Thomas R.

Covey
Edgar D. Lee
Andries P. B N h
Jack D. H e n h
Drug Testing and Toxicology

New Yo& Siate College of Veterinary Medicine

Cornel1 University
Ithaca. N.Y. 14850

The combination of hgh-performance liquid chromatography

(HPLC) and mass spectrometry (MS)
offers the analytical chemist one of
the most powerful analytical techniques of modern times. Both techniques have matured to the point that
they represent two of the most important tools available for the characterization of organic compounds. Unfortunately, coupling these techniques
(LC/MS) has been a more challenging
task than it was for gas chromatography/mass spectrometry (GC/MS).
MS provides high sensitivity together with a wealth of structural information for the analysis of samples containing organic compounds. A variety
of available ionization techniques together with recent improvements in
the mass range of new mass speetrometers enable us to study a wide range
of organic compounds. Although MS
analysis of mixtures can be done di.
rectly, the use of hyphenated systems,
e.g., GC/MS or LC/MS, is clearly the
preferred approach.
HPLC has advantages over GC in
that compounds are not exposed to excessive heat, less sample cleanup is required, and derivatization is usually
not necessary. The high chromatographic efficiency available from cap.
illary GC, however, is not currently
available from conventional HPLC.
Thus, HPLC chromatographic peaks
may contain unresolved components
that nonspecific detectors cannot differentiate. This, coupled with the
need for a more sensitive and specific
detector for HPLC, has generated
0003-27001861A358-1451$01.5010
@ I986 American Chemical Society

1
considerable interest in the development of routine LC/MS.
The potential benefits derived from
on-line coupling of LC to MS have
been known for about 12 years. The
problems associated with interfacing
these two techniques in a practical
way are considerable, but recently
there has been significant progress toward this end. An excellent review
covering the status of this subject
through 1978appeared in this JOURNAL ( I ) , and this article will attempt
to cover the state of the art through
early 1986.As with any rapidly developing area, the details of all aspects of
LC/MS cannot be covered in a short
overview. The reader may refer to the
references and recent reviews (2-5)for
more information on other aspects of
this subject.
Hlotalcal review
Transport. The f i t successful
commercially available LC/MS interface was the transport. or moving-belt,
system (I).The status of this interface
bas improved as a result of modifications that minimize its difficulties in
dealing with higher percentages of
aqueous eluents. The operating principle of the moving belt can be separated into three distinct steps: deposition
of the eluent, removal of the solvent in
vacuum, and volatilization of the 88111ple into the ion source.

The original suggestion of spray depcaition by Smith (6) was improved by

Hayes and co-workers so that improved ion current profiles of reasonably difficult compounds have been
reported (7).The improved performance of the cnrrent version of a
spray deposition moving-belt LC/MS
interface is shown in Figure 1.Reduced mixing of the ana& within the
film d e p i t e d on the belt and the fact
that the eluent is evenly deposited as
small droplets on the belt surface
make solvent removal and sample volatilization easier. Direct deposition
gives an irregular film with heads of
liquid on the belt surface that are difficult to remove by the vacuum system. The spray deposition modification appears to allow total effluent introduction of eluents containing
higher percentages of water. Those
eluents containing very high percentages of water, however, may still require either reduced chromatographic
flow rates or partial splitting of the effluent away from the interface.
The other significant improvement
to the moving-belt LC/MS interface
was reported by Games et al. (8).
These workers provided a comparison
of the two previous versions of the belt
with a modified version that placed
the terminus of the belt inside the ion
source close to the electron beam. In
this way analytes can be thermally desorbed from the belt surface and volatilized directly into the electron beam.
This system has displayed some of the

ANALYTICAL CHEMISTRY, VOL. 58. NO. 14, DECEMBER 1986

145lA

Time (rnir

Figure 1. Comparison of solvent deposition modes for moving-beR LClMS interface

[a)COnllnuwS flw, (b) s p a y depwition. Insets show typlaal flow panerm far ea& mthod. Figure shows
Peparalion of fluwene, pvene. biphenylens. and benzo[a]pvene: 20 cm X 4.6 mm. NucleDsil5-prn nilm
banded phase. 5% msthy!+nene chloriddhexane, 1 mllmin. (Repinted hom Referencn 7)

most impressive transport LC/MS results on, for example, digoxin, which is
notoriously difficult by conventional
approaches. Deactivation of the belt
surface with Carbowax 20M facilitates
volatilization of polar samples, whereas medium MS resolution (R = 4000)
can separate sample ions from the
background, further enhancing performance.
A major advantage of the transport
interface is its ability to provide the
analyst with more than one ionization
mode. Because solvents are almost
completely removed before the sample
is transferred into the ion source, the
belt allows a choice between electron
ionization (EI) and chemical ionization (CI), with a free choice of reactant gases. The combination of the
belt with the technique of fast atom
bombardment (FAB) allows the ionization and determination of involatile
high molecular weight compounds
that are not amenable to other modes
of analysis. Just as conventional direct
insertion probe MS techniques require
pure samples for reliable analyses,
FAB could benefit from an on-line
chromatographic separation prior to
ionization.
The technique of supercritical fluid
chromatographylmass spectrometry
(SFC/MS) using packed columns appears uniquely suited to the movingbelt LCIMS interface. Recent reports
by Games et al. suggest that deposition of SFC effluent onto the moving
1452A

belt is a convenient means of accomplishing packed-column SFC/MS

while obtaining either E1 or CI m a s
spectra. It seems likely that these and
other amlications will continue to
maintain the belt interface as a viable
tool for LC/MS.
Direct liquid introduction. The
other popular mode of on-line LC/MS
has been celled direct liquid introduction (DLI) ( I ) . I t is just what the term
implies in that the effluent from the
liquid chromatograph is introduced
directly into the MS ion source region.
This approach has been used in many
applications, including explosives (9),
drugs (10).and environmentally important compounds (11). It should he
realized.that the gas burden from conventional LC flow rates (1mllmin of
water produces 1.2 Llmin of gas) creates nearly 20 times more gas than a
cryopumped vacuum system can handle. Thus, the introduction of total
conveutional HPLC effluent into an
unmodified CI mass spectrometer is
not feasible. Therefore, DLI LC/MS
often uses a split such that only 1-5%
of the total effluent is introduced into
the mass spectrometer. This, of
course, sacrifices sensitivity such that.
detection limits for most compounds
are typically 0.1-1 fig.
An important aspect of DLI LCIMS
that should be appreciated is the fact
that the analyte is introduced in solution into the MS ion source. The volatilized eluent produces conditions in-

ANALYTICAL CHEMISTRY. VOL. 58. NO. 14, DECEMBER 1986

(rnin)

Dure 2. Chromatograms of a mixtwe

eight pesticides by (a) micro LClUV
and (b) micro LClMS DLI total ion current
me (yadierd was 45/55 CHsCNIH@to 100%
CbCN OMT 20 mln, maintained 61 a flow ram of
40 fiL/min though m O W , 11.d X 25cm
mlaDbDre wlmn (Whatman. Clifton, NJ.1

side the mass spectrometer that are

conducive to chemical ionization.
Thus, only CI mass spectra are available from DLI LC/MS experiments,
which often provide desired molecular
weight information. I t also means that
an analyte is ionized without directly
contacting a heated surface. This provides improved detection limits for labile cornpounds (12). In a sense the
analyte apparently never becomes
completely desolvated, $0 it always
has a blanket of solvent that proteds it from heated or catalytic surfaces.
Perhaps the most significant improvement in the DLI approach to
LC/MS involves the realization that
micro HPLC techniques can eliminate
the need to split the major part of the
chromatographic effluent from the
mass spectrometer. Because micro
HPLC typically nses 1-mm4.d.
packed columns with eluent flow rates

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F b r r 3. A therimspray LClMS Interlace equiwed for Wee modes of Ionization: direct or fiiament-off ionization, filamenton ionization, and discherge ionization
Vapw lhamcapls measva ma vapor tempgatwe. ( R e p r i m with pBrmisdon of Vssmc Cap.)

of 10-50 pLlmin, the total effluent

from these systems can he introduced
into a conventional CI mass spectrometer. If eluent flow rates are in excess
of 20 pL/min, a cryogenic vacuum
pump is required. Applications of this
approach have been reported by several researchers (Reference 10 and references cited therein). and these results
demonstrate that GC/MS-like detection limits can be obtained from DLI
micro LC/MS. It should also be realized that the transport L C N S interface described above also benefits
from the reduced solvent volume of
micro HPLC. The problems associated with volatilizing trace amounts of
labile compounds from the belt surface remain, however.
A representative application of DLI
micro LC/MS is shown in Figure 2.
The UV chromatogram (Figure 2a)
and total ion current (TIC) profile
(Figure 2b) clearly show the eight major components of a pesticide mixture.
Note that the UV base line rises
through the gradient, whereas the TIC
profile maintains a stable base line.
The CI mass spectrum for each component can he viewed, but often only
the (M + 1)+ion and perhaps just a
few fragment ions are observed. This,
unfortunately, is sometimes not sufficient for identification purposes; we
would like the structurally useful information usually provided by E1
mass spectra. Thus, a shortcoming of
1454A

DLI LC/MS is that only CI mass spectra are available.

Recent devdopmmts
The above discussion summarizes
refinements of the basic LC/MS interfaces that were available a t the time of
the last report in these pages ( I ) .
Since that time there has been exciting progress on more routine approaches to L C N S and on variations
in ionization techniques that allow for
the determination of more intractable,
higher molecular weight compounds.
Thermospray. Thermospray LCI
MS was born out of the desire and determination to develop a better way to
accomplish LC/MS. Vestal and coworkers progressed from early attempts to rapidly volatilize HPLC effluent with lasers to simple direct
heating of a metal capillary tube (13).
The latter resulted in the serendipitous discovery of thermospray ionization, which is the formation of ions
without the use of an external source
of ionizing electrons. Aqueous mobile
phases containing an electrolyte such
as ammonium acetate are passed a t
flow rates from 1to 2 mllmin through
an electrically heated stainless steel
capillary situated in a special heated
ion source with an auxiliary pumping
line situated opposite the capillary.
This results in the production of a supersonic jet of vapor containing a mist
of fine droplets or particles. At least

ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986

part of the droplet population in the

aerosol beam is electrically charged,
the number of positive droplets being
equal to the number of negative droplets. As the droplets travel through the
heated source they continue to vaporize because of the heat input from the
surrounding hot vapor. As the size of a
charged droplet is reduced, the electric field at the liquid surface increases until ions present in the liquid
phase are ejected from the droplet.
Ions are sampled through a conical
exit aperture in the mass analyzer.
Ions can be produced in the thermospray ionization process by direct
ion evaporation (14) of a sample ion or
in a two-step process analogous to
conventional CI, whereby an ion of the
electrolyte, e&, NH4+,ejected from a
droplet, reacts with a sample molecule
in the gas phase and generates a sample ion that is mass analyzed. This
process can he used with equal facility
to obtain positive- or negative-ion
mass spectra. Ammonium acetate has
emerged as the best general-purpose
electrolyte for ionizing neutral samples, although other volatile salts, acids, bases, or no electrolyte at all may
he preferred in certain applications.
Ionic samples are best analyzed without the use of ammonium acetate. Reversed-phase eluent mixtures containing a high percentage of water are preferred for good sensitivity in
thermospray ionization.
The thermospray system described
so far is a combined liquid introduction and sample ionization method.
The liquid introduction principle can
be applied to any HPLC eluent that
does not contain involatile buffers, but
the ionization process is by no means
universal. Therefore, an important
mode of thermospray LC/MS operation was introduced called filamenton thermospray, wherein an external
filament is turned on to generate ions
under CI conditions when insufficient
ions are produced by filament-off
thermospray ionization (15).In this
mode a form of hot DLI results. An
electric discharge can be used to generate reactant ions for CI (16). if filament-on ionization is less suitable as a
result of exposure of the heated filament to the highly oxidizing environment of aqueous eluents.
A typical thermospray LC/MS interface and ion source is shown in Figure 3. The heated capillary is housed
in a removable probe (17).This facilitates cleaning and maintaining the interface, which can occasionally become restricted or plugged. It also provides for adjustment of the probe tip
relative to the ion exit orifice, which
can sometimes help optimize the sensitivity of the system.
The thermospray system shown in
Figure 3 provides the analyst with LCI

MS data from three different ionization modes. These are the thermospray ionization or filament-off mode,
the filament-on mode, and the discharge ionization mode. Examples of
data obtained in each of these ionization modes are shown in Figure 4. In
Figure 4a the thermospray ionization
LC/MS mass spectrum of meclofenamic acid is shown using high percentages of water on a compound that generally benefits from the mild ionization conditions afforded under these
conditions. When nonaqueous HPLC
conditions are required, as with chiral
stationary phases (CSP) for the separation of enantiomers (B),
the filament-on thermospray LCIMS mode is
preferred. Individual enantiomers of
methamphetamine are identified in
Figure 4b (19). When HPLC conditions are not well suited for either
thermospray or filament-on ionization, discharge ionization (Figure 4c)
can be used. These three different ionization modes can accommodate most
HPLC eluent conditions, with the possible exception of involatile inorganic
modifiers.
The control of experimental temperatures during thermospray L C N S
is less of a problem now than it used to
be, but the analyst should he aware
that there are several temperatures
that are critical to optimum performance. These indude the temperatures of the vaporizer tip, the ion
source block, and the vapor. The vaporizer temperature in particular is
strongly dependent on the comhina-

tion of eluent flow rate, eluent composition, analyte composition, and the
diameter of the capillary vaporizer,
which may vary because of deposita of
nonvolatile materials. Gradient elution usually requires a temperature
program for the vaporizer. The commercial versions of most thermospray
LC/MS systems now automate most
controls, but the importance of correct
settings cannot be overemphasized.
Although some impressive detection
limits on certain compounds have
been reported using thermospray LC/
MS (13),we have found that practical
detection limits are routinely a factor
of 1%50 times higher than are customarilv obtained hv GCNS. This.
coupledwith significant variations in
response to different analytes (in filament-off or -on modes), causes some
problems when samples containing
unknown analytea are studied. For example, while analyzing a urinary extract for unknown metabolites, we
missed one major metabolite by both
filament-off and filament-on thermo.
spray LCMS. Later studies using a
heated pneumatic nebulizer LCmS
interface (see below) easily detected
this metabolite. Thus, the analyst
should be aware that thermospray LC/
MS may not display the universality
that might be desired.
The advantages of thermospray LC/
MS include ita commercial availability
and its freauent abilitv to emure molecular weight information. As with
any analytical technique, however, it
is not without limitations. These in-

clude limited structural information,

the need for extremely careful control
of relevant temperatures during analysis, and wide variation in response to
certain analytes. If the analyst is interested in studying or verifying
known compounds, thermospray LC/
MS may he well suited for this purpose. However, thermospray in any of
ita ionization modes provides very
mild ionization such that little fragmentation information is provided.
One does not obtain as much mass
spectral specificity from thermospray
or DLI LC/MS as is customary from
E1 mass spectra. But, thermospray
and DLI give far more information
than other conventional HPLC detectors and are therefore very helpful for
assisting in the characterization of unknown compounds along with other
analytical information that may be
available.
The development of the thermospray interface has significnntly increased the acceptance of L C N S for
at least two reasons. First, the thermospray interface readily accommodates conventional reversed-phase
HPLC flow rates, which represent the
majority of HPLC applications today.
Second, it is a simple, rugged system
that does not require unusual skill or
technique for ita operation. These
facts plus commercial adaptation to a
wide variety of different mass spectrometers have contributed to considerable interest in L C N S . The combination of filament-on, filament-off, and
discharge ionization modes for ther-

ANALYTICAL CtMISTRY. VOL. 58, NO. 14. DECEMBER 1988

1455A

Flgun 5. Simplified schematic diaoram of a heated pneumatic nebulizer LC/MS interfacecombined wilh an APi source
me hot nebulizer vapor en- llw r e g h ol llw c m d1-F
needle. laM farmed are fowsed thorn a &y nicurtain gas where lhey e m tha h i e
meum anawa region amuah a 100-pin orlfica. ions a e resolved via el* single u tripl%quadrupole msss analysis

mospray L C N S has indeed provided

the analyst with previously unavailable diversity for solving analytical
problems.
Atmospheric pressure ionization
(API). The fundamental problem of
L C N S with conventional flow rates is
the inability of a regular mass spectrometer vacuum system to pump the
full solvent vapor of the evaporated eluent. The thermospray L C N S comhination has cleared this hurdle hy connecting an additional pumping line directly to the ion source. No vacuum a t
all in the ion source clearly obviates
the problem altogether. Indeed, one of
the earliest reports of on-line LCNS,
published by Homings group in 1974,
described the use of an atmospheric
pressure ion source.
Understandably, interest in API has
been limited, because the traditional
manufacturers of analytical mass
spectrometers do not offer an API
source. In recent years, however, one

manufacturer (Sciex, Thornhill, Ontario, Canada) has produced an API

source incorporated into a triplequadrupole mass spectrometer. In the
following sections we will discuss three
LC/API MS systems, namely, the
heated pneumatic nebulizer, liquid ion
evaporation, and electrospray.
Heated pneumatic nebulizer. The
most common interface currently used
on an API source system is the heated
nebulizer. This probe-type interface
was developed by Thomson and is currently commercially available (20). It
takes full advantage of the large gas
and solvent vapor throughput tolerated hy the API source and provides
routine operation using HPLC flow
rates up to 2 mL/min with reversedphase eluents. It can tolerate volatile
salts, acids, bases, and other additives,
which may be necessary for the chromatographic separation.
Figure 5 shows the hasic components of the heated pneumatic nebu-

lizer L C N S interface and the atmospheric pressure chemical ionization

(APCI) source. The HPLC effluent
passes through the central microbore
throughput tube of the probe while
the nebulizer gas and makeup gas are
introduced coaxially into the heated
nebulization region. The combination
of heat and gas flow desolvates the
nebulized droplets, producing a dry
vapor of solvent and analyte molecules. Ionization of solvent molecules
is initiated by a corona discharge at
the discharge needle. The solvent ions
formed produce analyte ions by APCI
of the analyte. These ions are focused
and declustered through a dry nitrogen curtain gas and then through a
100-pm orifice into the high-vacuum
analyzer region of the mass spectrometer, where they are mass analyzed.
Control of the nebulizer temperature
is not critical because a wide variety of
analytes can be ionized with the same
heated nebulizer conditions. Typical-

B. Separation of five standard underivatlzedestrogenic substances by (a) LClUV and (b) API LC/MS (TIC), 6 0
CH,CNHD malntalnedat 1 mL/min thugh a Perkin-Elmer 4.6-mm X Xm, S-pm particle size C18 column
F

1456A

ANALYTICAL CHEMISTRY. VOL. 58, NO, 14. DECEMBER 1986

ly, a nebulizer vapor temperature of

125-150 C is maintained, which is
suitable for applications ranging from
involatile drugs to disulfonated azo
dyes in reversed-phase HPLC eluents.
An example of LC/MS data obtained from the heated nebulizer interface is shown in Figure 6. The mixture of estrogen-related compounds
and their metabolites shown in the
UV and TIC chromatograms of Figure
6 demonstrates excellent chromatographic fidelity; the mass spectra show
the abundant protonated molecules
characteristic of APCI mass spectra.
Unfortunately, the limited fragmentation resulting from this rather mild
ionization makes it difficult to identify unknown compounds by MS interpretation alone. This same feature is
also characteristic of both DLI and
thermospray LC/MS.
Liquid ion evaporation. Liquid ion
evaporation at atmospheric pressure
was reported in 1983 (14) but has not
enjoyed acceptance because a commercial interface was not available until late 1985. The mechanism of ion
production is emission of ions from
small, charged droplets. In liquid ion
evaporation a solution is dispersed
through a pneumatic nebulizer into air
at atmospheric pressure. Charges are
induced on sprayed droplets by means
of a small high-voltage electrode
placed close to the sprayer. There is
no need for elevated temperatures,
and conventional HPLC flow rates are
suitable. Only reversed-phase eluents
can be used, however, and the analyte
must be readily ionizable in the liquid
phase. This technique is well suited
for polar, ionizable compounds, such
as salts, acids, bases, amino acids, etc.,
but could not be considered universal
for many less polar compounds.
The unique feature of ion evaporation is its very mild ionization at room
temperature. We have been able to
produce doubly charged negative ions
of disulfonated azo dyes, whereas
thermospray ionization of the same
samples failed in our hands. Liquid
ion evaporation for LC/MS appears
worthy of further investigation.
Electrospray. The production of a
fine mist of charged droplets by electrospray was first demonstrated by
Zeleny in 1917. Dole and co-workers
have described their experiments,
which produced macro ions from polymer solutions by electrospray, in a series of publications, but mass analysis
of ions in the 50,000 dalton range did
not appear to be feasible. The use of
electrospray as an LC/MS interface
has been described in patents, but
publications in the refereed literature
have appeared only recently.
A solution of the sample is injected
into nitrogen gas at atmospheric pressure through a metal capillary tube

that is a t a potential of several kilovolts relative to the surrounding

chamber walls. Charge is deposited on
the surface of the emerging liquid, resulting in the production of coulomb
repulsion forces that are sufficient to
overcome surface tension so that the
liquid is dispersed in a fine spray. Ions
emitted from charged droplets are
transferred into the vacuum chamber
of a mass spectrometer and are mass
analyzed. The technique enables spectra to be obtained from mass spectrometrically difficult compounds, and
impressive results have been demonstrated for gramicidin S (21),for example. The technique works best with
flow rates in the range of 5-10 pL/min
and hence will require use of microbore-packed columns. We recently reported a form of nebulized electrospray that accommodates micro LC
flow rates up to 100 pL/min (22).This
homemade ion spray LC/MS interface was used on an API mass spectrometer and appears to have considerable analytical promise. The electrospray-type approach offers some
attractive features for LC/MS. These
include the absence of critical temperature control as in thermospray
LC/MS, GC/MS-like sensitivity, and
the absence of a small orifice, which
can cause practical difficulties.
Electrospray LC/MS has thus far
been demonstrated with homemade or
otherwise extensively modified mass
spectrometers, but its use with a commercially available API source appears straightforward (22) as these
systems are well suited for extraction
of ions into the vacuum system of the
mass spectrometer. The unique potential of such a combination suggests
that further effort should be made in
this area of LC/MS.
MAGIC. E1 mass spectra have not
been available from any form of DLI
LC/MS. The quest continues, however, for a means of obtaining E1 MS
data because of the rich structural information available from such spectra.
One of the recent approaches that has
provided E1 mass spectra is the monodisperse aerosol generator for introduction of liquid chromatographic effluents (MAGIC) reported by
Browner and Willoughby (23).This
device directs a nebulizing gas orthogonal to the flow of HPLC effluent,
which generates highly uniform-sized
droplets. A dispersion gas is used to
prevent clustering of the drops as they
are passed through a desolvation region on their way to the MS ion
source. The desolvation chamber is
maintained a t atmospheric pressure
while an aerosol beam separator connects it to the high-vacuum region of
the mass spectrometer. Flow rates of
0.1-0.5 mL/min are optimal, and the
compIete separation of the sample

from the HPLC eluent provides a free

choice between E1 and CI mass spectra.
If the MAGIC LC/MS system can
be demonstrated to be a rugged, routine technique, it could become an important analytical tool. Research is
under way to improve this system so
that it can handle less volatile analytes and provide lower detection limits.
LC/MS/MS. The reduced fragmentation resulting from mild ionization
provides insufficient structural information for identification of unknown
compounds. Fortunately, ionization
techniques that produce abundant
molecular or pseudomolecular ions are
well suited for tandem MS (MS/MS).
The key principle of MS/MS is collisionally activated dissociation (CAD),
which produces a family of daughter
ions ( 2 4 ) .For example, a protonated
molecule can produce a full-scan CAD
mass spectrum characteristic of the
structure of the parent compound.
When this is accomplished with
HPLC sample introduction, we refer
to it as LC/MS/MS. It is our contention that any of the forms of direct liquid introduction described so far, including thermospray LC/MS, would
benefit from this aspect of tandem
MS. This pertains because all these
forms of LC/MS provide minimal
fragmentation information and require beginning the MS scan at least
as high as m/z 100 and often as high as
m/z 200. Abundant solvent cluster
ions observed in the lower mass region
overwhelm lower mass analyte fragment ions that may be of interest. LC/
MS/MS techniques could provide fullscan mass spectra down to low mass
that are rich in structurally significant
fragmentation information. In addition, the mixture analysis capability of
MS/MS could facilitate the identification of components in unresolved
HPLC peaks.
Figure 7 shows results typical of
LC/MS/MS using daughter ion analysis techniques. The very simple LC/
MS mass spectrum (Figure 7a) is
clearly not as informative as the fullscan LC/MS/MS (or CAD) mass spectrum (Figure 7b). Proposal of plausible ion compositions may facilitate interpretation of the CAD mass
spectrum.
SFC/MS. The benefits of supercritical fluid chromatography (SFC)
have been described recently (25).
These basically include solubility behavior similar to liquid solutions but
gaslike diffusivity that can lead to
narrower peak widths than in HPLC.
These unique features could provide
the best of both worlds. In a chromatographic sense the advantages include improved ability to handle compounds that have a high molecular

ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986

1457A

Flgure 7. LCIMSIMS results

(a) API LClMS m a s w r u m of standard phenylbutarone. (b) API LCIMSIMS m886 SpecIrum of standard
phenylbutazone using a wiiision energy of 70 eV

weight and are thermally labile.

From the MS point of view SFC
sample introduction has some desirable features. As soon as the mobile
phase (typically supercritical carbon
dioxide) is introduced into the mass
spectrometer vacuum system, it undergoes rapid decompression to a gas
that can be readily pumped away. In
the case of DLI LCIMS the liquid mobile phase, which often contains water,
must he rapidly volatilized to quite a
large volume of gas. The use of capillary columns with supercritical fluids
avoids this excessive gas burden characteristic of packed-column HPLC. In
addition, the narrower chromatographic peak widths obtained with
SFC can provide lower detection limits than those typically associated
with packed-column DLI LCIMS.
Applications of capillary SFC/MS
have been reported hy Smith (25-27)
and Lee (28).These researchers have
shown convincing analytical potential
for this technique. Our interest in
SFCIMS began with packed columns
(29).followed hy studies on the feasibility of capillary SFCIMS on a commercial bench-top GC/MS (30). Preliminary results with a modified capillary S F C M S bench-top system under
CI conditions are encouraging, but our
goal is to do routine E1 SFC/MS. Further work is required to accomplish
this task, hut all indications are that
this goal is achievable.
Figure 8 shows an example of how
capillary SFC/MS could offer a
unique advantage over LC/MS, where
often only CI mass spectra are available. The upper TIC chromatogram of
Figure 8s shows the capillary S F C M S
separation obtained for an injection of
100 pg each of a three-component

Figure 8. SFCIMS
(a) SFCIMS total and e m e d ion cunenl chmmatcgams fw a loopa compmenl injmion of a wee-mmponenl mixhm. (b) Mass spectra (70 eV El) obtained
fMCaminoblphenyi, benzidine. and 3,3'dichiombenzidirm fcf Um 8eperalion in (a). (Reprinted lmm Reference 27)

1458A

ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER I986

mixture of 4-aminobiphenyl, benzidine, and 3,3-dichlorobenzidine.

These results were obtained using a
short 50-pm X 1.75-m column and MS
scans over the range of rn/t 90-350.
The E1 mass spectra for the three
compounds are shown in Figure 8b.
The quality of these data is acceptable, with little evidence for competing CI.
Although routine capillary SFC/MS
is still in its infancy, it seems clear
that it could grow to the point of commercial use. With a combination of
pressure programming and the addition of polar modifiers, more polar
compounds will be amenable to SFC/
MS. When it is demonstrated that
high-quality, full-scan E1 mass spectra
can be obtained from complex samples
containing polar, involatile, and high
molecular weight compounds, SFC/
MS could fill a niche between GC/MS
and LC/MS.
Areas of future research
LCiMS in its various forms has
clearly grown and matured since the
last report on the subject in these
pages (I). Although LC/MS had indeed been demonstrated by the late
1970s. it was not a routine technique
such as GC/MS was at that time.
Since then, however, advancements
including the thermospray and heated-nebulizer LC/MS interfaces have
significantly increased the ease of accomplishing LC/MS. A wide variety of
LC/MS applications have appeared,
and commercial mass spectrometers
dedicated to LCIMS are now available.
Although LC/MS is perhaps not yet
as routine or simple to accomplish as
bench-top GC/MS, increasing numbers of researchers are implementing
this technique in their laboratories.
Unfortunately, most of them may later be disappointed when they are
faced with the identification of unknown compounds by LC/MS. All of
the commercially available LC/MS
techniques, with the exception of the
moving-belt interface, provide only
mild ionization, which produces little
structurally useful fragmentation information. There is a practical need
for EI-like fragmentation from LC/
MS experiments.
There is also a need for a more universal response to different compounds. Thermospray ionization, for
example, provides excellent response
from adenosine but very poor response
from corticosteroids. If the latter are
important components in an unknown
sample they could go undetected in
such an experiment.
The need for increased structural
information could be met in several
ways. As described above, LCIMSIMS
using a mild ionization mode such as

thermospray ionization or API could

generate ions characteristic of the intact compound. Although MS/MS
techniques are well established at
present, the cost of such equipment is
too high for most laboratories. Thus,
what is needed is a low-cost, benchtop LC/MS/MS system with a rugged,
routine, and dedicated LC/MS interface and flexible software that provides access to all the popular modes
of MS/MS. The ion source should provide mild ionization with near universal detection. The system should be
fully computerized and should be
made to do just one thing very well,
rather than many different types of
experiments.
It should be noted that there may
be more rationalization for bench-top
LC/MS/MS than a corresponding GC/
MS/MS system. This is because capillary GC readily provides higher chromatographic resolution than does
HPLC so that MS/MS mixture analysis capability is not as necessary. In
addition, GC/MS readily provides E1
spectra, which is very useful for identifying unknown compounds. A benchtop LC/MS/MS system could make up
for the analytical deficiencies noted
for LC/MS.
Capillary SFC/MS is another possible solution to the problems of reduced chromatographic efficiencies
and limited structural information
currently afforded by LC/MS. The introduction of low gas flows of supercritical fluids directly into the mass spectrometer vacuum system can allow E1
ionization directly from SFC/MS. In
addition, the intermediate physical
properties of SFC between GC and
HPLC provide better chromatographic efficiencies from SFC than HPLC.
These two features make SFC/MS a
viable alternative, or perhaps a useful
complement, t o LC/MS. In addition,
as SFC becomes more widely used, it
could become a more inexpensive and
possibly easier means of obtaining
convenient mixture analysis with
structural information for components
not amenable to GUMS.
Finally, the limited mass range of
most commercial mass spectrometers
has precluded LC/MS applications
that include compounds with molecular weights in excess of 2000 daltons.
This, coupled with limited reports of
accurate mass measurements, has precluded work in important analytical
areas of LC/MS, including studies of
peptides and other high molecular
weight biological compounds. It does
not seem likely that quadrupole or
even magnetic-sector mass spectrometers will be amenable to the routine
LCIMS determination of compounds
with molecular weights in excess of
several thousand daltons. However,
Fourier transform MS (FTMS) could,

in principle, provide this capability

(31).FT mass spectrometers are capable of handling compounds up to
150,000 daltons and can provide highresolution capability upwards of several million. Thus, if LCDTMS, or
more likely, SFCIFTMS, were possible, we might be able to enjoy the best
of both worlds.
The most obvious hurdle to LC/
FTMS or SFC/FTMS is the realization that the FT mass spectrometer
operates with a pressure nearly 1000
times lower than conventional mass
spectrometers. Because any form of
LC/FTMS or SFC/FTMS introduces
an extra gas burden to the vacuum
system, the combination of these devices seems unlikely. However, a tandem ion source has recently been implemented in a commercial F T mass
spectrometer that could enable the
marriage of SFC to FTMS (32).With
the realization that capillary GC/
FTMS has been accomplished with
such a system, it seems likely that capillary SFC, for example, should be feasible.
We have, in fact, conducted preliminary capillary SFC/FTMS experiments on a dual-cell Nicolet 2000
FTMS instrument. Volatile compounds including decafluorotriphenylphosphine (DFTPP),methyl stearate,
and caffeine were successfully introduced via SFC into the high-pressure
source cell of the FT mass spectrometer. Low- and high-resolution mass
spectra were obtained under E1 and
CI conditions. Although these were
relatively simple experiments, they
demonstrate that SFC/FTMS is feasible. It remains to be seen whether very
high molecular weight compounds can
be successfully handled with such a
system. However, if this were indeed
possible, one would have virtually unlimited analytical capability if all the
features of both SFC and FTMS could
be utilized.
Finally, another potential area of
opportunity for successful LC/MS determination of high molecular weight
compounds is time of flight (TOF)
MS. The rapid scanning and high molecular weight capability of TOF MS
could offer some of the benefits of
FTMS without the considerable problems of maintaining extremely low
pressures in the vacuum system. Presently there are no reported applications of LC/TOF, but such a system
seems worthy of exploration and development.
It should be clear that LCIMS, in
one form or the other, is here to stay.
It is likely that even more impressive
developments than those of the recent
past will follow in the next 5-10 years.
Not only will refinements in existing
techniques occur, but totally new approaches should be invented that will

ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986

1459A

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m a k e LC/MS b o t h m o r e versatile a n d
routine. T h e ultimate goal would be t o
identify u n k n o w n compounds n o t
amenable t o GC/MS f r o m LC/MS
d a t a alone. Because it i s sometimes
n o t even possible t o p e r f o r m s u c h
i d e n t i f i c a t i o n s by GUMS, we s h o u l d
n o t e x p e c t t o t a l s a t i s f a c t i o n f r o m LC/
MS. But, we s h o u l d b e able t o o b t a i n
significant structural information for
a n y c o m p o u n d t h a t is p r e f e r e n t i a l l y
h a n d l e d by HPLC. As we s t r i v e f o r
t h i s a n a l y t i c a l c a p a b i l i t y , we w i l l dev e l o p b e t t e r a n d m o r e r o u t i n e LC/MS.

References
(1) Arpino, P. J.; Guiochon, G. Anal.
Chem. 1979,51,682-701 A.
(2) Vestal, M. L. Science 1984,226,27581.
(3) Henion, J. D. Micro LC/MS Coupling, in Microcolumn High-Performance Liquid Chromatography; Kucera,
P., Ed.; Elsevier: Amsterdam, 1984; J .
Chromatogr. Library, Vol. 28, pp. 260300.
(4) Eckers, C.; Henion, J. D. Combined
Liquid Chromatography-Mass Spectrometry of Drugs, in Therapeutic

Drug Monitoring and Toxicology by Liquid Chromatography; Wong, S.H.Y.,

Ed.; Marcel Dekker: New York, 1985;
Chromatogr. Sci. Series, Vol. 32, pp. 11549.
(5) Karger, B. L.; Vouros, P. J . Chromatogr. 1985,323,12-32.
(6) Smith. R. D.: Johnson. A. L. Anal
Chem. 198153,739-40.~
(7) Hayes, M. J.; Lankmayer, E. P.;
Vouros, P.; Karger, B. L.; McGuire, J. M.
Anal. Chem. 1983,55,1745-52.
18) Games. D. E.: McDowell. M. A.: Levsen, K.; Schafer, K. H.; Dobberstein, P.;
Gower, J. L. Bzomed. Mass Spectrom.
1984,11,87-95.
(9) Yinon, J.; Hwang, D. G. J . Chromatogr.
1985,339,127-37.
(10) Lee, E. D.; Henion, J. D. J . Chromatogr. Sei. 1985,23, 253-64.
(11) Parker, C. E.; Haney, C. A,; Harvan,
D. J.; Hass, J. R. J . Chromatogr. 1982,
242,77-96.
(12) Lant, M. S.; Martin, L. E.; Oxford, J.
J . Chromatogr. 1985,323, 143-52.
(13) Blakelv. C. R.: Vestal. M. L. Anal.
Chem. 1983,55, 750-54.
(14) Irabarne, J. V.; Dziedzic, P. J.; Thomson, B. A. Int. J . Mass Spectrom. Ion.
Phys. 1983,50,331-47.
(15) Garteiz, D. A,; Vestal, M. L. LC Mag.
1985,3,334-46.
(16) Vestal, C. H.; Garteiz, D. A.; Smit, R.;
Blakely, C. R. Presented at the 33rd Annual Conference on Mass Spectrometry
and Allied Topics, San Diego, May 27June 1, 1985; pp. 771-72.
(17) Henion, J. D. Presented a t the 31st
Annual Conference on Mass SDectrometry and Allied Topics, Boston,May 8-13,
1983; pp. 862-63.
(18) Pirkle, W. H.; Finn, J. M.; Schreiner,
J. L.: Hemaer, B. C. J . A m . Chem. Soc.
1981,103,3964-68.
(19) Lee, E. D.; Henion, J. D.; Brunner,
C. A.; Wainer, I. W.; Doyle, T. D.; Gal, J.
Anal. Chem. 1986,58, 1349-52.
(20) Thomson, B. A.; Danylewych-May, L.
Presented a t the 31st Annual Conference
on Mass Spectrometry and Allied Topics, Boston, May 8-13, 1983; pp. 852-53.
(21) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem.
1985,57,675-79.
(22) Bruins, A. P.; Covey, T. R.; Henion, J.
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1460A

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D. 34th Annual Conference on Mass

Spectrometry and Allied Topics. Cincinnsti, June a13,1986; paper-WPB 11.
(23) Willoughby, R. C.; Browner, R. F.
Anol. Chem. 1984.56.2626-31.
I

(24)

~~~

~~

Tandem Ma.& Spectrometry;McLaf-

ferty, F. W., Ed.; John Wiley and Sons:

NowVnrk
.
._.. .-.__,
-lPR2
(25) Smith, R. D.; Wright, B. W.; Udseth,
H. R. Caodlarv Suoercritical Fluid
Chromato&aphy-Mass S ectrometry,
in Chromatography and Eeparation
Chemistry;American Chemical Society:
Washington, 1986, pp. 261-93.
(26) Smith, R. D.; Felix, W. D.; Fjeldsted,
J. D.; Lee, M . L.Anol. Chem. 1982,54,
1883-85.

(27) Smith, R. D.; Udseth, H. R.; Kalinoski, H. T. And. Chem. 1984.56.297173.

(28) Fjeldsted, J. C.; Lee, M . L. Anol.
Chem. 1984,56,619-25 A.
(29) Crowther, J. B.; Henion, J. D. Anol.
Chem. 1985,57,2711-16.
(30) Lee, E. D.; Henion, J. D. J . High Res.
Chromatogr. Chromatogr. Comm. 1986,
9.172-74.

(31) Wilkins, C. L.; Grms, M . L. Anal.

Chem. 1981,54,1661-76 A.
(32) Ghaderi, S.; Littlejohn, D. P. Presented at the 32nd Annual Conference on
Mass Spectrometry and Allied Topics,
San Antonio, Tex., May 26-31,1984; pp.
727-28.

.
t

...

, . .

Jack Henion (left)is associate professor of toxicology at the New York

State College of Veterinary Medicine
of Cornell University in Ithaca, N.Y.
He is assistant director of the Equine
Drug Testing and Toxicology Program at Cornell and holds a B.A. from
Alfred University,an M.S. i n organic
chemistry from the Rochester Institute of Technology, and a Ph.D. i n
analytical organic chemistry from the
State Uniuersity of New York at Albany. After postdoctoral research in
ion cyclotron resonance MS at the
University of North Carolina at Chapel Hill, he joined the chemistry department of Cornell as director of the
NIH Biotechnology Resources High
Resolution Mass Spectrometry Facility. After two years he joined the veterinary college faculty, where he has
continued work in LCIMS as it relates to the real world of racehorse
drug testing and toxicology.
Edgar Lee (center left)is a secondyear graduate student interested in
capillary SFC combined with MS. He
is currently modifying a bench-top
capillary GCIMS for capillary SFCI
MS. He holds a B.A. from Brigham
Young University and is working toward a Ph.D. at Cornell.

,,,
,

Thomas Covey (center right) holds a

B.S. degree from Holy Cross College
and an M.S. from Cornell. Couey is
currently completing his Ph.D. i n analytical toxicology at Cornell in areas
including the ultratrace isolation and
identification of toxic substances
found in biological samples. He has
had extensiue experience with various
modes of LCIMS and LCIMSIMS.
Andries Bruins (right)received his
undergraduate training at the University of Amsterdam, the Netherlands, and earned his Ph.D. at the
same University on fragmentation
mechanisms and ion molecule reactions in MS. After postdoctoral research on negative-ion CIand linked
scans in MS at the University of
Warwick, England, he joined the Department of Pharmacy, State Uniuersity, Groningen, the Netherlands, as
a faculty member in charge of the MS
service facility, where he did research
on negative-ion C I , desorption C I ,
and LCIMS. He currently is on a oneyear leave as a visiting assistant professor a t Cornell University.

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