Atmospheric-pressure-ionization mass spectrometry

ken&

in analytical chemistry

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vol. 13, no. 2, 1994

Research Scientist at Abbott Laboratories, North Chicago (1984- 199 1). Currently he is a Research Scientist at the Biophysics Research Division and Professor at the Department of Biological Chemistry of the University of Michigan. Dr. Ananya Majumdar was educated at the Indian Institute of Technology-Kanpur (MS Chemistry)

and the Tata Institute of Fundamental ResearchBombay (PhD 1990). Currently he is Research Fellow of the Biophysics Research Division of the University of Michigan carrying out post-doctoral research on leave from the Tata institute where he is Fellow.

Atmospheric-pressure-ionization spectrometry

mass

II*. Applications in pharmacy, biochemistry and general chemistry A.P. Bruins Groningen, Netherlands Mass spectrometer ion sources are normally located inside a high-vacuum envelope. An ion source operating at atmospheric pressure is better suited, it not essential, for a growing number of applications. MS analysis of samples pyrolyzed under controlled conditions makes use of chemical ionization at atmospheric pressure. On-line LC-MS which employs the heated pneumatic nebulizer interface together with atmospheric pressure chemical ionization is suitable for moderately polar samples that are not too labile. Highly polar, thermolabile, and ionic samples and biolpolymers require the application of electrospray ionization at atmospheric pressure combined with on-line separation by LC or CE. The determination of the molecular mass of proteins, nucleic acids and other (bio)polymers by electrospray ionization has accelerated the formerly slow development of atmospheric pressure ionization. In recent years the atmospheric pressure ionization sauce has been transformed from am exotic research instrument into a standard tool for problems in (bio)chemistry, pharmacy and biotechnology. *For Part I, see Trends Anal. Chem., 13 (1994) 31.

D 1994

Elsevier Science B.V. All rights reserved

Introduction Air monitoring was one of the original fields of application of atmospheric-pressure-ionization mass spectrometry (API-MS). API instruments installed in vans have been used for the measurement of hazardous substances released in accidents [ 1,2]. API can be used successfully in combination with gas chromatography and supercritical-fluid chromatography, but these combinations are in service in only a limited number of research groups. The introduction of pyrolyzed samples into the API source allows rapid detection of targeted components in pharmaceutical formulations [3]. Most API applications in the biomedical area concern the study of samples in solution, where other standard ionization techniques, such as electron impact, do not provide molecular mass information. Many sample solutions that could be handled by ionization in a low-pressure ion source are more conveniently introduced into an API source. Necessity or convenience make API the method of choice in a growing number of applications in which mass spectrometry is connected on-line with liquid phase separation techniques such as HPLC, ion chromatography, capillary electrophoresis, and isotachophoresis. Electrospray ionization, with or without pneumatic or ultrasonic nebulization, represented a breakthrough for the molecular mass determination of biopolymers, and is receiving more attention as a tool for studies of ions in solution in general chemistry.

0165-9936/94/$07.00

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Glossary API APCI Electrospray

Electrospray ionization lonspray Ultraspray Ion evaporation

CE Heated nebulizer

Atmospheric pressure ionization in general. Atmospheric pressure ionization by chemical ionization. Dispersion of liquids into an electrically charged aerosol by the action of an electric field. Ion formation from samples in the use of solution by electrospray. Pneumatically assisted electrospray ionization. Ultrasonically assisted electrospray ionization. /Vow: one of the proposed mechanisms for the liberation of ions from an electrically charged droplet; In the past: used to denote the entire system of pneumatic nebulization with the aid of an electric field followed by ionformation from a droplet. Capillary electrophoresis. Pneumatic nebulizer combined heated desolvation with a chamber, for the introduction of a solution into an sample atmospheric pressure chemical ionization source.

On-line liquid spectrometry

chromatography-mass

LC-MS in general

The combination of GC with MS is easy, since sample components elute from the GC as vapour that can easily be introduced into the vacuum of the mass spectrometer, and ionized in the gas phase by electron ionization (El) or chemical ionization (Cl). In HPLC, on the other hand, the sample is in solution at atmospheric pressure, while the mass analysis of ions has to take place inside a vacuum system. One can identify two problem areas in LC-MS coupling. First, a large volume of solvent vapour is introduced into the vacuum system which has a limited vacuum pumping capacity. Secondly, the supply of heat for the evaporation of

solvent and sample may lead to thermal degradation of labile or highly polar components. The API source has no problems with a high gas load from solvent vapour or gas from a pneumatic nebulizer, since excess gas can easily be removed from the source by means of a simple centrifugal blower. The API source has problems of its own, however, related to the transport of ions from the source into the vacuum of the mass analyzer, as discussed in Part I. In the ideal situation, only ions should be introduced into the vacuum, but no neutrals (molecules or particulate matter) that can block a sampling orifice or contaminate ion optics. The introduction of the vapour of polar solvents together with ions may lead to the formation of large sample-solvent cluster ions during the freejet expansion into the vacuum. A nitrogen gas curtain or countercurrent flow of drying gas will block all neutrals other than dry nitrogen from entering the vacuum and will assist in the removal of solvent molecules from sample-solvent cluster ions. By using other techniques that prevent or cure the cluster-formation problem, API sources can be built which operate without a gas curtain [4]. LC-MS with chemical ionization at atmospheric pressure

Ionization in the gas phase by atmospheric pressure chemical ionization (APCI) follows the sequence: Sample in solution + sample vapour + sample ions The effluent from the HPLC is evaporated completely. The mixture of solvent and sample vapour is then ionized by ion-molecule reactions (chemical ionization), or by electron capture. Carroll and his co-workers [5] were the first to show the feasibility of atmospheric-pressure chemical ionization for LC-MS coupling. Either an electron-emitting 63Ni foil or a corona discharge was used to generate reactant ions. The vaporizer for sample and solvent was a heated glass tube filled with a plug of glass wool, directly attached to a small API source. Detection of fairly small and thermostable molecules was achieved. Henion et al. followed this by combining a Hewlett-Packard DLI probe [6] with a heated glass tube inserted into the ion source of a SCIEX API mass spectrometer originally built for air monitoring [7]. This LC-

a3

trends in analytical chemistry, vol. 13, no. 2, 1994

place: Nebullzer Gas

---A-e...

chemical

L

---

CH,OHl

lonizatlon

Vapour -

*‘,

-

sample

.’

sample

10”s

solvent

ions

solvents

-

MS

I Corona &charge Needle

760 Torr

Fig. 1. Heated nebulizer interface for LC-MS: a combination of a concentric pneumatic nebulizer, heated tube for evaporation of solvents and sample, and atmospheric-pressure ated by corona discharge.

chemical ionization initi-

MS interface is a combination of an aerosol generator with a desolvation tube. The same principle was developed further into a combination of a concentric pneumatic nebulizer with a desolvation tube, shown in Fig. 1: this is now commonly called a heated-nebulizer LC-MS interface. The operation of the heated nebulizer does not require critical adjustments. Pneumatic nebulization takes place at room temperature, and desolvation in the hot tube is complete if the temperature is high enough. There is no need for temperature tracking during gradient elution. Sample transport into the corona discharge region is further assisted by a flow of makeup gas. The heated nebulizer can accept the full effluent from a standard 4.6 mm I.D. column, and volatile salts such as ammonium acetate do not appear to present a problem at concentrations up to 0.1 M. Heated nebulizers have become accessories for API sources available from an increasing number of manufacturers. The pneumatic nebulizer can be replaced by a thermospray nebulizer [8]. All modem commercially available LC-MS systems with APCI sources make use of a corona discharge. LC-MS with APCI takes place with reactant ions generated from solvent components: H,O+, CH30H;,

+ NH, + NH: + CH30H

CH,CNH+

so that protonated methanol is removed and the ammonium ion becomes the reactant ion, which is suitable for many applications. If, however, an even more basic additive such as triethylamine or octylamine is used to improve the chromatographic behaviour of a sample, then the most abundant ions are the protonated amines. Since nearly all the original solvent-derived reactant ions will be consumed by these amines, and since these two protonated strong bases are not likely to transfer a proton to less basic samples, one may find that sample ions cannot be formed at all. So caution has to be exercised in the mixing of additives with eluents. The same holds for strongly acidic additives, if negatively charged sample ions are to be detected. Fig. 2 shows the simple APCI mass spectrum of trenbolone [9]. The sample solution was the eluate from a reversed-phase LC column. The mobile phase was water-methanol (70:30) containing 0.1 M ammonium acetate. A daughterion mass spectrum of the MH+ ion of a new drug is given in Fig. 3. HPLC combined with APCI and tandem mass spectrometry is becoming a widely used technique for the determination of concentration-time profiles of drugs in animals and humans [lO,ll], as shown in Fig. 4. An example of an application in environmental chemistry is the screening for organophosphorus pesticides [ 121. Negative-ion chemical ionization with chloride attachment appears to be well suited for LC-MS determination of mono and oligosaccharides [ 131.

4

In most cases the reactant ions are solvated by a few solvent molecules, e.g. CH,OH$(H,O);(CH,OI-I), When NH3 vapour is also present in the source, for example as a result of the evaporation of ammonium acetate, a rapid proton transfer takes

Fig. 2. Atmospheric pressure chemical ionization mass spectrum of Trenbolone. (Reproduced with permission from ref. 9.)

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trends in analytical chemistry, vol. 13, no. 2, 7994

L-365.260 100

3000

24!

l 2.5 mg

$

5 7 = $ F ‘Z 3

A 5.0 mg

1000

80

I

v

10mg

+

25mg

n 50 mg

221 60 40

a

3

20

,

0 100

146, I

-

200 1 I 0

Fig. 3. Daughter-ion mass spectrum of the MH+ ion formed by heated nebulizer APCI of the new drug L365,260. (Reproduced with permission from ref. 9.)

APCI is a selective ionization technique. In general, one can ionize samples that would be detectable by isobutane or ammonia chemical ionization in a low pressure source. Since highly reactive reactant ions such as CH$ cannot be generated in an API source in the presence of traces of water or other solvents, it is not possible to ionize saturated hydrocarbons or other compounds that have a low gas-phase basicity. For an indepth discussion of chemical ionization the reader is referred to the book by Harrison [ 141. APCI along with a suitable sample introduction method can in general handle those samples that are presently analyzed by thermospray LC-MS. Thermospray will probably be gradually phased out when API sources equipped with both APCI and electrospray (see below) find more widespead use. L C-MS with electrospray

ionization

Electrospray is most suited to compounds that exist as preformed ions in the LC eluent, to samples that can be ionized at the correct pH, or polar neutral molecules that can associate with small ions such as Na+, NH: or Cl-. As a rule of thumb, for positive ion MS, electrospray is suited for those neutrals that are basic enough to form MH+ or M.NH$ in a low-pressure chemical ionization source under ammonia-C1 conditions. Fig. 5 presents a simplified drawing of a pneumatically-assisted electrospray (IonSpray@) interface in an API source equipped with a gas curtain. Other arrangements are feasible as discussed ear-

4

6

12

16

20

24

Hours After Dosing

Fig. 4. Concentration-time profiles after single oral doses of L365,260: obtained by heated nebulizer and APCI, with selected ion monitoring in the MSMS mode. (Reproduced with permission from ref. 9.)

lier. A recent modification uses ultrasonically-assisted electrospray (Ultraspray@). As mentioned in Part I, electrospray becomes difficult for solvent mixtures that contain a high percentage of water and/or a high concentration of electrolytes. A sheath-flow helps, but pneumatic or ultrasonic assistance make the system much more rugged and reliable, and much less critical with respect to solvent composition and the positioning of the sprayer inside the ion source. Here the term “electrospray” will be used as a generic name that also covers modified electrosprays. Some manufacturers operate the electrospray interface at high voltage, which produces no problems since a connnection to the outlet of the HPLC is conveniently made from a flexible fused-silica capillary tube. Other

IONS TO MS

f--L NEBULIZING

O-----% + VE IONS

GAS

I ATM

Fig. 5. Pneumatically-assisted electrospray ionization system for the generation of positive ions, in an atmospheric pressure ionization source equipped with a dry nitrogen-gas curtain.

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trends in analytical chemistry, vol. 13, no. 2, 1994

manufacturers prefer to have the interface at ground potential and the source at high voltage. There has been no indication that one or the other configuration is to be preferred with regard to the quality of spectra or chromatograms. Electrospray can be carried out at room temperature so that thermolabile samples are not decomposed. In many instruments, the source is gently heated to about 70°C. In some instruments the ions pass through a hot zone into the vacuum system. Despite the supply of heat, all instruments appear to handle non-volatile samples such as proteins without thermal degradation. It is more difficult to assess the performance of different instruments for small labile molecules such as drug metabolites since such studies are mostly made by pharmaceutical companies. The compounds studied are not available off the shelf from chemical suppliers. Also, there is no “standard” small molecule that is used for performance specification by all manufacturers. In the protein field, in contrast, all manufacturers show their electrospray spectrum of myoglobin. Electrospray is a low flow-rate technique. When the flow-rate is increased for a given sample concentration, the sample ion signal does not increase. So, in terms of sample concentration the sensitivity remains constant, but in terms of mass-flow the sensitivity drops when the flow-rate of the sample solution is increased. This is an unusual situation in MS since a mass spectrometer operating in the EI or CI mode is a mass-flow-sensitive detector. The apparent concentration-sensitivity of electrospray can be attributed to a decrease in dropletcharging efficiency and a shift towards larger diameters in the droplet-size distribution if the liquid flow-rate is increased. Both effects lead to a lower ionization efficiency. The reduced efficiency of the release of ions from droplets is approximately compensated for by the introduction of more sample molecules per unit time into the interface, so that the signal level at the detector remains constant. If the liquid flow-rate is increased too much, the ion signals become smaller and less stable. The practical upper limit to flow-rate in pure electrospray is 10 to 20 ~Vrnin, depending on the composition of the solvent and on the use of a coaxial sheath-flow (see Part I). Pneumatically-assisted electrospray has been used up to 200 @/min. Manufacturers have claimed good performance of ultrasonically-assisted electrospray up to 1 ml/min. High-flow pneumatically-assisted elec-

trospray is another new development, which appears to require a heated zone in the source, or a heated tube for the transport of ions into the vacuum, in combination with a socalled liquid shield [ 151. It is not yet possible to judge the performance of high-flow electrospray in everyday use. So far, electrospray has been combined with 0.32 mm I.D. microbore LC columns without a split of the eluent. Columns of 1 mm I.D. can be used at 40 pl/min without a splitter, but with pneumatic assistance. In most cases, larger-bore columns are used in combination with a splitter. Two capillary tubes can be combined in a Tee piece at the end of the LC to form a splitter: one narrowbore capillary takes 5-10 @min into the electrospray interface, the other, short and wider-bore capillary, having a much lower resistance to flow, takes most of the effluent into a fraction collector, or to waste. A pneumatically-controlled splitter originally designed for LC combined with continuous flow fast atom bombardment mass spectrometry [ 161 has proved to be convenient and reliable in our hands. The performance of electrospray ionization in LC-MS is compromised if electrolytes are added LC/UV

9

11 10

I-

LC/MS

I _ 0 N C U

R R E N T IllI”

Fig. 6. Electrospray LC-MS: comparison of LC-UV (top) and LC-MS (bottom) of an extract of a municipal waste water fortified at the 5ppm level with eleven sulphonated azo dyes. The LC-MS trace is the total-ion-current profile obtained in the negative ion mode. (Reproduced with permission from ref. 18.)

trends in analytical chemistry, vol. 73, no. 2, 7994

to the eluent in order to improve chromatographic performance. Ammonium acetate can be tolerated at 1 rnil4, but sample-ionization efficiency becomes progressively poorer at higher concentrations. Often, the addidion of salts is a prerequisite for the ionization of neutral molecules by formation of MeNa+, M.NH$, or MCl- [ 171. If a salt or acid or base cannot be added to the HPLC eluent, post-column addition is a good alternative. A few examples of electrospray ionization in LC-MS are: the detection of sulphonated azo dyes in water [ 181 as depicted in Fig. 6, the separation of peptides in enzymatic digests of proteins [ 191, and the detection of metabolites of drugs which is facilitated by the use of stable isotopes [20,21]. The pharmaceutical industry makes use of electrospray LC-MS for the quantitation of drugs in body fluids [22]. Since electrospray ionization is at its best in the detection of ionic samples, it was obvious that electrospray could be combined with ion chromatography. This coupling is indeed feasible if the excess of electrolytes is removed by a micromembrane suppressor [23].

On-line capillary spectrometry CE-MS

few picomol of sample) for the recording of fullscan mass spectra. Laser-based fluorescence detectors can be used with much lower concentrations. There is an increasing number of applications of CE in protein chemistry and pharmaceutical chemistry 1251. On-line CE-MS offers the same benefits for detection and identification as experienced in GCMS and LC-MS. In LC-MS coupling with electrospray the flow from the column is more than the electrospray interface needs for optimal performance. In capillary electrophoresis the flow through the column is very low, or zero, so that a makeup flow of a few l.Wnin is needed. Devices providing such makeup flow are the coaxial [24], and liquid junction [26] interfaces. The makeup liquid has to be chosen with care: it should have a high enough conductivity, since it serves as an electrical contact that closes an electrical circuit of which the CE column is the essential part. Its composition and pH can be selected such as to promote ionization of samples migrating out of the CE column, and it should be remembered that electrolyte ions from the makeup solution may migrate back into the CE column and

electrophoresis-mass

with electrospray

ionization

Capillary electrophoresis allows high resolution separation of small amounts of material and especially ions in solution. It is therefore considered ideal for coupling with an electrospray mass spectrometer [24]. The volume injected is a few nanoliters of a solution with sample components at a concentration of ca. 10m3M, which is sufficient (a

Make up solution

1

,mx-

N+L I+1

18935232

L

5

/

10 migration

Spray capillary, stainless steel 0 4 mm id. x 0.7 mm o.d. connected to HV power supply

Fig. 7. Pneumatically-assisted coaxial interface for the coupling of capillary electrophoresis with electrospray ionization. (Reproduced with permission from ref. 25.)

I

15 time

Fig. 8. Capillary electrophoretic separation of synthetic conjugates of naproxen (N) with lysozyme (L); detection by electrospray mass spectrometry: left, total ion current profile, right, profiles of eight-timescharged ions (M+8H)8+ of native lysozyme (L) and conjugates (N+L 1+I, N+L 2+1, N+L 3+1).

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adversely affect separation conditions. CE-MS interfaces are becoming commercially available. In our laboratory, a simple home-made coaxial system equipped with the pneumatic nebulizer shown in Fig. 7, gave satisfactory performance in the separation and detection of chemically moditied lysozymes [27] (see Fig. 8). The purpose was to separate native lysozyme from lysozyme coupled with one, two, and three naproxen molecules. The stainless-steel spray capillary in Fig. 7 serves as the cathode of the CE separation system. Fig. 9 shows the separation and detection of three myoglobins at the 100 fmol level, as part of a performance test of CE-MS carried out by Smith and his co-workers [28].

Electrospray separation Electrospray

ionization

without

(M+41H)“+

h I

m/z

on-line

ionization of biopolymers

The demonstration by Fenn and co-workers that electrospray can measure the high-molecularmass of a protein from a series of multiply charged ions was a landmark in the development of this technique [29]. It changed the minds of manufacturers who had not considered electrospray a viable approach for LC-MS. The error in molecular mass measurement is less than 1 in 10 000 when a quadrupole mass spectrometer is used. Fig. 10 shows the electrospray mass spectrum of bovine

Fig. 10. Electrospray mass spectrum of bovine serum albumin (WA); charge states ranging from 28+ to 54+are observed. Measured I!&66 431.5 k 1.3 mu for MO. Insert: expanded mass scale around the (M +41H14’+’ Ions. MO: major component, true BSA. MI and M2: unexpected minor components, possibly formed by posttranslational modification. (Reproduced with permission from ref. 28.)

serum albumin [30]. At first sight, an electrospray mass spectrum of a biopolymer seems confusing: how does one know the charge state of each multiply charged ion? Determination of the charge state is based on two assumptions. First, that the charge states in a series of multiply charged ions in a series derived from a biopolymer always differ by one between two neighbours (there are no charge states missing in the middle of a series). Secondly, the charge of positive ions is caused by protonation. In this case, the mass-charge ratio of a multiply charged positive ion is m -z-

M,+n n

and the mass-charge m -ZTime

(min)

Fig. 9. CE separation with UV and electrospray MS detection of three myoglobins. (Reproduced with permission from ref. 26.)

(hl+%H;8

ratio for its neighbour is

M,+n+l n+l

wherenisthenumberofcharges(protons)andM, is the molecular mass of the biopolymer. The equationscaneasilybemodifiediftheioncharge is due not to protonation, but to the addition of

88

trends in analytical chemistry

sodium or ammonium ions. After careful measurement of both m/z values, these two equations with two unknowns are solved for n and Mr. From all pairs of neighbouring multiply-charged ions a series of molecular masses is calculated and averaged. This method requires nothing more than a simple calculator. However, when a biopolymer is not pure, the electrospray mass spectrum will consist of two or more overlapping series of ions that may be impossible to unravel. Molecular mass calculation is then impossible without help from computer programs [31] in the data systems of electrospray mass spectrometers. Mathematical techniques such as Maximum Entropy are used to increase the resolution of the molecular mass determination [32]. Electrospray

of complexes

vol. 13, no. 2, 1994

(2xHIV PR+JG~~IS+IOH)“~ 2235.5

A.

c2tHlV PR+JC;OS+I IH)“’ 2032 5

I’100

IXIH)

2(X10

m/l

7100

2300

2200

H. IlKI,

in solution

Covalent bonds are not broken upon electrospray ionization since electrospray is a very soft ionization technique. Weak bonds, such as hydrogen bridges, can also survive. Often unwanted sample cluster ions such as 2M.H+ are observed, with high relative abundance at high sample concentration. More interesting is the observation of complexes between metal ions and ligands [33], between enzymes and substrates, enzymes and inhibitors, receptor and ligand, and complexes

21.750

22.000 22.250 22.500 ,Molecular Weight

22.750

23.0(X)

Fig. 12. Electrospray mass spectrum of a non-covalently bound complex between the dimeric enzyme HIV protease and an inhibitor. A = ions with 10 and 11 charges; B = computer-constructed molecular mass scale. (Reproduced with permission from ref. 33.)

formed in supramolecular chemistry. Fig. 11 gives an example of an air- and moisture-sensitive organometallic compound complexed with tetrahydrofuran. Ganem, Li and Henion were the first to report the observation of non-covalently bound complexes [34]. A ternary complex between the dimeric enzyme HIV- 1 protease and the inhibitor, desmethyl-JG365 is observed intact by electrospray [35], as shown in Fig. 12. II

0 350

1

400

450

m/z

500

Electrospray-mass-spectrum Fig. [CPiZrC~~(THF)]+ an air- and moisture-sensitivoef organometallic complex existing as an ion in solution: Cp* = pentamethylcyclopentadienyl; THF = tetrahydrofuran; THF is partially removed by collision induced dissociation in the nozzle-skimmer region. (Reproduced from J. Eshuis, PhD thesis, Inorganic Chemistry Department, University of Groningen, 1991.)

Electrospray kinetics

as a tool in general chemistry and

Since electrospray ionization is based on the liberation of ions or ion-molecule complexes from solution, it can be used as a tool in studies of ions in solution in general chemistry. Charge transfer complexation is a technique for the formation of ion pairs from two neutral molecules, by transfer of an electron. The radical-cation

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vol. 13, no. 2, 1994

and radical-anion in the ion pair can be observed by electrospray mass spectrometry [36]. Formation of radical-cations by electrospray may find application in studies of vibrationally-cold molecular ions that are difficult to generate by lowvoltage electron-impact ionization. Radicals in solution and radical ions in solution can be identified by the combined use (on-line or off-line) of electron spin resonance and electrospray mass spectrometry [37,38]. Direct and continuous introduction of a sample stream from a reactor into an electrospray mass spectrometer can be used for monitoring the kinetics of a reaction. The consumption of the reaction mixture is negligible at the low pl/min flow-rate required for electrospray. An enzyme reaction has been followed in this manner [39].

267

A

loo-

:

IO eV

I. A

(M-H)-

T I : R : N: N : w...-.-.-,...,.,.,-,..4.-..... 100

50

100

: L

A T : E R :

n :: n :

C

L 300

200 m/z 267

I I 1

k-

Fig. 13. Fragmentation of diethylstilbestrol in the free-jet expansion region between the atmosphericpressure ion source and the quadrupole mass analyzer. The (M-H)- ion was formed by electrospray (T.R. Covey, A.P. Bruins and J.D. Henion, unpublished).

Fragment ions in atmospheric-pressureionization mass spectra Under the mild ionization conditions of APCI and electrospray, fragment ions are not normally generated inside the ion source. If a fragment ion is observed when a sample is introduced into the source by means of a heated nebulizer interface, it is more likely that some thermal degradation has taken place, followed by the ionization of decomposition products. While fragmentation inside the source is uncommon, it is quite easy to generate fragment ions once sample ions have been drawn into the vacuum system. Ions are entrained in a large excess of gas when they pass through the ion sampling orifice of the atmospheric pressure ion source [4]. The application of an accelerating field inside the vacuum system, in the expansion region where the gas pressure is still relatively high, creates the conditions for collision induced dissociation (CID) and efficient collection of fragment ions. This CID “up front” is, of course, not true tandem mass spectrometry, but is very easy and reproducible. It can be used together with any atmospheric-pressureionization technique: APCI, electrospray, or new methods to be developed in the future. Nomenclature is confusing: high orifice potential, high cone voltage, or high nozzle voltage, are the most widely-used keywords in descriptions of CID “up front”. Mild CID is sufficient for breaking of the noncovalent bond in a complex of an enzyme with an inhibitor [35]. More energetic CID is suitable as an aid to structure elucidation, or for the generation of a fragment ion spectrum shown in Fig. 13 that can be used as a fingerprint” for identification of targeted compounds.

References D.A. Lane, B.A. Thomson, A.M. Lovett and N.M. Reid, Adv. Muss Spectrom., 8B (1980) 1480. J.B. French, B.A. Thomson, W.R. Davidson, N.M. Reid and J.A. Buckley, in: F.W. Kerasek, 0. Hutzinger and S. Safe (Editors), Mass Spectrometry in Environmental Sciences, Plenum Press, New York 1985. A.P. Snyder, Trends Anal. Chem., 12 (1993) 296. A.P. Bruins, Muss Spectrom. Rev., 10 (1991) 53. D.I. Carroll, I. Dzidic, R.N. Stillwell, K.D. Haegele and E.C. Horning, Anal. Chem., 47 (1975) 2369.

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Dr. A.l? Bruins is at the University Centre for Pharmacy, A. Deusinglaan 2, 9713 A W Groningen, Netherlands.

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