499
Anal. Chem. 1982, 54, 499-503
the Cd net concentration in the lake samples. If one assumes that contamination affected individual samples and blanks equally on average, then most of the irreproducibility in the MSID determinations is attributable to variability in the extent of contamination.
CONCLUSIONS The ED-MSID combination proved to be a suitable method for determining Cd at very low concentrations in freshwater. Once the proper precautions are taken, the procedure yields a concentrate that is free of significant interferences and which gives steady ion currents from samples and blanks containing 25-100 ng total Cd. In practice the lower working limit is determined by the blank value, rather than inherent sensitivity. And it is the variability introduced by contamination that appears to controll the overall precision. In our experience, tlhe most persistent and troublesome source of this contamination is Cd-laden particulate matter in the laboratory and shipboard atmospheres. Airborne Cd often reaches several micrograms per cubic meter around urban-industrial centers (14) and can be higher in dusty environments or where tobacco is being smoked. These particles readily contaminate not only open liquid samples but also the surfaces of containers and reagents exposed to unfiltered air. Much of the associated Cd is found near the surface of these particles (15) and is readily available for solubilization. Our use of closed conlahers and clean air work stations was effective in dealing with these problems. In some control samples, Cd was undetectable even by ED/MSID, indicating that further experience with the technique will lead to even lower mean blank values. However, other reports ( 1 , Z ) indicate that clean room conditions may still be required if metal concentrations below 10 ng L-l are to be studied routinely. Unrecognized difficulties in controlling contamination and/or lack of sensitivity appear to have plagued earlier efforts
to determine Cd coincentrationsin the Great Lakes. After a critical review of the historical data, Rossman (16) concluded that none was reliable. For Lake Michigan, the Cd data reported here are the first to be gathered with sufficient attention to contamination control, using independent methods of sufficient sensitivity, to provide a glimpse of the actual present concentrations. In a separate report (17), we have shown how these rrneasurements are crucial to arriving at a budget for Cd in Lake Michigan and to establishing the rate of its accumulation in the future.
LITERATURE CITED (1) Settle, D. M.; Patterson, C. C. Science 1980, 207, 1167-1176. (2) Rosman, K. J. R.; DeLaeter, J. R. J. R . SOC.West. Aust. 1977, 59, 91-96, (3) Batley, 0. E.; Malousek, J. P, Anal. Chem. 1977, 49, 2031-2034. (4) Torsi, 0. Ann. ChJm. (Rome) 1977, 67, 557-566. (5) Brandenberger, H.; Bader, H. At. Absorpt. Newsl. 1987, 8 , 101-104. (6) Falrless, C.; Bard, A. J. Anal. Left. 1972, 5433-437. (7) Fairless, C.; Bard, A. J. Anal. Chem. 1973, 45, 2289-2291. (8) Lund, W.; Larsen, B. V. Anal. Chim. Acta 1974, 72, 57-64. (9) Catanzaro, E. J. ,/.-Water Pollut. Control Fed. 1975, 47, 203-204. (IO) Elzerman, A. W.; Armstrong, D. E; Andren, A. W. Environ. Sci. Techno/. 1979, 13, 720-725. (11) Allen, H. E; NOH, K E.; Jamljun, 0.; Boonlayangoor, C. Radlological and Environmental Research Dlvision Annual Report, Jan-Dec 1977, Argonne National Laboratory, ANL-77-65. Part 111, pp 70-73. (12) Cameron, A. E.; Smith, D. H.; Walker, R. L. Anal. Chem. 1989, 4 7 , 525-529. (13) Bickford, M. E.; Sllka, L R.; Shuster, R. D.; Anglno, E.;Ragsdale, C. R. Anal. Chem. 1978, 50, 469-472. (14) Thompson, R. Adv. Chem. 1979, 772, 54. (15) Natusch, D. F. S. Environ. Health Perspect. 1978, 22, 79. (16) Rossman, R. Absiracts of the 24th Conferance of Great Lakes Research, 28-30 April 1981, Columbus, OH, p 34. (17) Muhlbaler, J.; Tisue, G. T. Water, Air, Soil Pollut. 1981, 15. 45-59.
RECEIVED for review October 7,1981. Accepted December 8, 1981. A preliminary account of this work was presented at the 2nd Midwest Water Chemistry Workshop, Minneapolis, MN, 8-9 Oct 1979.
Direct Chemical Ionization Mass Spectrometry with Polyimide-Coated Wires V. N. Reinhold” and !Steven A. Carr Department of Biological Chemistry and Laboratory of Human Reproductlon and Reproductive B/ology, Harvard Medical School, Boston, Massachusetts O.? 115
A procedure is descrlbed for improving the results obtalned in the analysts of thermally labile organic compounds by direct chemlcal Ionization (DCI)mass spectrometry. The nichrome wire sample support is coated wlth a polyimide material (Pyre-ML) In a two-step procedure uslng an eiectrlcal current to induce polymeriratlon. Comparison of the DCI mass spectra of numerous bioioglcaliy Important compounds (lncluding oilgosaccharides and peptldes) obtained with polyimide-coated wire vs. bare metal Indicates a dlminutlon in pyrolysis and a correspondlng Increase In the intensity of ions related to molecular welght for samples desorbed from the coated wlres. Avolding metal-sample interactton by coatlng the wire with a polylmide surface extends the DCI approach to compounds of greater polarity and higher molecular weight.
A major goal of mass spectrometric research is to develop ionization methods which will enable the determination of 0003-2700/82/0354-0499$0 1. W O
highly polar and thermally labile compounds with a minimum of decomposition. One approach has been to rapidly heat nonvolatile samples coated on an extended direct insertiton probe within a chemical ionization reagent gas plasma. This technique, known as direct chemical ionization (DCI), has evolved through the contributions of a number of research groups into a useful alternative approach for obtaining structural information on thermally labile samples. For a recent review of this topic see ref 1. In contrast to conventional solids probe electron impact or chemical ionization mass spectrometry, the spectra of polar compounds obtained by direct sample insertion into the reagent gas plasma contain (and are often dominated by) ions indicative of the molecular weight as well as structurallly significant fragment ions. The DCI technique is more sensitive than field desorption (FD) mass spectrometry, and invariably more fragmentation is observed, if only, in some cases, a spectrum of the products of pyrolysis. However, a number of involatile compounds fail to exhibit ions diagnostic of 0 1982 American Chemical Society
500
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982 8000
200
I
Total
Ion
,:'"',.. . /Current I
175
.
-
150
5000 -
I25
6000
I=(342+360)
R =
4000
-
IO0
3000
-
75
2000
c
50
,
I
,
,
25
I
@,r,*W--fl
0
0
50
55
60
65
Scan
70
75
80
85
Number
+
Figure 1. Total and selected ion current profiles obtained by DCI of kojibiose from uncoated nichrome wire. Profile I = C [ m / r342 m / z NH,]' and (M NH,)', respectively) which are ions related to the intact or nearly intact molecule. Curve R is the ratio of pyrolysis ions m / z 180 and m l z 198 to I as a function of heatlng current and mass spectral scan number. The wire was heated linearly at 2 mA/s.
3601 ([M - H20)
+
+
molecular weight by DCI but do by FD. Sample analysis by DCI is most frequently initiated by a programmed heating current through a resistive wire (such as rhenium or nichrome) on which the sample has been coated. Since heating and sample-surface interactions are involved, pyrolysis is a constant problem and the DCI technique must contend with this fact. As would be anticipated, pyrolytic products are enhanced over molecular weight related ion products as the sample increases in molecular weight, decreases in amount, and possesses greater ionic character. Thus, obtaining molecular weight information on nanogram amounta of large thermally labile compounds by DCI can be exceedingly difficult. We report here the use of polyimide-coated (PIC) wires, a technique which extends the limits of the DCI approach.
EXPERIMENTAL SECTION Polyimide Wire Coating. A 5 cm length of nichrome wire (0.0125 in. diameter) bent into a "U" shape is attached to a variable ac power supply and the voltage is adjusted to obtain between 0.7 to 0.9 A flowing in the circuit (- 1 V ac). The wire is then immersed briefly in polyimide primer (primer and topcoat are available from American Durafilm Co., Inc., Newton Lower Falls, MA, under the trade name Pyre-ML) diluted 1:l with N-methylpprolidone. Upon removal of the wire from the primer the current initiates polymerization (5-10 8); droplets which may form on the wire are removed by touching with a glass rod. This step is repeated. the polymerized primer covered wire is then dipped into a topcoat solution and treated identically. Fully coated wires are attached to the DCI probe, inserted into the CI chamber, and programmed slowly through a heating cycle to remove interfering background. Although a relatively large amount of material desorbs during this initial "burn-in", we have experienced no loss in sensitivity or increase in the rate of ion source contamination. To initiate desorption a linear programmed heating current (mA/s) was started coincident with repetitive scanning. Current applied to the PIC wire during an analysis should not exceed that which is sufficient for sample desorption, since excess heating shortens the usable life of the polyimide coating. The coated wires are reusable (up to 10 different samples) with no problem of cross-contamination. Instrumentation. The mass spectrometer utilized in this study was a Finnigan-MAT 312 instrument with reverse geometry
(magnetic sector preceding electric sector) and fitted with a combined CI/EI ion source. The programmable DCI probe power supply unit for ramping the wire current was obtained from Finnigan-MAT, Sunnvale, CA. Modifications were made to read applied DCI wire current through an ammeter on the ion source control unit (2). RESULTS AND DISCUSSION The ideal mass spectrum for an unknown material would be one that yields ions indicative of the molecular weight as well as structurally informative fragment ions. Unfortunately for many classes of compounds, including carbohydrates, glycoconjugates, amino acids, and peptides there is often a disproportionate distribution of ions when using electron impact (EI) or field desorption (FD). Conventional chemical ionization (CI) and electron impact ion sources are designed to analyze samples in the gas phase with sample material vaporized external to the ionization chamber. Under these conditions, molecular weight related ions can only be observed for polar and thermally labile materials which have been derivatized prior to their analysis. The mass spectra of compounds ionized by FD are usually dominated by the molecular weight related ions with very few (if any) fragment ions. In contrast, vaporization of involatile samples by DCI gives rise to intact or nearly intact neutral molecules which are subsequently ionized to produce both molecular and fragment ions ( I ) . Ions indicative of the molecular weight are observed very early in the desorption profile and usually last for only a few seconds ( I , 3 ) . Desorption and subsequent ionization of neutral pyrolysis products formed on the support results in an abundance of low mass ions which become dominant in the latter part of the total-ion profile. These factors can be demonstrated with the disaccharide kojibiose and a plot of the ratio of ions associated with glycosidic bond rupture ( m / z 180 and 198) to those ions related to the molecular weight ( m / z 342 = [(M - HzO) + NH4] and m / z 360 = (M NH4)+)against the repetitive scan number (Figure 1). This ratio (R) is observed to increase dramatically on the descending side of the selected ion profile for the intact disaccharide (I). The abundance of ions desorbing after the initial molecular weight related ion plume (i.e., total ion plot
+
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
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For compounds more polar than simple saccharides (such as amino acids and peptides) the total-ion current profile usually maximizes well after the molecular weight related ion current profile and recovers only slowly to base line. Furthermore, for these materials the percentage of the total-ion currenit carried by molecular weight related ions is much smaller and is observed for a shorter duration. Other workers have indicated that the relative ratio of pyrolysis products to intact neutrals depends largely on two factors: the nature of the surface on which the sample is coated and the rate of sample heating (3-7). To reduce sample-surface interactions and, therefore, increase the desorption of intact neutrals, experimenters have used glass (51, quartz (7), Teflon (:I), silicone gum (IO), and Vespel ( 4 ) . However, these materials are not good conductors and are difficult to heat rapidlly in a controlled fashion. In an effort to combine the conductivity of wire with the inert surface properties of the above materials, we explored various thermally stable polymeric coatings. The polyimide material used in a two-step coating of primer and topcoat proved most satisfactory. The influence of wire heating and the resulting pyrolysis can be decreased by desorbing samples from this PIC wire. The strength of sample-surface binding is probably much lower for these polymer-coating wires than for bare metal andi, therefore, the heat of vaporization is now more related tio overcoming sample-s,ample interaction. This can be demonstrated by desorbing equal amounts of the pentapeptide Phe-Asp-Ala-Ser-Val(mol w t = 537) from bare wire and PIC wire and assessing the results by plotting the selected ion profiles of m / z 537 ([(M - H20) ",It) and the total ion profiles, (Figure 2) (11). The peptide desorbs intact from both substrates at essentially the same heating current but is sustained as such for a longer period of time with greater molecular weight related ion intensity in the PIC wire experiment. Not only is there a quantitative increase in the ion
Flgure 2. Total ion Intensity and selected ion plot of m l r 537 obtained from equal amounts of the pentapeptide (Phe-Asp-Ala-Ser-Val) desorbed from (a) bare, wire and (b) PIC wire as a function of increasing scan number and linear heating current. Mass 537 corresponds to [(M - H20) NH,]' rather than M', as demonstrated by using "NH3 as the reagent gas ( I ? ) .
+
+
and R, Figure 1)indicatee that sample remaining on the wire is pyrolyzing as a result of the increased heating current and the closer proximity to the metal surface. Mass spectra observed from this trailing edge of the total-ion profile consist of a series of high intensity low mass ions formed by pyrolysis. 105
13'5 280
I
'i I I
188
I
M W = 537
537
135
(M-17,-
373 393
55
le@
150
200
3 0
16
358
400
18)'
(M-17)'
427 449 458
500
550
608
/z Flgure 3. Mass spectrum obtained at the maximum of m l z 537 for the pentapeptide (Phe-Asp-Ala-Ser-Val) using (a) bare wire (scan no. 36, Figure 2a) and (b) PIC wire (scan no 38, Flgure 2b).
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
'
C0.e
:N
AAI
+
YH if
*'
IS88
ZAl
M W = I490
Reduced Perrnethylated Heptasaccharide
+
ElgUre 4. Mass spectra obtained at the polnt of maximum Intensity of (M NH4)+ for the reduced and permethyiated glucose polymer isolated from corn syrup and purified by HPLC. The A series ions correspond to cleavage of the glycosidic llnkage with charge retention on the reducing end whereas the 2 series ions correspond to charge retention on the nonreducing portion of the molecule.
MW = 481
34 1
B
Flgure 5. Mass spectrum of 1-0-stearoyi-2-hydroxy-sn lyceryl-3-phosphorylcholine(lyso platelet activating factor) taken at the point of maximum molecular weight related ion intensity ( m / z 482 = (MH) ).
522
/
N-Palmi toy1 DI h y d r o l a c t o c e r e b r o s i d e
Flgure 6. Mass spectrum of N-palmitoyl dlhydrolactocerebroside taken at the polnt of maxlmum molecular weight related ion intensity ( m / z 864 = (MH)' and m / z 881 = (M 4- NH4)+).
current carried by the molecular weight related ion but there is also a qualitative difference in the spectra with the appearance of more ions related to sequence structure when using the PIC wires (Figure 3).
We have compared DCI using PIC and bare wire for a number of compound classes and noted in every case two major advantages with the PIC wires: (1)A larger percentage of the ion current within each scan is carried by molecular
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982 578
1La-
Flaure 7. +NH,)+).
Mass sDectrum of
5031
12.13-dihexanoate ahorbol ester taken at the polnt of maximum molecular weight related intensity ( m / z 578 = (MI
weight related ions [(M t- H)+ and (M + NHd)+], and (2) a greater number of scans within the total-ion profile contain this molecular weight information. A comparison of the limits of sample detectability for coated and uncoated wires has not been pursued extensively in out laboratory. However, preliminary results by single-ion monitoring of the disaccharide melibose and the phorbol diester 12-myristate-13-acetatehave indicated sensitivities down to the 2-5 ng level using the PIC wires. Similar experiments on these compounds using bare wires were unsuccessful at the 50-ng level. Earlier studies with FDMS had indicated that derivatization aids in sample analysis by yielding a more lasting and stable ion current (8). For DCI analysis of polar compounds derivatization is also helpful and becomes mandatory at higher molecular weights. As an example, 100 ng of the underivatized trisaccharide raffinose yielded a protonated or cationized molecular weight related ion which was only 5% of the base ion. The molecular ion was not detectable using a bare wire. Using the combined approach of a PIC wire and derivatization, a permethylated heptasaccharide of a(1-4) linked glucose residues yielded a base ion at m / z 1508 corresponding to the ammonia cationized molecular ion (Figure 4). It is important to note that the fragment ions produced in this spectrum allow the sequence to be “read” from either end of the molecule whereas both the field desorption and fast atom bombardment spectra of this same compound consist almost entirely of ions indicative of the molecular weight. Further comparison of PIC and bare wires for two classes of lipids, and the very labile phorbol diesters indicated again that desorption from PIC wires provides greater intensity of high mass ions with a minimum of pyrolysis. One class of lipids, the phosphatidylcholines, were evaluated by using as an example l-O-stemoyl-2-hydroxy-sn-glyceryl-3phosphorylcholine (lyso platelet activating factor). This zwitterionic material gave an intense protonated molecular ion with numerous structural fragments which allowed excellent characterization of‘ the lipid and phosphorylcholine moieties (Figure 5). In a contrasting lipid class, the neutral but very polar cerebrosides were analyzed. The DCI mass spectrum from PIC wire of one example in this lipid class (lacto-N-palmitoyl cerebroside) is presented in Figure 6. Again, the protonated molecular ion (MH+ = 864), the ammonium attachment ion ( m / z 881), and the dehydration product [ m / z 846 = (MH - HzO)’] aid in molecular weight characterization. Additional fragments characterizing loss of the galactose (m/z 702) and glucose (m/z 522) moieties as well as ions related to the fatty acid and sphingosine residues are also observed. The phorbol esters, potent tumor promoters isolated from croton oil, are a class of compounds which show
extreme lability of the ester side chains and field desorption or chemical ionization (9) is required to determine their molecular weights. Using PIC wires these tumor promoters showed a threefold enhancement of the molecular weight related ion intensity over bare wires and a detectability to the low nanogram range. ‘Theresults obtained for 12,13-dihexanoate phorbol ester were typical (Figure 7 ) . From an instrumental standpoint, DCI is simple to set up, easy to perform, and inexpensive to operate. The use of polyimide-coated wires as the emitter substrate instead of the commercially supplied bare metal wires extends mass spectral analysis to higher masses with greater sensitivity and at a minimum of cost. We would expect this technique to be most helpful for materials of high polarity which are often available only in trace amounts.
ACKNOWLEDGMENT The authors thank C.E. Costello for providing the phorbol ester samples and for excellent assistance in gathering and interpreting the data. We also thank Bruce Stratton, President, American Durafdm, for the samples of Pyre-ML topcoat and primer, Peter Blmberg for the samples of phorbol diester, and Edward Goetzl for the sample of lyso platelet activating factor. LITERATURE CITED Cotter, R. J. Anal. Chem. 1980, 52, 1589 A. Kaufman, H., Flnnigan-MAT, Bremen, Germany. Hansen, G.; Munson, 13. Anal. Chem. 1880, 52, 245. Cotter, R. J.; Fenselau, C. C. Blomed. Mass Spectrom. 1979, 6 , 287. Baldwin, M. A,; McLafferty, F. W. Org. Mass Spectrom. 1973, 7 , 1353. (6) Beuhler, R. J. Flanagan, E.; Greene, L. J.; Friedman, L. J. Am. Chem. Soc . 1874, 96, 3990 (7) Ohashi, M.; Nakayamti, N. Org. Mass Spectrom. 1978, 73,642. (8) Costello, C. E.; Wilson, B. W.; Biemann, K.; Reinhold, V. N. I n “Cell Surface Glycolipids”; dweeley, C. C., Ed.; American Chemical Society: Washington, DC, 1980; p 35; ACS Symp. Ser., No. 128. (9) Solomon, J. J.; Van Duuren, B. L; Tseng, S A . Biomed. Spectrom. 1978, 5 (2), 164. (IO) Howlln, J. G.; Carroll, D. I.; Dzldlc I.; Hornlng, M. G.; Stillwell, R. H.; Hornlng, E. C. Anal. Lett. 1979, 72, 573. (11) Carr, S. A.; Reinhold, V. N. I n “Proceedings of the I V International Conference on Methods in Protein Sequence Analysis”; Elzlnga, M., Ed.; The Humana Press: Clifton, NJ, 1981, in press. (I) (2) (3) (4) (5)
RECE~VED for review September 24,1981. Accepted November 24, 1981. A preliminary account of this work was presented at the 29th Annual American Society for Mass Spectrometry meeting held in Minneapolis, MN, May 1981, papers numbered RPA 12, RPA lB, and RPA 14. This work was supported by the National Institute of General Medical Sciences, National Institutes of Health (Department of Health and Human Services, USA), Grant No. 5 RO1 GM2625.
