Anal. Chem. 1989, 6 1 , 1013-1016
of the major species obscures the signal of interest. This can degrade limits of detection by several orders of magnitude and, thus, can severely limit detectability in real samples. The applicability of the technique will depend upon the details of which species are to be discriminated, and a t what levels. In the most general terms, the greatest problems are posed by the detection of a trace of a species of much higher ionization potential than that which is present in excess (such as zinc in seawater or biological fluid). If the excess species ionizes by nonresonant two-photon ionization, its signal will tend to mask the resonant signal. In such a case, only an experiment involving a mass spectrometer or photoelectron spectrometer is plausible. When the ionization potentials are more similar there is much less interference as resonant photoionization is several orders of magnitude larger than the nonresonant process, with proper choice of resonance. Two-color experiments should allow even more sensitive and selective detection of many species.
LITERATURE CITED (1) Hurst, G. S.; Payne, M. G.; Kramer, S. D.; Young, J. P. Rev. M o d . Phys. 1979, 57, 767-819. (2) Hurst, G. S.; Nayfeh, M. H.; Young, J. P. Appl. Phys. Left. 1977, 3 0 , 229-23 1. (3) Hurst, G. S.; Nayfeh, M. H.;Young, J. P. Phys. Rev. A 1977, A75, 2283-2292. (4) Whltten, W. B.; Koutny, L. B.; Nolan, T. G.; Ramsey, J. M. Anel. Chem. 1987, 59, 2203-2206.
1013
(5) Ramsey, J. M.; Whitten, W. B. Anal. Chem. 1987, 59, 167-171. (6) Nolan, T. G.; Koutny, L. B.; Blazewlcz, P. R.; Whltten, W. B.; Ramsey, J. M. Appl. Spectrosc. 1988, 42, 1045-1048. (7) Apei, E. C.; Anderson, J. E.; Estler, R. C.; Nogar, N. S.; Miller, C. M. Appl. Opt. 1987, 26, 1045-1050. (8) Levenson, M. D. Introduction to Nonlinear Laser Spechoscopy; Academic Press: New York. 1982. (9) Lucatorto, T. 8.; Clark, C. W.; Moore, L. J. Opt. Common. 1984. 4 8 , 406-410. (10) Engleman, R.; Keller, R. A.; Mlller, C. M.; Nogar, N. S.; Paisner, J. A. Nucl. Instrum. Methods Phys. Res., Sect. B 1987, 826, 448-451. (11) Mallard, W. G.; Miller, J. H.; Smyth, K. C. J. Chem. Phys. 1982, 76, 3483-3492. (12) Smyth, K. C . ; Mallard, W. G. J. Chem. Phys. 1982, 7 7 , 1779-1787. (13) Bagratashvili, V. N.; Ionov, S. I.; Mishakov. G. V.; Semchisen. V. A.; Masalov, A. V. J. Opt. SOC.Am. B: Opt. Phys. 1987,B4, 129-132. (14) Marr, G. V.; Wherrett, S. R. J. Phys. B 1972, 5 , 1735-1743. (15) Koch, M. E.; Collins, C. B. Phys. Rev. A 1978, 79, 1098-1105. (16) Cheret, M.; Lindinger, W.; Barbler, L.; Deloche, R. Chem. Phys. Left. 1982, 88, 229-232. (17) Cheret, M.; Barbier, L.; Linger, W.; Deloche, R. J. Phys. 8 1982, 75, 3463-3477. (18) Barbler, L.; Cheret, M. J. Phys. B 1983, 76, 3213-3228. (19) Travis, J. C. Anal. Chem. 1987, 59, 909-914. (20) Morellec, J.; Normand, D.; Petite, G. Phys. Rev. A 1976, A M , 300-31 1.
RECEIVED for review November 17,1988. Accepted February 9, 1989. P.R.B. acknowledges a Postgraduate Research Trainee Fellowship from Oak Ridge Associated Universities. This research was sponsored by the U S . Department of Energy, Office of Energy Research, under Contract DE-ACOB840R21400 with Martin Marietta Energy Systems, Inc.
Quantitative Analysis of Low Molecular Weight Polar Compounds by Continuous Flow Liquid Secondary Ion Tandem Mass Spectrometry Tao-Chin Lin Wang,* Ming-chuen Shih, and Sanford P. Markey Laboratory of Clinical Science, National Institute of Mental Health, 9000 Rockville Pike, Bethesda, Maryland 20892
Mark W. Duncan Intramural Research Program, National Institute of Neurological Disorders and Stroke, 9000 Rockville Pike, Bethesda, Maryland 20892
Quantitative analyses of low molecular weight ( 100-200) polar compounds [ 1-methyl-4-phenyipyridine (MPP'), 2amlno-3-(methylamino)propanoic acid (synonyms, @-(methylamino)+-alanine or BMAA), and tryptophan] were conducted on a triple-stage quadrupole mass spectrometer configured for contlnuous flow liquld secondary ion mass spectrometry ionization (CF L-SIMS). I t is shown that quantlflcatlon by CF L-SIMS at subnanogram sensltivlty can be preclse (correlation coefficients > 0.99), accurate, speclflc, and routine for compounds not measurable by static L-SIMS. Successful analyses, however, are strongly dependent upon the stability of the film formed by the mobile phase on the probe tip. I n our system, film stability is affected by mobile phase composltlon and flow rate, ion source and probe tip temperature, probe-tip and capillary alignment, film thlckness, and sample composltlon.
INTRODUCTION The techniques of fast atom bombardment (FAB) (1,2)and liquid secondary ion mass spectroscopy (L-SIMS) (3) have
been widely accepted for their ability to analyze polar organic analytes. Effective ionization of polar compounds in both techniques is attributed to the use of low volatility, viscous solvents as a liquid matrix ( 4 , 5 ) . Recently, Ito et al. (6) and Caprioli et al. (7) introduced a refinement to FAB and L-SIMS which offers a number of potential advantages. Rather than external application of sample to the probe tip and reinsertion into the ion source, this approach, now known as continuous flow (or dynamic) FAB or L-SIMS, conveniently introduces the analyte into a continuous stream of mobile phase containing the low volatility, viscous matrix and applies it, via capillary tubing, directly to the surface of the probe tip in the ion source (6-10). This technique combines the convenience of direct liquid introduction characteristic of most chromatographic techniques with the power of FAB or L-SIMS ionization technique. While continuous flow FAB or L-SIMS is finding applications in many areas of qualitative and quantitative analyses, we were interested in applying this technique to the accurate quantification of low molecular weight polar biologicals with minimum chemical modification and sample work-up. In contrast to static FAB or L-SIMS, it was reasoned that CF techniques would permit an increase in signal to noise ratio in the low mass range where analyte
This article not subject to U S . Copyright. Published 1989 by the American Chemical Society
1014
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
mlz : 110
Lens probe
nnn
n
8
0 '
1
mlz :
173
\
Capillary
Figure 1. Structure of continuous flow
L-SIMS
interface.
ions are frequently obscured by matrix ions. In this paper the utilization of a continuous flow L-SIMS probe on a triple-stage quadrupole mass spectrometer will be described. Quantitative analyses of several polar compounds extracted from biological fluids and using deuteriated isotopomer internal standards are included as examples for performance evaluation. Factors affecting the feasibility of CF liquid SIMS/MS and MS/MS as a routine device for quantitative applications are discussed. EXPERIMENTAL SECTION Materials. Glycerol was purchased from J. T. Baker Chemical Co. and used without further purification. All other reagents and solvents were of analytical grade. Rat Adrenal Extracts. Single adrenal glands were dissected from adult male Sprague-Dawley rats (220-260 g) which had (MPTP, received l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine 10 mg/kg, sc). The adrenals were then sonicated in 0.2mL of 0.1 N HCIOl containing l-methyl[2H3]-4-phenylpyridine ([2H3]MPP+, 10 ng/pL) as an internal standard. Sonicated samples were centrifuged for 20 min at 13000 rpm, and 0.15 mL of the supernatant was transferred to centrifugal filters (Centrifree, Amicon, Danvers, MA) and spun at 4000 rpm for 20 min. The clear solution was used directly for mass spectrometric analyses. Plant Extracts. Whole seeds of Macrozamia lucida (1.085g) and the gametophyte tissue of Cycas rumphii seeds (2.813g) were homogenized in dilute HCl (0.1 M, 10 mL) in the presence of [2H3]BMAAas internal standard. Samples were then centrifuged to remove particulate matter (30min, 3000 rpm), and a portion of the clear supernatant (2 mL) was taken to dryness under nitrogen. The residue was then esterified by treatment with ethanolic HCl(500 pL, 65 OC, 2 h) and taken to dryness. Samples were reconstituted in water (2mL) and washed with chloroform (1 mL), and the aqueous phase was separated by centrifugation and basified by the dropwise addition of NH40H. The free bases were back-extracted into fresh chloroform (1mL). The extract was dried under nitrogen, and the residue redissolved in 400 pL of 2.5% trifluoroacetic acid water/glycerol (80/20).The acidic water/glycerol solution was used directly for mass spectrometric analyses. Construction of the Continuous Flow L-SIMS Interface and Experimental Parameters. Mass spectra were acquired on a Finnigan Mat TSQ-70 triple stage quadrupole mass spectrometer (Finnigan Mat, San Jose, CA) equipped with a Finnigan Mat prototype Bioprobe ion source and a Phrasor cesium ion gun. An HPLC pump (Gilson, Model 302, Middleton, WI) with a microflow adaptor and pulse dampener (Gilson, Model 802B) was used to deliver mobile phase through a 0.5-pL loop injector (Rheodyne, Model 7410,Cotati, CA) to 75 pm i.d. fused silica capillary tubing (Polymicro, Phoenix, AZ). The fused silica tubing passes through a vacuum manifold flange into the ion source. In the ion source, capillary tubing is further guided through an aluminum cylinder, which is vertically screwed onto the bottom plate of the ion source block, to lay in contact with a standard probe tip and release liquid onto the parallel surface where it is bombarded with 5-6 keV cesium ions (Figure 1). The thermal energy required to prevent the liquid stream from freezing due to quick evaporation in vaccum at the capillary exit is provided by direct contact of the capillary tubing with the aluminum cylinder as well as the probe tip. The length of capillary exposure
ot---------T-
"
100
200
'
,
"
300
"
,
400
"
"
I
'
,i
500
Time (sec) Figure 2. Repetitive injections of MPP' (50 ng) and [2H,]MPP+ (125 ng) mixtures indicate system stability for repeated measures. is therfore limited to 2-4 mm beyond the top end of the aluminum cylinder. An external telescope was required for mechanical alignments including probe tip orientation and tubing position so that a stable film could be obtained. Film stability is a crucial factor in precise quantitative analyses. Samples were injected into the mobile phase (water/glycerol 80/20) through the loop injector. With the unheated probe tip and source block at 40-45 OC, a stable film was obtained at a flow rate of 5.5-6.5 pL/min. Typical analyzer manifold pressures were 6-7 X lo4 Torr for MS experiments and 1 X lod Torr for MS/MS due to the presence of collision gas (e.g. argon gas, 0.5 mTorr). In the single MS scanning mode, the [M + H]+ peak was scanned over a narrow mass range at a scan rate of 5-10 daltons/s. In the MS/MS daughter scanning mode, argon gas was introduced as collision gas to produce fragments from a selected parent. The MS/MS experiment requires a collision gas pressure of 0.5 mTorr in the collision chamber. Coupled with the pressure caused by the continuous liquid flow into the mass spectrometer, minimization of the gas load was achieved by using a cesium ion gun rather than the conventional saddle field fast atom source. For ethyl ester (BMAA-Et)daughter the p-N-(methylamino)-L-alanine scan, [M + H]+ at m / z 147 and its [Q3] isotopomer standard (i.e. [2H3]BMAA-Et;m / z 150) were selectively transmitted through the first quadrupole every 0.1 s, and their corresponding daughter fragments at m / z 44 and 47,respectively, were then alternatively monitored. Centroid data were collected and stored in the data system for analysis. The integrated ion current peak areas were obtained via routine chromatographic data processing techniques. RESULTS AND DISCUSSION Precision and Accuracy of Quantitative Analysis of MPP+. The reproducibility of the continuous flow L-SIMS system was found to be consistent as demonstrated by the repeated injections ( n = 10,0.5pL) of a solution containing 50 ng of MPP+ (m/z 170)and 125 ng of [q3]MPP+(m/z 173). As shown in Figure 2,the ion current response was symmetrical with minimal tailing. The sample ion current peaks were 50 s wide at base line and 15 s wide at half height, thereby allowing sample introduction every 2 min. Since the sample was eluted in a discrete manner, background subtraction and peak ratio calculation as routinely applied in chromatographic techniques were employed for data analysis. The mean ratio integrated for ion current peak areas for this mixture of MPP+ isotopomers (i.e., m / z 170/173) was found to be 0.3983 (10.0020,standard deviation) with a range of 0.3955-0.4011. The quantitative analysis of MPP+ incorporating [2H3]MPP+ as internal standard gave a linear standard curve ion abundance ratio, m / z 170/173 = 0.8717( [ MPP+] / [ ['HS]MPP+]) - 0.3214 with a correlation coefficient of 0.999 over the range 200 pg
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
(a) Macrozamia lucida 147
I
I
150
-
-
44
47
1015
(b) Cycas rumphii
! 1
14’
150
-I +
44
47
0
so loo 50 100 Time (sec) Time (sec) Figure 3. Reconstructed ion chromatogram at m / z 170 and 173 for (a) rat control adrenal gland extracts without MPTP treatment and (b) rat adrenal gland extracts 4 h after injection of 10 mg/kg, sc, MPTP. A fixed amount of [*H,]MPP+ was added as internal standard. MPP’ ( m / z 170) signal intensities were normalized with respect to the internal standard intensities for easy comparison. Note high interfering chemical background at mlz 170 in part a indicates requirement of additional separation procedure for quantification by single-stage MS. to 200 ng. A detection limit of 200 pg was routinely obtainable a t a signal to noise ratio of 3:l. For quantitative analyses in the low to subnanogram range of MPP’, and possibly for all adsorbable compounds, our data indicate the need for deactivation of materials in contact with the sample. That is, adsorption of subnanogram quantities of analyte on the valve surface and transfer line may be minimized by including a structural analogue, homologue, or isotopomer in the mobile phase, or by prior saturation of active sites by injection of a “bolus” of analyte. Semiquantitative Analysis of MPP+ in Rat Adrenal Gland Extracts. The quantification of MPP+ in perchloric acid extracts of rat adrenals, following pretreatment of the animals with MPTP, is shown in Figure 3. MPP+ is known to be a major metabolite of MPTP (11). Figure 3a shows the results obtained by the analysis of extracts, doped with [2H3]MPP+,from the adrenal glands of animals not exposed to MPTP. Extracts from MPTP-treated rat adrenals exhibit a substantial increase in the signal at m/z 170 (Figure 3b) as compared to that obtained from controls (Figure 3a) when normalized to the internal standard at m/z 173. In Figure 3a the background signal at m/z 170 is not due to isotopic contamination but results both from other components in the complex crude extract and from an increase in background chemical noise due to perchloric acid. Thus, simple acid extraction of tissues is suitable for semiquantitative analyses, but additional sample pretreatment would be required for specific MPP+ quantitation. CF L-SIMS/MS/MS Analysis of BMAA in Plant Extracts. Linear standard curves over the range 5-250 ng were routinely obtainable with the underivatized amino acids, tryptophan, and BMAA. CF L-SIMS procedures were extended to the quantification of BMAA in plant extracts. As expected from the above example of MPP+, there was a poor correlation between capillary GC-selected ion assay for derivatized BMAA (12) and direct CF L-SIMS/MS analysis of crude plant extracts. However, a simple and direct approach to resolving the discrepancy, which apparently resulted from insufficient selectivity of CF L-SIMS/MS analysis, was found to be CF L-SIMS/MS/MS. Formation of BMAA-Et ( m / z 147) permits acid-base extraction as well as concentration in chloroform prior to drying and dissolution in glycerol-water.
800 200 400 Time (sec) Time (sec) Figure 4. CF L-SIMS MS/MS analyses of BMAA-Et indicate the absence of BMAA in the plant extracts obtained from (a) Macrozamia spp. relative to the amount detected in (b) Qcas spp., both having been spiked with identical amounts of [*H,]BMAA-Et.
600
These steps were therefore performed prior to mass spectrometric analysis to provide the requisite sample concentration and to enhance specificity. The MS/MS results were obtained by monitoring alternatively m / z 147 fragmenting to m / z 44 and for the [2H3]isotopomer, m/z 150 to 47. Comparable linear standard curves produced by CF L-SIMS/MS and MS/MS of BMAA-Et using [2H3]BMAA-Et as internal standard were obtained with correlation coefficients of 0.999 ion abundance ratio, m / z 147/150 = 1.486(BMAA-Et pg/kL) 0.047
+
ion abundance ratio, m / z 44/47 = 1.253(BMAA-Et kg/kL)
+ 0.070
Excellent selective and quantitative results were obtained from plant extracts that contain BMAA (Cycas spp., Figure 4b) in contrast to those containing trace levels (Macrozamia spp., Figure 4a), consistent with the quantitative values obtained by GC/MS of the same plant samples (12). With both GC/MS and CF L-SIMS assays for BMAA now available to us, we find each approach has its advantages. While CF L-SIMS offers a significant reduction in sample run time and requires minimal chemical derivatization (i.e., esterification to aid cleaning), these benefits are currently offset by a higher limit of detection and the increased hardware costs. Consequently, precise and selective determination of BMAA content in the microgram to nanogram per sample range is most expediently accomplished through CF L-SIMS whereas the need for lower detection limit (i.e., pg to ng per sample range) currently requires the routine application of our GC / MS-based methodology (12). A Stable Film. We have found that for optimum quantitative analyses and detection of low to subnanograms levels of analytes of low m / z values, the liquid film coating the probe tip must remain stable. A stable film is defined as no more than a 5% peak height or area variation in the signal at mlz 185, which derives from glycerol (protonated glycerol dimer). When such a condition prevails, we observe constant pressure, consistent background spectra, and stable ion current. However, in order to establish a stable film with the current prototype design, there must be a delicate equilibrium between the rate of solvent delivery and evaporation. Achievement and maintenance of a good stable film are thus far the most
1016
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
mlz :
147
0
200
400
600
800
Time (sec) Flgure 5. Repetitive injections of BMAA-Et made while increasing mobile phase flow rate from 6 to 6.5 FL/min show that spurious base-line rise detected at m l z 150 is due to film instability at the time when sample is eluted. At higher flow, signal at m l z 147 is less intense, but base line at m l z 150 is stable. crucial factors for performing routine quantitative analyses. Factors for Obtaining a Stable Film. First, the mobile phase composition, ita flow rate, and the temperatures of the ion source and probe tip are interrelated factors for achieving a stable film. For example, when mobile phase composition must be changed (i.e., to increase solubility or sensitivity), a series of testa are required to optimize these key variables and to establish a new equilibrium state. Before equilibrium is achieved, flow instability may be experienced caused either by freezing (i.e., if the heat supplied to the probe tip region is less than the heat required for evaporation) or dripping (i.e., if the mobile phase delivery rate is faster than the evaporation rate or, if the temperature is too high and hence the viscosity is reduced). These types of instability can cause the film to "puff" or "bubble". However, it is worth noting that "puffing" may be beneficial in the beginning of the experiment to help establish a channel of flow along side the probe tip so that excess solvent can flow away. We have also observed instability which produces a sinusoidal-like oscillation of pressure and signal intensity. The amplitude of the oscillation can be as high as the base line of the glycerol background signals or as low as a few percent but can persist for hours if the probe position, flow rate, and probe temperature are not altered. While the origin of this type of pulsation is still under investigation, we have found that stability can be reestablished by changing one of the principal factors, most conveniently the flow rate to the probe tip. When a stable film is obtained, the sensitivity and peak shape can be further optimized by fine adjustments of the probe-tip orientation relative to the capillary position.
Important Factors for Maintaining a Stable Film. Maintenance of a stable film requires that the mobile phase solvent is properly degassed, filtered, and delivered to the probe tip at a constant flow rate. Additionally, excess mobile phase should be removed from the region of the probe tip by an absorbent material (i.e., to prevent any pool of liquid formed near the probe tip area and in the ion source). This becomes important because a puddle of liquid can easily pop and thereby cause pressure fluctuations and sudden jumps or drops in the base line ion current. Furthermore, to be able to maintain a stable film during the time when sample elutes
is equally important. In Figure 5, the first three injections of BMAA-Et ( m / z 147) indicate that the film stability was apparently disturbed when the sample flowed onto the probe tip and changed the film surface tension, and thereby produced a spurious increase at m/z 150. On the other hand, salt content and co-emerging surface active compounds may cause an overall background decrease at all masses, or even cause the film to become unstable. Stopping and restarting the flow, or cleaning the probe tip, may be required to regain film stability. In general, sensitivity is highest when a thin stable film is obtained. Film instability caused by differences in surface tension between sample and mobile phase becomes more pronounced when operating with a thin film. It may therefore be necessary to sacrifice the optimal sensitivity possible with thin films so that samples containing salts or surfactants very different from that of the mobile phase will produce signals free from artifactual base-line shifts. In the example in Figure 5, a stable signal at m / z 150 indicative of the absence of base-line fluctuation was obtained as the film thickness increased by slightly increasing the mobile phase flow rate from 6 to 6.5 kL/min.
CONCLUSION Continuous flow L-SIMS offers a direct, precise, and accurate method for quantification of polar compounds of low molecular weight, as well as being a convenient introduction system for qualitative purposes. We have also demonstrated that increased analytical specificity can be achieved by CF L-SIMS/MS/MS. Due to the sensitivity and ability to directly analyze conjugated species, many clinical research applications are anticipated. However, a better wetting mechanism which will improve film stability with respect to mobile phase composition and sample property change is highly desirable. Furthermore, lowering the detection limit by using a focused cesium ion beam or other methods to produce a more effective sample-tc-ion yield are obvious areas of development to make CF L-SIMS a more attractive analytical tool. Registry No. MPP', 48134-75-4; BMAA, 15920-93-1;tryptophan, 73-22-3.
LITERATURE CITED (1) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. Soc.. Chem. Commun. 1981, 7 , 325-327. (2) Surrnan, D. J.; Vickerman, J. C. J . Chem. Soc.,Chem. Commun. 1981, 7,324-325. ( 3 ) Aberth, W.;Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 5 4 , 2029-2034. (4) Barber, M.; Bordoli, R. S.; Elliott, G. J.; Sedgwick, R. D. Anal. Chem. 1982. 59. 645A-657A. (5) Surmin, 6. J.; Vickerman, J. C. J . Chem. Res., Synop. lB81. 6 , 170-172. (6) Ito, Y.; Takeuchi, T.; Ishii, D.; Goto, M. J . Chromatogr. lB85. 346, 161 - 166. (7) Caprioli, R . M.; Fan, T.; Cottrell, J. S. Anal. Chem. 1986, 58 2949-2954. (8) Caprioli, R. M.; Fan, T. 6lOchem. 6iophys. Res. Commun. 1986, 1 4 1 , 1058. (9) Ashcroft, A. E.; Chapman, J. R.; Cottrell, J. S. J . Chromatogr. lB87, 15, 394. (10) Ito, Y.; Takeuchi, T.; Ishii, D.; Goto, M.; Mizuno, T. J . Chromatogr. 1888,358, 201-207. (1 1) Markey, S. P.; Johannessen. J. N.; Chiueh, C. C.; Burns, R. S.; Herkenham, M. A. Nature 1984,311 (5985). 464-467. (12) Duncan, M. W.;Crowley, J. S.; Jones, S. M.; Kopin, I. J.; Markey, S. P. Anal. Toxicol., in press.
RECEIVED for review October 6,1988. Accepted January 23, 1989.
