various mercaptans were bought as neat liquids (Aldrich). All other reagents were of analytical reagent grade. Deionized distilled water was used throughout. “Regular”grade gasoline was purchased from local filling stations. Mercaptans were removed from gasoline by extracting with 5 M NaOH twice. This “clean”gasoline was used for establishing blank responses and as a medium for preparing mercaptan standards. Mercaptan standards in gasoline are not stable for more than 24 h. The standards were kept in ice during use.
Flow injection system configurationsinvolved a continuously flowing sample stream (rFIA, see ref 16);this arrangement is often advantageous for process applications. Experimental System. The mercaptan determination system is shown schematically in Figure 1. Gasoline, with or without added thiols, flows through a electropneumatically actuated rotary valve V1 @heodyne Type 5020, Cotati, CA) equipped with a WpL sample loop and a microcolumn (1.7 mm i.d. poly(tetrafluor0ethylene) (PTFE) tube, packed with a strong base anion ex0 1989 American Chemlcal Society
