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Eric C. Hung, Timothy Wachs, James J. Conboy, and Jack D. Henion Drug Testing and Toxlcoiogy New York State College of Veterlnery
Medicine Cornell uliversliy 925 Warren Dr. ihaca, NY 14850
Atmospheric pressure ionization (API) combined with mass spectrometry (APIMS) can provide many benefita, including analytical ruggedness as well as enhanced sensitivity and selectivity. Although some people are just learning ahout APIMS, the concept originated morethan30yemago(I). At fmt it may appear impractical to couple ionization conducted at atmospheric pressure with mass analysis and detection carried out under high vacuum. In certain situations, however, there are distinct advantagesto decoupling the sample inlet from the h i h vacuum that is necessary for reliable mass analysis. One important example of this situation is when it is desirable to interface a liquid inlet system such aa a liquid chromatograph to a mass spectrometer. Other important separation science techniques with inlet systems that can he interfaced with MS include supercritical fluid chromatography (SFC), capillary electrophoresis 000~2700/9010362-713A1$02.5010 0 ISSO American Chemicei Society
(CE), and ion chromatography(IC). In this article we will briefly describe some important fundamentals of APIMS that provide a practical and versatile approach to the routine coupling of these modern separation techniques with MS. Figure 1 shows a schematic of a generic liquid inlet and APIMS system. The API ion source region (center) is separated from the high-vacuum mass analyzer region by a small orifice. The orifice must he large enough to introduce as many ions as possible from the atmospheric pressure region into the
counter a free-jet expansion and undergo considerable adiabatic cooling, which ions in conventional mass spectrometer ion source systems do not experience This region can contribute to the formation of cluster ions that results from the weak attachment of protonated water or solvent ions to the analyte(s) of interest. Focusing lenses are also required to contain ion transmission as the ions are focused to the mass analyzer. These lenses, combined with an acceleration region, can effectively dissociate cluster ions prior to directing them toward the mass analyzer.
lNS7RUMEN7ArION vacuum region while maintaining a low enough pressure mallow efficient operationofthe mass spectrometer. There are several ways to achieve this goal; for example, two or more differentially pumped stages with a carefully aligned nozzle and ion "skimmer" (not shown here) can be used. In another approach (Figure 1). ions are sampled from atmoepheric pressure directly into the high vacuum, and either a relatively small orifice (10-25 pm) or a very high pumping capacity (> 100,ooO L/s) is required. In either case, as the sampled ions enter the high-vacuum region, they en-
Historically, quadrupole maas spectrometers have been used for AF'IMS because they operate at the low translational ion energies that result in the AFI' source when it is housed at ground potential. However, APIMS using magnetic sector mass speetrometers has been described (2). and researchers have reported sampling ions from atmospheric pressure into a Fourier transform mass spectrometer (3) and an ion trap mass spectrometer (4). Gas-phase ionization Ions can be formed at atmospheric pressure by a variety of means. Two
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historically important approaches involve the use of a radioactive foil or a corona discharge needle (5), and both provide a source of ionizing electrons that produces ions from nearby gases. These ions readily undergo ion-molecule reactions with air and other gaseous molecules because of the short mean-free path between molecules at atmospheric pressure. This produces reactive intermediate ions that readily interact further with analyte molecules, resulting in atmospheric pressure chemical ionization (APCI), a very mild form of ionization. A thin foil that could emit energetic 5- particles was an early source of ionizing electrons for APCI. For example, a 63Ni foil housed in a cylindrical tube maintained at ambient temperature produces a range of electron energies approaching20 keV and a broad spatial distribution of reactant ions in the e~urce.Ionization is easily accomplished; a continuous source of electrons is available from the 5- emitter without any external power supply. Under normal operating conditions, chemical and thermal equilibrium are attained before ions are sampled. Becauae the ions possess low internal energies, fragment ions are rarely observed. Proctor and Todd reported that for trace components, the ionization efficiency of APCI is 103-104 times greater than that of electron ionization (EI)at reduced pressures (5). It appears that in favorable cases almost 100% ionization efficiency of a trace amount of sample can be achieved in an APCI source (5).Unfortunately, all of this ion current is not sampled into the mass analyzer, so the overall sensitivity is not necessarily better than with EI. The corona discharge method of ionization, which occurs through an electrical discharge at the tip of a needle held at high voltage (e.g., 3-6 kV), reportedly produces reactant ion spectra that essentially are identical to those produced by the 63Ni5- emitter source.
However, the corona source has approximately 100 times the reactant ion intensity of the 63Ni source. This results in a greatly increased dynamic range that is desirable for liquid inlet system applications (6). The reaction volume in the corona discharge system is defined by the region between the corona point and the sampling orifce. This region is typically only a few centimeters in length and has been referred to as a “wall-less” reactor because it is continuously swept by the carrier gas to exclude molecules that may desorb from the distant walls. The ion chemistrythat takes place in both of these APCI ion sources can be very complex (7).Adduct or cluster ions of air and water, which may be observed through a mass range of several hundred Daltons, can be formed. This formation process results from the high reactivity of the ions and the high concentration of other nearby gaseous molecules. The cluster ions formed produce a high chemical background that reduces the signal-to-noise ratio of analyte signals present; thus, there is a practical need for suppressing cluster ions or precluding their formation without losing analyte ions. Several approaches have been used to deal with cluster ions. In some instances heating the ion source region to 260 “C reportedly reduced cluster ions (8). In another case (9), a combination of elevated temperature and careful cleaning and drying of the carrier and sample gas reduced clustering in a “terminal” plasma. Kambara and Kanomata (10, 11) attacked the clustering problem hy using collision-induced dissociation (CID) in an intermediatepressure, differentially pumped stage between the ion source and the mass spectrometer. By using accelerating voltages of -10 V/cm at pressures of -1 Torr, they demonstrated the stripping of water molecules from various molecular and quasi-molecular ions. An alternative solution to the ion
Figun 1. Schematic of a generic liquid inlet APiMS system. 714A
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clustering phenomenon has been presented by French‘s group (12). A gas curtain or membrane between the ion source and the vacuum expansion region together with an applied electric field produces a CID or a “cluster buster” feature during the free-jet expansion. This momentum transfer of clustered product ions into a dry curtain gas shifts the cluster equilibrium distribution toward the low-cluster (lower mass) members and reduces the possibility of further clustering that occurs during the rapid adiabatic cooling encountered in the expansion. The gas curtain also helps maintain cleanliness by bathing the ion sampling orifice with excess dry Na which effectively precludes excess solvent or other molecules from entering the vacuum system. When this approach is coupled with cryogenic pumping, very high pumping speed is available with a minimum of the contamination that is characteristic of more conventional pumping systems. Although this pumping system may be more expensive, it potentially provides the optimum sensitivity and simplicity of operation. The single stage of ion sampling avoids the transfer losses that are characteristic of staged systems with a nozzle and ion skimmers, and in principle it should provide better detection limits. Further developments in both the French et al. and the Kambara and Kanomata approaches,or other novel approaches, are needed. Liquld-phase ionization In the systemsdescribedabove, there is a complex array of competing ion-molecule reactions. Positively and negatively charged ions are formed simultaneously in the APCI ion source; positive ion formation is favored by chemical ionization of strong gas-phase bases, whereas negative ion formation is favored by chemical ionization of strong gas-phaseacids. Analyte volatility is required, or detection of one component in a mixture may predominate. Prior separation via chromatography will be beneficial (and perhaps necessary)to allow detection of lower volatility or lower concentrationcomponents. Alternatively, for LCiMS, it is desirable to transfer ionic species in the HPLC effluent solution directly into the gas phase for mass spectrometric analysis. Ion evaporation, as its name implies, accomplishes this. It is an ionization process whereby ions are emitted from solution in charged liquid droplets directly into the gas phase. This process and its application to combined LCiMS have been described by ThomsonandIribarne (13)andVestal et al. (14). More recently Fenn and
Yamashita have demonstrated its importance in electrospray ionization for the analysis of solutions containing very polar, intractable compounds heretofore considered unlikely candidates for mass spectral analysis (15). The mechanisms involved in emission of ions from a liquid droplet into the gas phase at atmospheric pressure are not fully understood. Researchers have suggested that as a charged droplet evaporates in air under spraying conditions, a critical point may be reached where it is kinetically and energetically possible for ions at the surface of the liquid to evaporate into the air (16).It appears that the mechanism is independent of how the droplets or the charge are originally formed. A charged droplet consists of the solvent containing positive and negative ions, and the predominant charge depends on the polarity of the induced potential. Two mechanisms have been proposed for gas-phase ion formation from very small charged droplets: droplet fission at the Rayleigh limit (17) and direct field-induced ion evaporation (13).It is believed that the excess charges reside at the droplet surface. Convective heat exchange and interaction with air cause rapid size reduction of the droplets to a point where repulsive coulombic forces approach the level of droplet cohesive forces. As the radius becomes very small, the increased repulsive forces between like charges become very high. When the Rayleigh limit is reached, the larger droplets appear to “explode” to produce a cascade of fission products comprising smaller and smaller droplets. Repulsive forces within these tiny droplets appear to cause a field-induced “ion evaporation” of the dissolved analyte ion, which effectively transfers the ion from the condensed to the gas phase (13). This situation is desirable because it precludes introducing liquids directly into the mass spectrometer vacuum system; instead, gas-phase ions are preferentially sampled at atmospheric pressure into the mass spectrometer at the expense of the liquid. The details of the mechanisms for these processes are not well understood, and experiments to characterize them continue. For the separation scientist, this situation offers the prospect of mass spectrometric analysis of liquid effluents by producing gas-phase ions of low internal energy at atmospheric pressure without excess heat or other conditions that are detrimental to analyte stability. Droplet formatlon
It is apparent that the need to form small droplets is a phenomenon com-
mon to LC/MS and related liquid inlet systems coupled to MS. In most cases it is desirable to form small, uniformsized droplets from which analyte ions may be desorbed or ionized. This process can be accomplished in several ways, all of which involve disruption of the bulk liquid surface with an energy source that is sufficient to overcome the surface energy of the solvent. Three common methods use heat for vaporization, pneumatic nebulization incorporating a high-velocityjet of air to shear droplets from a liquid stream, or an electrostatic potential to create an electric stress to generate droplets (16). These three approaches used alone or in combination succeed in varying degrees to form small droplets. However, the size distribution of droplets depends not only on the mode of formation but also on the flow rate and solvent composition of the flowing stream. For example, thermospray produces a spray from liquid flows of 1-2 mL/min when heat is applied. The composition of the spray is believed to be at least 90% vapor, and the remainder is small droplets. The measured droplet size distribution ranges from about 5 to 200 pm for thermospray vaporization at atmospheric pressure, whereas under the same conditions a combination of heat and pneumatic nebulization produces smaller sized droplets over a narrower size distribution (18). In contrast, the electrospray process produces droplets that are < 10 pm with a narrow distribution in size (< 1 pm to -20 pm), albeit only from liquid flows typically < 5 pL/min (18).
In our experience, pure electrospray LC/MS with HPLC flow in the range 1-5 pL/min is not analytically rugged enough to be practical for solving realworld problems. Existing commercial HPLC pumps will not reliably form the wide range of gradients at 1-5 pL/min that are often required for many HPLC separations. In our laboratory, we have incorporated the concept of pneumatically assisted electrospray originally reported by Dole et al. (19),which we refer to as ion spray. This approach provides electrospray ionization while using a 40-50 pL/min HPLC flow, which is compatible with 1-mmi.d. columns (20). Alternatively, when sufficient sample is available, a postcolumn split from higher HPLC flows may be used with pure electrospray. Because electrospray produces droplets with a higher ratio of charge to analyte than any combination of techniques used to form droplets (161,in principle the high sensitivity of electrospray can ac-
commodate the postcolumn split while still using conventional HPLC hardware. In practice, however, there may be problems with this approach. The most important problem is the difficulty in maintaining a stable spray when highly aqueous eluents are used unless very slow flows are maintained. This situation commonly occurs at the beginning of gradient programs required for the separation of protein enzymatic digests. In addition, the change in eluent viscosity and surface tension during a gradient program causes practical problems in flow splitting that may produce spray instability in the pure electrospray process. There is little difficulty in maintaining a stable ion spray with good sensitivity at flow rates of 40-50 pL/min, even when the eluent consists of 100%water. This eluent or aqueous eluents containing trifluoroacetic acid and other buffers is necessary for the liquid chromatographic separation of components in biological samples. Tandem MS
Although the very mild ionization conditions common to APIMS can usually guarantee the determination of molecular weight for unknown compounds, the lack of fragmentation precludes useful structural information. A solution to this problem is the use of tandem mass spectrometry (MS/MS) with on-line HPLC (LC/MS/MS) to gain fragmentation information. These combined techniques are useful for the characterization of smaller molecules (21) or even the larger biomolecules that can be ionized by soft ionization techniques (22). LC/APIMS combined with M S N S provides complementary information. The former gives molecular weight determination and on-line separation as well as a molecular ion that indicates the molecular weight of the compound in question. When this molecular ion species is focused into the collision cell by the first mass analyzer, it may be subjected to ion-molecule interactions with a collision gas. These interactions cause subsequent fragmentation into product ions indicative of the chemical structure of the molecular ion. The population of fragment ions thus produced may then be passed into a second mass analyzer region to produce a full-scan product ion mass spectrum. The resulting mass spectrum may be interpreted by reference to model compounds and related mass spectral behavior from known structures. In addition, a variety of related MS/MS experiments can be used to further characterize structural features (23). Thus LC/MS/MS complements the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 13, JULY 1, 1990
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mixture analysis demands that are ebaraeteristic of incomplete HPLC separations, in addition to the mild ionization conditions afforded hy APIMS.
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knty-tnree chapters dexnbe a vanety of iew applications of mineral speamvopy to lkterrnine purity. interacton wth energ. characterizatbn of acdw centers. md adsorbate intermions A discussion of ionoptical methods inclLdes instructon on low to d a n k a mineral and Its surface be ore studylng R. Mher chapters focus on en!rqy storage Whin minerak. A wition on ac. re centers uses clays as a model. because in p a of its complexity. it is one of the most rnportam c a s e of natural rea& minerals. If you are a physicist. chemist. geologist. or uel. soil. agrkuhral. and enumnmental sci. !mist interested in interfacial chemistry of pological surfaces. this book will sfimulate our thinBng and inspire you to try these new :harmerizanon methods. alia M. Coyne. Editor, San Jose State Universn Stephen W.S. M t k r . Editor, Ok!ahoma %ate Unlvemj David P. Blake. Edtor. NPSA.Ames Research Mer k w p d horn a s y n p u m .yamred kq the Dm-
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Applications with llqukl hkt SyslemS APIMS can be adapted to a variety of modern separation sciences inlet systems, including HPLC using conventional 4.6-, 2-, and 1-mm i.d. columns as well as SFC, CE, and IC. To illustrate, we will show how each of these separatbn techniques may be coupled to an API mass spectrometer (TAGA 6000E APIMS, Sciex) with either the heated pneumatic nebulizer LC/MS interface using corona discharge ionization or the pneumatically wisted electrospray (ion spray) LC/MS interface (20)using electrospray ionization. Detection limits in the picomole range or lower are possible for compound claases ranging from those that are nonpolar to the highly polar ionic compounds that are separated as ions in solution via CE or IC. Heated pneumaticnebulizer Interface LC/MS characterization of benzodiazepines. The heated pneumatic nebulizer interface is a simple device that combines heat and pneumatics to volatilize the sample and prcduce an effluent spray (Figure Za). For combined LC/MS applications with 2- or 4.6-mm i.d. HPLC columns, this inter-
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face handles reversed-phaseHPLC effluent flows ranging from 0.1 to 2.0 mL/ min with essentially the same temperature setting (250 "C measured vapor temperature). The LC/MS separation and detection of benzodiazepines in a synthetic mixture using the heated pneumatic nebulizer is shown in Figure 3. The full-scan LC/MS background subtracted total ion current (TIC) chromatogram and positive ion APCI mass spectra for 25 ng of each of five benzodiazepines are shown in the upper and lower portions, respectively, of the figure. Lower detection limits are available via selected ion monitoring (SIM) of only the protonated molecular ion of each compound, so LC/SIM MS analyses can provide low-picogram detection of these compounds. Fullscan LC/MS/MS analysis of mixtures can provide structural characterization of these compounds and their metaholites. SFC/MS determination of corticosteroids. If the heated pneumatic nebulizer LC/MS interface described above is modified to incorporate a pinhole restrictor suitable for maintaining supercritical fluid conditions in a packed-column SFC system, the total effluent from such a chromatographic system may he directed through the heated restrictor region to the corona discharge area of the APIMS ion source to accomplish SFC/MS. Figure 4a-d
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ANALMiCAL CMMISTRY, VOL. 62. NO. 13. JULY 1, I990
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I F l m 8. LClAPlMS separation and detection of a synthetic mixture containing 25 ng each of five benzodiazepines using a heated pneumatic nebulizer. (a) Fllldo~nLCIMS background submcted mtsl ion cunnt(TIC) chmnnlcgm and (b)posllve Ion A w l IWU) spurn. The total laooratlc flow of 1.2 mllmln CH&NICH30HIH20 (40:2535, vlv). 10 mM ammp nlm h n n a m ~ l r o m a 4.6 mn x 250 mn Zabax.Rxmlmn was passed- me LCIW Interlam vrhlb tho m a s specbome(er VIVI scanned horn m/z 100 10 m/z 350 at a scan rme of 3 a l m wnh wmnadischerge i o n l m . The A w l m a p S a r a In (b) weretaken lrcmme mnespondlngchomato. @aphkpaks In (a). Peak Wntukatlon: 1, oxa2qmm; 2. chlordlaurwxlds: 3.rodlarepem: 4, a l m e lam; and 5, b e P a m .
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shows the TIC chromatogram, UV chromatogram, and two plotted fullscan maen spectra for the packed-column SFC separation and APIMS detection of the components in n synthetic mixture of five corticosteroids. Approximately 25 ng of each component were injected to obtain the TIC
chromatogramshown in Figure 4a For additional structural details, SFC/MS/ MS experimenta were performed to provide the full-scan product ion maea spectra for each of these compounds, which yield abundant ions indicating the structure of these compounds (not shown here). This interface is also
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ANALYTICAI; CHEMISTRY, VOL. 82, NO. 19, JULY 1, 1990
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INSTRUMENTATION amenable to capillary SFC/MS applications with minimum modification. Ion spray LClMS interface The pneumatically assisted electrospray or ion spray LC/MS interface is particularly useful when the organic or biological compounds of interest exist as ions in solution during the separation process. This is the situation when drugs, peptides, or proteins, for example, are separated by reversed-phase HPLC as ions in solution at low or high pH, or for CE or IC separations when
the buffer or eluent contributes to separating the organic compounds of interest as organic ions. The ions in the condensed, liquid droplet phase may be evaporated by the electrospray process to produce gas-phase ions suitable for mass analysis. Figure 2b shows a schematic of the ion spray LC/MS interface. This device may be used without alteration for coupling HPLC, CE, or IC to APIMS,as we will show in the following examples. LC/MS/MS analysis of tryptic digests. HPLC analysis of protein tryptic digests is an established method for
producing so-called tryptic maps. To identify the chromatographic components in the mixture, this approach requires careful control of retention times for all components of the digest and comparison with authentic samples of each peptide. In contrast to tryptic mapping with UV or fluorescence detection, LC/MS analysis with the ion spray interface provides the molecular weight of each mixture component in addition to the corresponding retention time. If the retention times of the various components of the digest vary as a result of the vagaries of HPLC (e.g., nonequilibrium column conditions), it is still possible to distinguish the various components by their molecular weights. Figure 5a shows the LC/MS analysis of a tryptic digest of hemoglobin normal p chain. Figure 5b shows the mass spectrum for the tryptic fragment T-3 (MW 13141, which is observed at a retention time of 12 min in Figure 5a. (See caption for experimental conditions.) The signal observed at m/z 658 represents the doubly protonated molecule ion for the known peptide with the sequence Val-Asn-Val-Asp-
Glu-Val-Gly-Gly-Glu-Ala-Leu-Gly-
Flgure 4. Packed
