Anel. Chem. 1991, 63, 1482-1487
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Quaternized Nylon as a Support for Plasma Desorption Mass Spectrometry: Adsorption Mechanisms and Analytical Applications M.P.Lacey and Thomas Keough* The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, Ohio 45239-8707
A novel quaternired Nylon surface has been prepared to aid the study of analyte adsorptkn and desorptkn to and from the soiM supports used in plasma deswptlon mass spectrometry. At low lonk surfactant coverage onto an opposnely charged surface, binding occurs by an lon-exchange mechanlm that Is largely hwlependent of alkyl chain length. At higher cwrlace coverage, the longer chain homologues are preferentially adsorbed due to hydrophoblc forces. The nature of the adsorption lnteractlon can be varled in PDMS, simply by changing the sign of the charge on the solid support. Thls capability has important analytlcal implications that will be discussed. The quaternited Nylon surface has not proven to be useful for the characterization of peptides or proteins. However, for negativakn studies with varkus other classes of compounds, ll provk!es 10-100 tima higher sendWty than obtained from nitrocellulose surfaces. Finally, an affinity wparatlon of ionic surfactants containlng hydrophobic alkyl chains, from a mlxture also containing hydrophilic nonionic surfactants, is also demonstrated.
INTRODUCTION
Sample preparation plays a critical role in plasma desorption mass spectrometry (PDMS). Initially, thin films of nonvolatile analytes were prepared by an electrospray technique (I). Subsequently, Macfarlane and co-workers (2) proposed the idea of selectively binding an analyte from solution onto a thin solid support suitable for PDMS analysis. This idea was demonstrated by binding polarizable cations from aqueous solution onto a cation-exchange surface (2). In the ensuing search for improved substrates for PDMS applications, several other supports were developed including polyethylene terephthalate (Mylar) and polypropylene (3), Mylar impregnated with hydrophobic cationic surfactants (4, 5), nitrocellulose (6,7),silicon (8), Nylon, octadecane and polybrene (9),and anthroic acid (10). Alternatively, mixing analytes with suitable small-molecule matrices such as glutathione (11) and sucrose octaacetate (12)have also been demonstrated to be useful PDMS sample preparation approaches. Several reviews of PDMS sample preparation techniques have been published recently (5,7,13). Nitrocellulose, electrosprayed over aluminized Mylar, is currently the support of choice for the characterization of peptides, proteins and other polar biological molecules (6, 7). This surface, which is negatively charged when exposed to aqueous media, offers several benefits relative to the initial PDMS sample preparation techniques. They include simple sample preparation, easy removal of alkali-metal salts that hinder the desorption process, sensitivity enhancement of 2 orders of magnitude, and improved peak shape, which increases the practical mass resolution achieved in the PDMS experiment. We sought to develop a complementary positively charged surface possessing some of the analytical attributes of the negatively charged nitrocellulose surface. We prepared a 0003-2700/91/0363-1482$02.50/0
positively charged surface by electrospraying Nytran, quaternized Nylon-66, onto aluminized Mylar. We then studied the binding of cationic and anionic surfactants, from aqueous solution, to both Nytran and nitrocellulose. The mechanism of surfactant binding onto oppositely charged surfaces is dependent upon bulk solution concentration. Binding occurs by ion exchange (14-161,a process that is largely independent of alkyl chain length, below about lo4 M. Adsorption driven by hydrophobic interactions (14-161,a chain length dependent process, becomes important with bulk concentrations above ca. lo4 M. Of course, surfactant ion exchange does not occur with surfaces of like charge. Nytran has not proven analytically useful for the characterization of peptides and proteins by PDMS. However, it appears to be more generally useful for negative-ion PDMS studies than nitrocellulose. Negative-ion response for various compound classes such as anionic surfactants, triglycerides, phospholipids, and cholesterol esters is enhanced 10- to 100-foldfrom Nytran, relative to the response observed from nitrocellulose. This is particularly useful for the characterization of "real" samples, such as isolates from biological matrices, where the complementary information from positive and negative-ion spectra is often needed for analyte identification. Furthermore, we have demonstrated that Nytran is useful as an affinity surface, to selectivelybind both cationic and anionic surfactants containing hydrophobic alkyl chains from detergent formulations also containing hydrophilic nonionic surfactants (neodols). E X P E R I M E N T A L SECTION
Reagents. The analytes were obtained from various commercial sources and were used without additional purification. The homologous series of akyltrimethylammoniumchlorides had stated purities >95%. However, these materials, which were not specifically dried, probably contain variable amounts of water since they are hygroscopic. Instrumentation. All mass spectrawere obtained on a BieIon 20 (Applied Biosystems Swglen AB, Uppsala, Sweden) PD mass spectrometer that has been previously described (17-19). Mass calibration was accomplished by using H+ and Na+ as reference ions. Surface Preparation. A portion of a Nytran membrane (0.45-wm pore size, Schleicher & Schuell, Inc., Keene, NH) was dissolved in concentrated,reagent grade formic acid (88%, J. T. Baker Chemical Co., Phillipsburg, NJ) overnight at ambient temperature. The insoluble portion of the membrane was discarded and the formic acid solution concentrated to dryness. The residue was reconstituted in formic acid at a concentration of 10-12 mg/mL prior to electrospraying onto aluminized Mylar sample foils (Applied Biosystems Sweden AB, Uppsala, Sweden). The resulting Nytran foils were considerably less uniform in appearance than commercial (Applied Biosystems Sweden AB) electrosprayed nitrocellulose foils, because of the difficulty in electrospraying formic acid. However, the foil surface was readily covered. Typical Al-containing background ions, from the aluminized Mylar surface, were absent from the PD mass spectra of Nytran-covered foils. Furthermore, an infrared reflection absorption spectrum (Digilab SPS 40, Cambridge, MA) of the Nytran-covered foil was virtually identical with one obtained from 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
Table I. Summary of Positive- and Negative-Ion PD Mass Spectra of Crl Obtained from Nitrocellulose and Nytran Substrates substrate
measd intensa Cs+ I-
1-/Cs+
6130 89 0.02 nitrocellulose 456 1071 2.35 Nytran a Number of ions detected after integrating for 2 OOO OOO primary
events.
analysis of the original membrane. Sample Preparation. The Nytran surface charge was verified by positive- and negative-ion PDMS analyses of CsI. Separate nitrocellulose and Nytran foils were loaded with 10-rL portions of a lo-* M solution of aqueous CsI. After 10 min the excess solution was blown off the foil, which was then washed with deionized water prior to analysis. Positive- and negative-ion PD mass spectra were obtained for 2000000 primary events with ion-accelerating voltages of 1 1 2 kV. Ionic binding mechanisms to nitrocellulose and Nytran were investigated by analysis of an approximately equimolar (1@M) mixture of Cg- to C18-trimethylammonium chlorides (C8- to C18TMAC’s). For these studies, 10 pL of a solution containing about 10 pmol/pL of each surfactant was loaded onto the surfaces and allowed to equilibrate for 10 min. The excess solutions were blown off the foils, which were then washed with deionized water prior to analysis. We analyzed CsI from a nitrocellulose foil after the surface had been completely covered with C18TMAC. This was done to demonstrate that surface charge reversal occurred after loading M ClsTMAC. For this experiment we loaded 10 ML of a aqueous CIBTMACsolution onto nitrocellulose. After equilibration, the excess solution was blown off and the foil was water washed. On top of this modified foil was loaded 10 MLof a M solution of CsI. Again, after equilibration, the foil was water-washed prior to analysis by positive- and negative-ion PDMS (*12 kV). Spectra were integrated for 2000000primary events.
RESULTS AND DISCUSSION Verification of Surface Charge. Following Macfarlane and co-workers (3),we chose CsI as an analyte to probe the electrostatic component of the adsorption interaction. It is ideal for this purpose, since it is composed of a cation and anion having comparable charges, sizes, and polarizabilities. Therefore, cation and anion binding can be investigated simultaneously with ions having very similar propertiea (3). The positive- and negative-ion PD mass spectra are summarized in Table I. The nitrocelldosecoated foil exhibits a Cs+ cation that is more than 10 times larger than that observed from the Nytran-covered foil. On the other hand, the I- signal from the Nytran-covered foil is substantially greater than that observed from the nitrocellulose-coated foil. The measured intensity ratio of I-/Cs+ was 0.02 from nitrocellulose and 2.35 from Nytran. Clearly, the Nytran surface contains positively charged binding sites while nitrocellulose contains negatively charged surface sites. Ionic Surfactant Binding Mechanisms. Binding of ionic surfactants from aqueous solution onto surfaces with strongly charged sites may occur by a variety of mechanisms including ion exchange, ion pairing, hydrogen bonding, and hydrophobic adsorption (14,151.At low bulk concentration of surfactant, binding is thought to occur mainly by exchanging surfactant ions for the counterions orginally associated with the surface. A dramatic increase in surfactant binding is observed at higher bulk concentrations, as a result of hydrophobic interactions. After complete surface coverage haa been achieved, the original charge on the surface may actually be reversed. This results because the surfactant molecules, adsorbed by hydrophobic interactions involving their alkyl chains, have their charged
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head groups pointed up from the surface toward the aqueous medium. At low bulk concentrations, surfactant binding to an oppositely charged surface occurs by an ion-exchange mechanism. In this regime, the number of moles of surfactant adsorbed per unit area will be largely controlled by the number of surface binding sites and the size of the hydrophilic group and will be independent of alkyl chain length. In the specific case of alkanesulfonates adsorbed onto alumina (pH = 7.2), for example, the adsorption of nonassociated sulfonate ions results in an isotherm characterized by a single straight line that is independent of alkyl chain length (16).Similarly, the PD mass spectrum obtained from analyais of an approximately equimolar mixture of C8- to ClsTMAC’s, loaded onto a negatively charged nitrocellulose surface, shows comparable response for each homologue (Figure la). This spectrum is completely consistent with our expectations based on analyte binding through an ion-exchangemechanism. When thissame mixture is loaded onto the Nytran surface, surfactant binding by ion exchange is eliminated because the surfactant head group and the surface binding sites are both positively charged. Adsorption might still occur, but by hydrophobic interactions. Under these circumstances, the surface concentration of the surfactant should be strongly dependent upon alkyl chain length because of the increased change in free energy associated with removal of the longer chains from solution, and because of the increased van der Waals attraction with increasing chain length (14,151.Furthermore, the hydrophobic binding energy of short-chain homologues may not be large enough to offset the electrostatic repulsion between the head groups of the analyte and the surface. Results obtained from analysis of the equimolar alkyl-TMAC mixture, conducted on the Nytran surface, are given in Figure lb. Considerable discrimination against the short-chain homologues is evident. Only CIS- and C18TMAC’s are observed above background. At higher bulk concentrations, we expect increased surfactant adsorption to the solid surface. Furthermore, the longer chain homologues should be preferentially adsorbed (14-16).The results of the PDMS analyses of several equimolar mixtures of Clz- and C16TMAC,adsorbed onto nitrocellulose, are summarized in Figure 2. The observed ratios of molecular cations are reasonably constant and approximately unity at bulk concentrations below lo4 M. At higher bulk concentration the observed response for the homologue increases dramatically relative to that of the C12 homologue. The large increase in the slope of the curve marks the end of the ion-exchangeregime and the onset of surfactant adsorption by hydrophobic binding. The same trends were observed from the analyses of several equimolar mixtures of Cg-and Clcalkylbenzene sulfonates adsorbed onto positively charged Nytran. After complete coverage of the surface, we expect the original surface charge to be reversed to that of the polar head group of the adsorbed surfactant. After coverage with a cationic surfactant, the Aurface charge of nitrocellulose should be reversed from negative to positive. Under these conditions, we expect greatly enhanced adsorption of I- relative to Cs+ in the analysis of CsI. Indeed, the measured ratio of I-/&+ increased to 0.42, which is more than 20 times larger than the I-/Cs+ ratio obtained when CsI was analyzed from an untreated nitrocellulose foil (see Table I). Analytical .Implicationsof the Surfactant, Adsorption Mechanisms. Both the static-FABMS and PDMS methods detect molecules that have been adsorbed to surfaces (Figure 3). In the case of FAB, analytes adsorbed to the surface of a liquid are sampled. In PDMS, analytes that have been adsorbed to a solid surface are subsequently sampled. Analyte surface activity differences,which are strongly dependent upon
ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
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a> 1
Nitrocellulose
6-
cu
5 -
7
2
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5
-
-
.
.2? 3 v)
c12
c14
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"
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-4
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Log [conc.] Figure 2. Observed PD mass spectral intensity ratio from equimolar mixtures of CieTMAC and C,,TMAC, as a functlon of bulk solution concentration.
Vacuum Liquid
(b) + Nytran
PD
Liquid Solid
Figwe 9. schemetic Mustrating the different surfaces that are sampled in static-FABMS and PDMS.
c18
I
1
c16 I
m (WZ) Fburr 1. Positlveion PD mass spectra of an approxknatelyequkrolar IBO
Kx)
220
240
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mixture of alkyl-TMACs bound to (a) nitrocellulose and (b) Nytran surfaces. The x axis corresponds to mass/charge ratio, while the y axis corresponds to intensity.
0
alkyl chain lengths for a series of homologous surfactants, often exhibit prominent effects on relative response in staticFABMS (20-22). From a practical standpoint, this leads to the selective detection of the more surface-active components in mixtures and precludes the use of homologues as internal standards for quantitative FABMS applications (23). Quantitative applications of static-FAB typically require the availability of stable isotopic intemal standards. Furthermore, it may be difficult to obtain accurate quantitative surfactant homologue distributions by static-FABMS unless several stable isotopic internal standards, chosen to bracket the distribution, are used for calibration (24). The relative insensitivity of PDMS response to alkyl chain length, at least at bulk concentrations within the ion-exchange regime, suggests that PDMS might provide semiquantitative
Concentration C12/C16 Figure 4. Observed PD mass spectral intensity ratio of C,,TMAC/ C1eTMAC as a functlon of the bulk solution concentration ratio of these
cationic surfactants.
homologue distributions without the use of intemal standards and that homologues might generally serve as calibration standards for quantitative PDMS studies. The feasibility of quantitative PDMS using a chemical internal standard has already been demonstrated. Jungclas and co-workers (12) have quantitated the cytotoxic drug etoposide from the serum of cancer patients using teniposide as a internal standard. In the present study, the calibration curve shown in Figure 4 was constructed by using C,,TMAC as an internal standard for
ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991 %ui
7w
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500
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Figure 5. Negativalon PD mass spectra of 50 pmois of LAS bound to (a) nitrocellulose and (b) Nytran surfaces. The x axis corresponds to mass/charge ratlo, while the y axis corresponds to intensity.
CI1TMAC. Linear response was observed for 2 orders of magnitude variation in concentration of ClZTMAC. Comparison of Nytran and Nitrocellulose for Selected Analytical Applications. Negative-Ion Mass Spectra. The Nytran surface is completely ineffective for the PDMS characterization of peptides and small proteins such as synthetic pentapeptides, insulin, and lysozyme. Positive-ion PD mass spectra can be routinely obtained on 10 pmol of insulin from the nitrocellulose surface. However, no response was observed from 100 pmol of insulin loaded onto a Nytran foil. Positively charged Nylon membranes have a higher proteinbinding capacity than nitrocellulose (%), so the peptides and proteins may simply bind so tightly that subsequent desorption of the intact molecular ions is highly unfavorable. The negative-ion PD mass spectra obtained after loading 50 pmols of sodium tetradecylbenzeneadfonate(C,,LAS) onto nitrocellulose and Nytran are compared in Figure 5. The spectrum obtained from nitrocellulose (Figure 5a) shows no response for the molecular anion a t m/z 353. The corresponding spectrum obtained from the Nytran foil (Figure 5b) shows an abundant molecular anion as well as an expected
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fragment ion at m/z 183. Molecular anions of LAS have been readily detected to the 1.5-pmol level (signal to noise ratio >5) from the Nytran surface. Incidentally, aluminized Mylar is another surface that offers improved negative-ion sensitivity relative to nitrocellulose. Unfortunately, its background spectrum,which is dominated by AlzO3 cluster ions below m/z 1O00, is so intense that it strongly interferes with analyses of low levels of analytes in this molecular weight range. Ionpairing CllLAS with hydrophobic counterions such as tetrabutylammonium cations enhances its negative-ion PDMS response off the nitrocellulose surface, an effect similar to that previously noted in static FABMS (21). Ion-pairing has no effect on the LAS response when the analysis was conducted from the Nytran surface. Phospholipids are an important class of nonvolatile and thermally labile molecules that are amenable to other desorption mass spectrometry techniques such as field desorption (26) and static-FABMS (27,28). This class of compounds can also be readily analyzed by PDMS and we have obtained both positive- and negative-ion spectra from 100 pmol of phosphatidylcholine loaded onto both nitrocellulose and Nytran (data not shown). The positive-ion spectra are virtually identical for the two surfaces, both showing comparably abundant MH+ ions a t m / z 790. However, the negative-ion spectra were quite different. The spectrum obtained from the nitrocellulose surface showed no signals above background in the m/z 200-1000 range. The spectrum from the Nytran surface showed, in addition to a weak (M - CH3)- molecular anion, a prominent Cl7H&O2- fragment ion that defiies the fatty acid composition of the molecule. The negative-ion data are essential for defining the fatty acid composition of phospholipids,and in the case of static-FABMS,negative-ion detection is considered to be the method of choice for structural characterization (27). The negative-ion sensitivity enhancements observed when ionic compounds are analyzed off Nytran, rather than nitrocellulose, are also observed for nonpolar, nonionic species such as triglycerides and sterol esters. The negative-ion PD mass spectrum obtained from 100 pmol of 1,2-dipalmitoyl-3-myristoylglycerol, evaporated from benzene/hexane (30/70) onto Nytran, showed relatively strong signals for both C13HnC02and C15H31C0c.The corresponding spectrum from the nitrocellulose surface showed no response for the expected RCOZ-fragment ions and provided no direct confirmation of the fatty acid composition of the analyte. Similar results were obtained from sterol esters. The positive- and negative-ion spectra obtained after 5 nmol of cholesterol palmitate, loaded onto nitrocellulose and Nytran foils, are given in Figure 6. The positive-ion spectrum from nitrocellulose exhibits a weak MH+ molecular ion and is dominated by the (cholesterol OH)+fragment ion, which confirms the sterol part of the molecule. Confirmation of the fatty acid is not obtained from the negative-ion spectrum. The positive-ion spectrum obtained from the Nytran surface is similar to that obtained from nitrocellulose. However, the negative-ion spectrum exhibits an intense C1SH31C02-fragment ion, confirming the identity of the fatty acid. The need for complementary positive- and negative-ion mass spectra becomes particularly apparent in the analyses of samples isolated from biological matrices. During the course of this work, we analyzed a series of HPLC isolates from the sterol ester fraction obtained from hamster feces. The positive-ion mass spectra were dominated by (sterol - OH)+ ions. However, molecular weight confirmation was sometimes difficult because the HPLC isolates were often not single compounds and the spectra exhibited a number of potential molecular ions in the 500-800 range. The corresponding negative-ion spectra, obtained from the Nytran surface, ov-
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
+ Nitrocellulose
+ Nytran
- Nitrocellulose
TNYran
(b)
+ Nytran background ion
6. poslthre (+) and negative (-) ion FD m888 spectre of 5 n& of cholesterol palmttate from nitrocellulose and Nytran surfaces. The x axis corresponds to mass/charge ratio, while the y axis corresponds to intensity.
ercame this problem because they were dominated by RCOZfragment ions. Combination of the two pieces of complementary data allowed identification of the sterol esters contained in each of the HPLC isolates. Electron transfer to the surface provides one possible explanation for the poor negative-ion sensitivity obtained from the nitrocellulose support (29).Electron transfer from analyte anions to the electronegative surface nitrate groups, according to eq 1, would result in analyte ion neutralization and sen-
sitivity loss. Several studies with Nytran, aluminized Mylar and nitrocellulose foils revealed that analyte negative-ion response was consistently lower for analyses carried out on nitrocellulose than for those analyses carried out on either aluminized mylar or Nytran. These results suggest the involvement of the nitrate groups in the process leading to reduced negative-ion sensitivity. Furthermore, in accordance with eq 1, an abundant NO3- anion is observed in the negative-ion PD mass spectra obtained from nitrocellulose-coated sample foils. Selective Adsorption of Hydrophobic Surfactants from Complex Mixtures. The Nytran surface has been shown to adsorb both anionic surfactants (Figure 5 ) and cationic surfactants having a hydrophobic alkyl chain (Figure lb). Hydrophilic nonionic surfactants apparently do not strongly adsorb from aqueous solution, and this fact can be used to analytical advantage in the characterization of commercial surfactant formulations. The point is illustrated in Figure 7 by comparison of the PDMS results obtained from an artificial detergent formulation containing cationic (ditallowdimethylammonium chloride, DTDMAC), anionic
Figure 7. Positive-ion mass spectra of an artificial detergent formulatiin bound to (a) nitrocellulose and (b) Nytran surfaces. The x axis corresponds to masslcharge ratio, while the y axis corresponds to Intensity.
(LAS), and nonionic (neodol) surfactants. The positive-ion spectrum obtained from the nitrocellulose surface exhibits cationized (M- + 2Na+) LAS as the most abundant ions (m/z 371,385,399). The mass range from 400 to 900 Da is dominated by cationized neodols. The cationic surfactant (molecular cations at m / z 494,522, 550) is largely obscured by the strong response from the nonionics. The corresponding positiveion spectrum from Nytran show no response for U S because small positively charged ions like Na+, needed for cationization, do not adsorb to this surface and are not available for ionization. The neodols are not detected either. We see no evidence for the MH+ molecular ions that dominate neodol’s PD mass spectrum obtained from the nitrocellulose surface when Na+ has been washed off the foil. Obviously, this hydrophilic nonionic surfactant does not adsorb to the Nytran surface from aqueous solution. The only ions observed in the positive-ion spectrum are the DTDMAC molecular cations, except for a common background ion at m/z408. Of course, the LAS molecular anions were readily detected in the negative-ion spectrum obtained from this Nytran foil. This particular example illustrates the use of the PDMS sample foil to provide a crude “affinity” chromatographic separation of the hydrophobic ionic surfactants from the hydrophilic nonionics.
CONCLUSIONS A novel quatemized Nylon substrate has been prepared and evaluated for plasma desorption mass spectrometry. This
ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
substrate has been quite useful for studying adsorption mechanisms of ionic surfactants onto charged surfaces. Furthermore, it has also been shown to provide 10-100 times better negative-ion response than is obtained from nitrocellulose surfaces for several classes of compounds. Finally, it has proven effective as an “affinity surface”, allowing the separation of anionic and hydrophobic cationic surfactants from mixtures also containing hydrophilic nonionic surfactants. Nytran foils are more difficult to prepare than conventional nitrocellulose foils and much less uniform in appearance. Whether the additional foil preparation effort (relative to nitrocellulose) is justified depends entirely on the analytical problem at hand. It should also be noted that other cationand anion-exchange surfaces, such as Mylar and polypropylene (3) or Mylar impregnated with cationic surfactants (4, 51, might also exhibit some of the beneficial attributes of Nytran that have been outlined in this paper. These other surfaces were not specifically investigated here. In the case of Mylar impregnated with a cationic surfactant, one should anticipate a significant positive-ion background response (potential interference) due to detection of the parent cationic surfactant and a number of structurally significant fragment ions.
ACKNOWLEDGMENT We thank Gloria Story and Curtis Marcott for obtaining the infrared reflection absorption spectra from the nytran membrane and the nytran-coveredsample foil. We also thank Ron Jandacek, Tony Steimle and Alan McDowell for providing the HPLC isolates of the sterol ester fraction from hamster feces. Finally, we thank Phil Brode and Robert Laughlin for critically reviewing the manuscript prior to submission. LITERATURE CITED (1) McNeal, C. J.; Macfarlane, R. D.; Thurston, E. L. Anel. Chem. 1979, 51, 2036-2039. (2) Jordan, E. A.; Macfarlane, R. D.; Martin, C. R.; McNeal, C. J. Int. J. Mass Spectrom. Ion Pmcesses 1983, 53, 345-348. (3) Macfarlane, R. D.; McNeal, C. J.; Martln, C. R. Anal. chem.1986, 58, 1091-1097. (4) McNeal, C. J.; Macfarlane, R. D. J. Am. Chem. SOC. 1986, 108, 2132-2139. (5) McNeal, C. J. I n Mass Spectrometry ofhrge Non-Voktik h4o4cules for Marine Organic Chemistry; Hllf. E. R., Tuszynskl, W., Eds.; World
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Sclentlflc Publlshlng Co.: Singapore, 1090 pp 51-60. (6) Jonsson, 0. P.; Hedkr, A. B.; Hekansson, P. L.; Sundqvist, 8. U. R.; Save, B. G. S.; Nlelsen, P. F.; Roepstorff, P.; J o h a n w , K. E.; Kamensky, 1.; LIndbWg, M. S. L. AMI. chem.1986, 58, 1084-1087. (7) Roepstorff, P. ACC. chem.Res. 1989, 22, 421-427. (8) Save, 0.; Hakansson, P.; Sundqvist, B. U. R.; Jonsson, U.; Olofsson, G.; Malmqulst, M. Anal. Chem. 1987, 59, 2059-2063. (9) Roepstorff. P. I n Mass Spectrometry of Large Non-VoktMe Molecuks for Marine Organic Chemktty; Hllf, E. R.. Tuszynskl, W., Eds.; World Scientific Publishing Co.: Singapore, 1990 pp 23-32. (IO) Wolf, B.; Macfarlane, R. D. Presented at the 38th Annual Conference on Mass Spectrometry and Allied Topics, Tuscon, AZ, June 3-8, 1990. (11) Alal, M.; Demlrev, P.; Fenselau. C.; Cotter, R. J. Anal. Chem. 1986, 58, 1303-1307. (12) Jungclas, H.; Frits&, H. W.; Kohl, P.; Schmidt, L. I n Mass Specbwnetry of Large Non-Volatile h4o4cules for Marine Orgenic Chemistry; Hllf, E. R., Tuszynski, W., Eds.; World Sclenttflc Publlshlng Co.: Singapore, l9gO pp 96-102. (13) Cotter, R. J. Anal. Chem. 1988. 60, 781A-793A. (14) Rosen, M. J. J . Am. ONChem. Soc. 1975, 52, 431-435. (15) Rosen, M. J. Swfect8nts and Interfeclal Phenomna; John Wlley and Sons: New York, 1978; pp 26-55. (IS) Wakamatsu, T.; Feurstenau. D. W. In Advances in Chemistry Serbs No. 79; Weber, W. J., Matljevls. E.. Eds.; American Chemlcai Soclety: Washlngton, DC. 1968; pp 161-172. (17) Lacey, M. P.; Keough, T. RapU Commun. Mass Spectrom. 1989, 3 , 323-328. (18) Taklgiku, R.; Keough, T.; Lacey. M. P.; Schneider, R. E. RapUCommun. Mass Spectrom. 1980. 4 , 24-29. (19) Loo, J. A.; Edmonds, C. E.; Smith. R. D.; Lacey. M. P.; Keough. T. BEomed. Envkon. Mass Spectrom. 1990, 19, 286-294. (20) Ligon, W. V.; Dwn, S. B. rnt. J . Mass Spectrom. Ion Processes 1984, 57, 75-90. (21) Ligon, W. V.; Dorn, S. 8.. Int. J . Mess Speclrom. Ion Processes 1984, 61, 113-122. (22) Lacey, M. P.; Keough, T. Raw Commun. Mass Spectrom. 1989, 3 , 46-50.
(23) Simms, J. R.; Keough, T.; Ward, S. R.; Moore, B. L.; Bandurraga, M. M. Anal. Chem. 1988, 60, 2613-2620. (24) Wernery, J. D.; Peake, D. A. RapU Commun. Mass Spectrom. 1989, 3 , 369-399. (25) BloRad Laboratories, Product Bulletin 1080, 1983; pp 1-12. (26) Keough, T.; DeStefano, A. J. Anal. Chem. 1981, 53, 25-29. (27) Munster, H.; Steln, J.; Budzklewlcz, H. Biomed. Envlron. Mass Spectrom. 1986, 13, 423-427. (28) Welntrab, S. T. In Mass Spectrometry of B&bgicalMatenlais; M e w en, C. N., Larsen, B. s., Eds.; Marcel Dekker, Inc.: New York. 1990 pp 257-286. (29) Nord, R. F.; Macfarlane, R. D. Presented at the 37th Annual Conference on Mass Spectrometry and Allied Topics, Mlaml Beach, FL, May 21-26, 1989.
RECEIVED for review February 5, 1991. Accepted April 18, 1991.
