243
INSTRUMENTATION AND APPLICATIONS OF MICRO-LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY JACK HENION, EQUINE DRUG TESTING AND TOXICOLOGY, NEW YORK STATE COLLEGE OF VETERINARY MEDICINE, CORNELL UNIVERSITY, ITHACA, NEW YORK 14850 INTRODUCTION Micro high performance l i q u i d chromatography (micro HPLC) i s f i n a l l y enjoyi n g increased consideration and applications.
Several manufacturers a r e now
marketing e i t h e r modified conventional HPLC equipment, o r e n t i r e l y new hardware designed t o d e l i v e r micro LC f l o w r a t e s ranging u s u a l l y from 10-100 micro1 it e r s per minute. There are a v a r i e t y o f reported b e n e f i t s o r reasons f o r u t i l i z i n g micro LC r a t h e r than conventional HPLC ( f l o w r a t e s ranging from 1-3 mL/min), b u t u s u a l l y the most j u s t i f i a b l e reasons f o r "bothering" w i t h micro LC i n c l u d e HPLC solvent savings and o n - l i n e h i g h performance l i q u i d chromatography/mass spectrometry (LC/MS).
The l a t t e r i s the focus o f t h i s b r i e f review on micro
LC/MS w i t h emphasis placed upon p r a c t i c a l a p p l i c a t i o n s which demonstrate t h e s e n s i t i v i t y gains a v a i l a b l e when t o t a l i n t r o d u c t i o n o f micro LC e f f l u e n t i s provided t o the mass spectrometer. Combined o n - l i n e LC/MS has been demonstrated i n a v a r i e t y o f novel ways. Recent reviews discuss t h e r e l a t i v e m e r i t s and fundamental problems t h a t a r e associated w i t h LC/MS ( r e f s . 1 ,2). The seemingly d i f f i c u l t successful marriage o f a high-performance l i q u i d chromatograph (HPLC) t o a mass spectrometer (MS) has reached the p o i n t , however, where i t has become an established, a l b e i t n o t u n i v e r s a l l y accepted, l a b o r a t o r y technique ( r e f . 3). The most important d i f f e r e n c e (and problem) between the technique o f modern gas chromatography/mass spectrometry (GC/MS) and LC/MS i s t h e mobile phase utilized.
I t should be noted t h a t i t took several years t o develop a GC/MS
i n t e r f a c e which simply had t o remove s i g n i f i c a n t p o r t i o n s o f helium from t h e GC c a r r i e r gas.
The LC/MS i n t e r f a c e must e i t h e r r a p i d l y remove l a r g e p o r t i o n s
of the LC eluent, which may include components as diverse as water and b u f f e r s , o r a l l o w d i r e c t i n t r o d u c t i o n o f t h e LC e f f l u e n t i n t o the h i g h vacuum system of a mass spectrometer.
Thus, u n l i k e the problem encountered w i t h GC/MS,
the
LC/MS i n t e r f a c e must u n i t e the HPLC instrument, which normally operates i n t h e l i q u i d phase, w i t h t h e MS instrument'which normally operates a t high vacuum (ref. 3). I n a d d i t i o n t o t h i s seemingly d i f f i c u l t "marriage" t h e successful LC/MS i n t e r f a c e must e f f e c t i v e l y t r a n s f e r t h e LC s o l u t e from s o l u t i o n t o the vapor
244
phase without thermal degradation.
I f t h e conventional chemical i o n i z a t i o n
(CI) o r e l e c t r o n impact i o n i z a t i o n ( E I ) modes are n o t used, presumably, some a l t e r n a t i v e form o f i o n i z a t i o n i s necessary which w i l l be amenable t o these experimental conditions.
A v a r i e t y o f d i f f e r e n t approaches t o t h i s problem
are c u r r e n t l y under i n v e s t i g a t i o n . Perhaps the concept o f an i n t e r f a c e between two instruments i s p u t i n t o the best perspective when i t i s r e a l i z e d t h a t the "best i n t e r f a c e i s no This p o i n t i s supported by recent c a p i l l a r y GC/MS work wherein
interface."
f l e x i b l e fused s i l i c a c a p i l l a r y columns have been "snaked" d i r e c t l y t o the mass spectrometer i o n source r a t h e r than v i a heated t r a n s f e r 1i n e connections which r e s u l t i n a c t i v e s i t e s , e x t r a column voids, and hotspots ( r e f . 4). E x i s t i n g LC/MS i n t e r f a c e s include, however, varying degrees o f complexity. One o f the f i r s t r e p o r t s o f LC/MS consisted o f a stop-flow device wherein LC solvent was f l a s h evaporated p r i o r t o i n t r o d u c t i o n i n t o the mass spectrometer ( r e f . 5).
This device was r a t h e r elaborate and does n o t seem t o have enjoyed Following t h i s work were r e p o r t s o f LC/MS i n t e r f a c e s which
further interest,
include a moving w i r e o r b e l t concentrator ( r e f s . 6,7),
a s i l i c o n membrane
separator ( r e f . 8), an atmospheric pressure i o n i z a t i o n source ( r e f . 9), t h e d i r e c t i n t r o d u c t i o n o f a p o r t i o n o f t h e column e f f l u e n t i n t o a chemical i o n i z a t i o n source (refs. l O , l l ) ,
a f i x e d , heated concentration w i r e ( r e f . 12), and
a novel thermospray technique of LC/MS which shows g r e a t promise ( r e f . 13). The d i f f e r i n g approaches t o LC/MS have c o n t r i b u t e d t o e s t a b l i s h i n g t h e technique o f LC/MS as a v i a b l e t o o l i n the l a b o r a t o r y worthy o f a d d i t i o n a l work so i t can achieve the p o t e n t i a l t h a t GC/MS has enjoyed. However, the HPLC experiment includes: (a.) a wide v a r i e t y o f solvents and b u f f e r s , (b.) medium t o high molecular weight s o l u t e molecules t h a t may be i n v o l a t i l e and/or thermally l a b i l e , and (c.)
l i q u i d e l u e n t f l o w r a t e s t h a t have r e c e n t l y
increased t o s i x m i l l i l i t e r s / m i n u t e ( r e f . 14).
Each o f these f a c t o r s o f f e r s
a challenging problem t o the mass spectrometer which could p o t e n t i a l l y o f f e r the u l t i m a t e combination of s e n s i t i v i t y and s p e c i f i c i t y as an HPLC detector. A t the time o f t h i s w r i t i n g , the two most popular LC/MS i n t e r f a c e s (due i n p a r t t o t h e i r commercial a v a i l a b i l i t y ) are the moving b e l t t r a n s p o r t
(refs. 15,16) and the d i r e c t l i q u i d i n t r o d u c t i o n systems (DLI) ( r e f s . 17,18). Both of these interfaces r e q u i r e a maximum i n p u t o f l i q u i d s o l u t i o n a t a f l o w r a t e much lower than t h a t c u r r e n t l y used i n HPLC instrumentation as defined 4 mm and output flowrates by packed columns w i t h an i n t e r n a l diameter
ca 1 mL/min.
Reduced eluent f l o w i s e s s e n t i a l f o r t h e DLI systems, and
t r a n s p o r t i n t e r f a c e users have a l s o found t h a t performance i s improved when reduced flowrates are used, e s p e c i a l l y f o r operations i n v o l v i n g e l e c t r o n impact i o n i z a t i o n ( r e f . 19) because i t i s important t o remove a l l solvent
245
gases i n these experiments. S p l i t t i n g conventional HPLC e f f l u e n t f o r i n t r o d u c t i o n a t about 10 pL/min i n t o t h e DLI LC/MS i n t e r f a c e i s p r a c t i c a l o n l y when adequate sample i s available. This i s o f t e n n o t t h e case f o r a n a l y t i c a l problems i n drug metabolism and o t h e r areas o f t r a c e analysis. I t has been proposed t h a t workers i n such f i e l d s w i l l r e j e c t LC/MS as l o n g as throwing away 60-80% o f the sample i s r e q u i r e d ( r e f . 3). Since compromise i s essential f o r any successful marriage, one p o t e n t i a l s o l u t i o n t o LC/MS coupling i s m o d i f i c a t i o n o f the HPLC system such t h a t a constant reduced f l o w r a t e o f l i q u i d (ca 10-60 pL/min) i s maintained throughout t h e whole HPLC system. system (e.g.,
Minor m o d i f i c a t i o n o f t h e mass spectrometer vacuum
a d d i t i o n o f a cryopump) can a l l o w i n t r o d u c t i o n o f the t o t a l
micro HPLC e f f l u e n t t o the mass spectrometer i o n source i n such instances. A c u r r e n t t r e n d i n HPLC i s so-called " f a s t LC" which u t i l i z e s s h o r t columns containing 3-5 micron p a r t i c l e s .
Flowrates i n the neighborhood o f 3-6 mL/min
are used w i t h these columns and a f f o r d narrow chromatographic peaks i n s u r p r i s i n g l y s h o r t analysis times. Unfortunately, these increased f l o w r a t e s are n o t compatible w i t h modern MS pumping systems and appear unsuitable f o r LC/MS applications. Narrow-bore packed columns w i t h an i n s i d e diameter o f ca. 0.5 o r open t u b u l a r columns w i t h an i n s i d e bore o f . -ac
- 1 mm,
10 microns and t h e associ-
ated hardware f o r micro LC appear t o represent a reasonable p o t e n t i a l compromise o f the chromatographic aspect o f LC/MS. The optimum f l o w r a t e s from such "micro LC" systems are compatible w i t h some conventional ( r e f . 20) and modified ( r e f , 21) chemical i o n i z a t i o n mass spectrometer pumping systems. Thus, so long as r e a l i s t i c separation e f f i c i e n c y can be achieved i n reasonable analysis times by these systems, micro LC implementation i n t o LC/MS might provide the combination o f s e n s i t i v i t y and s p e c i f i c i t y so badly needed i n HPLC. This review w i l l cover the developments i n micro LC/MS reported through August, 1982. FLEXIBLE PACKED PDLYTETRAFLUOROETHYLENE (PTFE) MICROBORE COLUMNS By the mid-seventies, HPLC was a developing science and l i t t l e was known
about micro LC.
Some LC/MS work had appeared ( r e f s . 5,7,10)
utilizing
conventional HPLC, b u t the r e l a t i v e l y poor s e n s i t i v i t y necessitated by s p l i t t i n g up t o 99% o f t h e LC e f f l u e n t away from t h e MS made t h i s approach u n a t t r a c t i v e f o r t r a c e analysis.
I n l a t e 1975, a new commercially a v a i l a b l e
micro LC system was announced ( r e f . 24).
The JASCO FAMILIC 100 micro l i q u i d
chromatograph provided flowrates ranging from 2-16 pL/min d e l i v e r e d by a d i g i t a l l y d r i v e n syringe pump through a 0.5 mm i.d.
PTFE hand-packed column
246
connected t o a 0.3 pL v a r i a b l e wavelength micro UV detector.
This system
provided an o p p o r t u n i t y t o i n v e s t i g a t e the f e a s i b i l i t y o f i n t r o d u c i n g t h e e n t i r e micro LC e f f l u e n t continuously i n t o t h e MS, e.g.,
micro LC/MS.
Although the FAMILIC 100 and i t s predecessors have n o t o f f e r e d separation e f f i c i e n c i e s comparable t o conventional HPLC columns, j u d i c i o u s choice o f eluent and column packing does o f f e r considerable micro LC c a p a b i l i t y . This section reviews some o f the h i g h l i g h t s reported f o r t h e JASCO FAMILIC l O O N u t i l i z e d f o r micro LC/MS. The success o f our e a r l i e r DLI LC/MS work prompted t h e c o n s t r u c t i o n of a low dead volume connection between a JASCO FAMILIC 100 UV d e t e c t o r and the a l l - g l a s s c a p i l l a r y DLI LC/MS i n t e r f a c e reported p r e v i o u s l y ( r e f . 11). Figure 1 shows a schematic drawing o f t h e polypropylene glass-to-Teflon union which provided a convenient connection o f t h e PTFE column t u b i n g and t h e glass c a p i l l a r y LC/MS interface.
This simple, c o m e r c i a l l y a v a i l a b l e union formed
a l e a k - t i g h t connection which provided t o t a l t r a n s f e r o f t h e micro LC column e f f l u e n t t o t h e LC/MS i n t e r f a c e .
Since the DLI LC/MS technique e f f e c t i v e l y
"sprays" micro d r o p l e t s o f t h e LC e f f l u e n t d i r e c t l y i n t o the MS i o n source, t h i s micro LC/MS i n t e r f a c e allowed continuous C I monitoring o f t h e t o t a l micro LC/MS e f f l u e n t and a s i g n i f i c a n t c o n c o m i t a n t s e n s i t i v i t y increase. Figure 2 shows a schematic drawing o f t h e DLI micro LC/MS system.
I
I
I
I
I
Fig. 1. Schematic drawing o f t h e polypropylene t o glass-to-Teflon union which provides a convenient connection o f t h e PTFE tubing and the glass c a p i l l a r y probe. A: 75 pm x C.25 mm x 25 cm; B: glass-to-Teflon bushing; C: v i t o n O-ring seal; D: glass-to-Teflon cap; E: PTFE tube end f i t t i n g ; F: 0.5 mn x 1.5 mm T e f l o n tubing; G : f l a r e d end o f PTFE t u b i n g mating t h e f l a t surface o f t h e glass c a p i l l a r y probe. Reproduced w i t h permission from Ref. 25. Copyright 1981 Preston Publications, Inc.
241
Fig. 2. Schematic drawing of t h e m i c r o LC/MS system used i n t h i s work. 1: pump; 2: 250 u l g a s - t i g h t s y r i n g e ; 3: PTFE tubing, 0.5 mm i.d.; 4: sample i n l e t ; 5: micro column; 6: UV v a r i a b l e wavelength d e t e c t o r ; 7: m i c r o UV c e l l , 0.3 p1 volume; 8: s t a i n l e s s s t e e l c a p i l l a r y ; 9: PTFE tubing; 10: g l a s s - t o - T e f l o n connector; 11 : g l a s s c a p i l l a r y m i c r o LC/MS probe; 12: d i r e c t i n s e r t i o n probe i n l e t of MS; 13: C I mass spectrometer i o n source. Note t h e c l o s e p r o x i m i t y o f t h e UV e x i t and t h e m i c r o LC/MS glass c a p i l l a r y probe. Reproduced w i t h permission from Ref. 25. C o p y r i g h t 1981 Preston P u b l i c a t i o n s , Inc.
During t h e course o f e a r l i e r work w i t h t h e FAMILIC l O O N m i c r o LC, o t h e r s c r i t i c i z e d t h e system f o r i t s reduced s e p a r a t i o n e f f i c i e n c y compared t o conventional HPLC.
Although t h i s p o i n t i s w e l l taken i n c e r t a i n instances,
nevertheless, t h e 0.5 mm i.d.
packed microbore columns p r o v i d e adequate
separation i n many s i t u a t i o n s . To g i v e some i d e a o f t h e chromatographic d i f f e r e n c e s between m i c r o HPLC
and conventional HPLC, reference i s made t o t h e separations shown i n F i g u r e 3. The UV t r a c e i n F i g u r e 38 shows t h e s e p a r a t i o n o b t a i n e d from a t h r e e component m i x t u r e of a c i d drugs on a 4.6 mm x 25 cm C18 ODS Waters u Bondapak column u s i n g 50:50 CH3CN/1% a c e t i c a c i d a t a f l o w r a t e o f 1 mL/min w i t h a UV d e t e c t o r s e t a t 235 nm.
These i n s t r u m e n t a l c o n d i t i o n s p r o v i d e d marginal
separation f o r t h e a c i d drugs, phenyl butazone, oxyphenbutazone, and indomethac i n , w i t h a d e t e c t i o n l i m i t of about 10 ng.
The UV t r a c e shown i n F i g u r e 3A
was obtained from t h e JASCO FAMILIC w f t h a 0.5 mm i.d.
x 5 cm C18 ODS column
u s i n g 50:50 CH3CN/l% a c e t i c a c i d a t a f l o w r a t e o f 8 p l / m i n and t h e UV d e t e c t o r s e t a t 235 nm.
The s e p a r a t i o n achieved f o r t h e same t h r e e a c i d drugs
mentioned above was acceptable and t h e d e t e c t i o n l i m i t f o r these drugs on t h e m i c r o HPLC was about 0.7 ng.
I n general, most o f t h e i s o c r a t i c chromato-
g r a p h i c separations achieved u s i n g conventional equipment can be approximated w i t h some minor e l u e n t composition changes on t h e m i c r o LC.
248
0
4
8
12
Fig. 3. L i q u i d chromatograms a t 235 nm o f A: 10 ng l e v e l s o f phenylbutazone, oxyphenbutazone, and indomethacin v i a micro LC using t h e JASCO FAMILIC l O O N w i t h 8 pl/min o f 50:50 CH3CN/1% a c e t i c a c i d on a 0.5 mm i.d. x 5 cm JASCO SC-01 micro LC column and B: 50 ng l e v e l s o f phenylbutazone, oxyphenbutazone, and indomethacin v i a conventional LC using a Waters ALC-202 1i q u i d chromatograph w i t h 1 ml/min 50:50 CH3CN/l% a c e t i c a c i d on a Waters 4.6 mm x 25 cm C18 ODs P Bondapak column. Reproduced w i t h permission from Ref. 25. Preston Pub1 i c a t i o n s , Inc.
Copyright 1981
Figure 4 shows t h e C I micro LC/MS t o t a l i o n c u r r e n t (TIC) f o r t h e separation o f cortisone and dexamethasone obtained using a 0.5 mm x 7.0 cm C,8 LC column w i t h 40:60 CH3CN/H20 f l o w i n g a t 8 ul/min.
ODS micro
The Finnigan 3300 C I mass
spectrometer scan r a t e was 8 sec/scan from m/z 120-450 and t h e peak volumes were l e s s than 10 pL.
Thus, 20 ng l e v e l s o f c o r t i s o n e and dexamethasone were
r e a d i l y detected and t h e i r respective C I micro LC/MS mass spectra a r e shown i n Figures 5A and 58.
249
Fig. 4. T o t a l i o n c u r r e n t p r o f i l e p l o t f o r 20 ng of c o r t i s o n e and 20 ng dexamethasone. The separation and micro LC/MS a n a l y s i s was achieved using a 0.5 mm i . d . x 7 cm SC-01 column w i t h 8 pl/min 40:60 CH3CN/H20 as micro LC e l u e n t / C I reagent gas. Reproduced w i t h permission from Ref. 25. 1981 Preston Pub1 i c a t i o n s , Inc.
Copyright
250
80-
z
60
li
-
I
4020
-
I W 380
I .
3 nn A"
I
33
I
Fig. 5. Micro LC/MS C I mass spectra o f A: 20 ng c o r t i s o n e and B: 20 ng dexamethasone using a 0.5 mm i.d. x 7 cm SC-01 column w i t h 8 pl/min 40:60 CH3CN/H20 as micro LC/CI reagent gas. These mass spectra were obtained from the corresponding spectrum numbers i n Figure 4. Reproduced w i t h permission from Ref. 25. Copyright 1981 Preston Publications, Inc. There i s an unexpected p o t e n t i a l advantage o f micro LC/MS.
The determina-
t i o n o f a c i d i c hydrogens i n a s o l u t e molecule may be made by u t i l i z i n g e x o t i c solvents o r deuterated micro LC eluents.
A comparison of t h e C I micro LC/MS
mass spectrum o f sulfadimethoxine u t i l i z i n g 60/40 CH3CN/H20 as LC eluent and
C I reactant gas was made wherein the LC e l u e n t and C I reactant gas was CD3CN/D20 (ref.
25).
Figures 6A and 68 show t h e micro LC/MS C I mass spectra
obtained f o r sulfadimethoxine i n these two experiments. (M+H)+
I n Figure 6A, t h e
i o n a t m/z 311 v e r i f i e s t h e molecular weight o f 310 f o r t h i s drug.
Since there are three hydrogen atoms i n sulfadimethoxine t h a t are p o t e n t i a l l y exchangeable, the deuterated C I reactant gas o f 60/40 CD3CN/D20 could be expected t o exchange each of these hydrogens f o r deuterium under the conditions o f the experiment ( r e f . 26).
I n addition, t h e t r a n s f e r o f an a d d i t i o n a l
deuterium atom t o the deuterium exchanged sulfadimethoxine molecule i n the C I process e f f e c t i v e l y contributes f i v e atomic mass u n i t s t o the 310 molecular
weight o f t h i s drug. 315 ion.
This i s corroborated i n Figure 68 by t h e abundant m/z
The low solvent volumes u t i l i z e d i n micro LC/MS a l l o w one t o use
r a t h e r expensive deuterated solvents f o r the purpose o f LC separation and MS determination o f a c i d i c hydrogens.
261
Fig. 6. Micro LC/MS C I mass spectra f o r sulfadimethoxine using A: 60:40 CH3CN/H20 as micro LC eluant/CI reagent gas and B: 60:40 CD3CN/D20 as micro LC eluant/CI reagent gas. The SC-01 column was e q u i l i b r a t e d w i t h 60:40 C03CN/0,0 f o r 1 h r p r i o r t o o b t a i n i n g t h e data i n B. Reproduced w i t h permission from Ref. 25.
Copyright 1981 Preston Publications, Inc.
We continued t o i n v e s t i g a t e t h e u t i l i t y o f micro LC/MS v i a t h e LC/MS i n t e r f a c e shown i n Figure 1.
This system provided low nanogram, f u l l scan
C I mass spectra from an unchanged commercially a v a i l a b l e C I quadrupole mass
spectrometer ( r e f . 25).
Representative examples o f these data a r e shown
i n Figure 7 wherein mass spectra obtained v i a micro LC/MS o f t h r e e t o x i c o l o g i c a l l y important m a t e r i a l s a r e shown.
The upper mass spectrum represents
a t y p i c a l micro LC/MS mass spectrum o f a f l a t o x i n B1 (MW 312) using 40% acetonitrile/water.
I t s protonated molecular i o n a t
nJr
313 i s r e a d i l y
apparent and t h e spectrum i s e a s i l y obtained from 45 ng o f m a t e r i a l i n j e c t e d onto t h e microbore column. The second mass spectrum i n Figure 7 was obtained by i n j e c t i n g 10 ng o f the potent n a r c o t i c analgesic, fentanyl, on column. o f 336 i s r e a d i l y revealed by t h e nJi
the base peak i n t h e mass spectrum.
The molecular weight
337 protonated molecular i o n representing Routine f u l l spectrum a n a l y s i s o f t h i s
m a t e r i a l can be obtained from on column i n j e c t i o n o f as l i t t l e as 750 pg o f material.
262
18
z
60
I
40 20 140
160
200
180
220
240
260
280
aoo
320
340
..,I.
1
40 20
100
80
z
60
, -
LCSULb
SCAN
I
24
...,
-
v o 4 q
I:
2,.
40-
Fig. 7.
1
$1
Micro LC/MS C I mass spectra of A:
45 ng o f a f l a t o x i n B1; 8:
10 ng
fentanyl; and C: 3.5 ng sulfamethazine. The micro LC eluant/CI reagent gas was 20:80 CH3CN/H20 f o r A and 40:60 CH3CN/H20 f o r B and C. Reproduced w i t h permission from Ref. 25.
Copyright 1981 Preston Publications, Inc.
The sulfadrug, sulfamethazine (MW 278), i s q u i t e amenable t o LC/MS assay and e a s i l y displays i t s protonated molecular i o n as t h e base peak from o n l y 3.5 ng i n j e c t e d on column of t h e micro LC as shown i n the lower mass spectrum o f Figure 7.
The thermally l a b i l e sulfonamide bond o f t h i s m a t e r i a l precludes
i t s r o u t i n e assay from b i o l o g i c a l f l u i d s by GC/MS.
Thus, LC/MS o f f e r s a useful a l t e r n a t i v e r o u t e t o t h e successful a n a l y s i s o f such sulfadrugs a t l e v e l s comparable t o those formed i n b i o l o g i c a l f l u i d s . The packed PTFE microbore columns and t h e associated JASCO micro LC
equipment was a l s o useful f o r the micro LC/MS determination o f a t e r n a r y mixture o f phenothiazine t r a n q u i l i z e r s ( r e f s . 20,2728).
Figure 8A shows the UV chromatographic trace a t 254 nm f o r t h e i n j e c t i o n and separation o f a mixture o f 2-hydroxypromazine (2-OH-Pro), 2-chlorpromazine (2-C1-Pro).
acepromazine (Ace Pro) and
The separation i s accomplished by i n j e c t i o n
263
uv
1
5
I
10
1
MIN
15
1
20
Fig. 8. ( a ) L i q u i d chromatogram a t 254 nm f o r t h e micro LC separation o f 2-hydroxypromazine (35 ng), acepromazine (20 ng) and chlorpromazine (30 ng); (b) T I C p l o t from t h e micro LC/MS analysis o f t h e promazine m i x t u r e was accomplished under t h e same micro LC conditions described i n Figure 9. Reproduced w i t h permission from Ref. 20. Copyright 1980 John Wiley and Sons, Ltd.
264
o f 0.1 pL o f s o l u t i o n onto a 0.5 nnn x 7.0 cm SC-01 ODS C18 reversed phase column using a f l o w r a t e o f 8 p1 min-'
o f 90/10 a c e t o n i t r i l e / w a t e r c o n t a i n i n g
The amount o f each material a c t u a l l y i n j e c t e d on column and
0.1% TMA.
assayed by micro LC/MS was 35 ng 2-OH-Pr0,
20 ng Ace Pro, and 30 ng 2-C1-Pro.
The UV chromatogram reveals three major components which are r e f l e c t e d by the TIC chromatogram shown i n Figure 8B.
The presence o f t h e 0.1% TMA
i n the eluent was essential f o r good chromatographic peak shapes f o r t h e promazines on t h e SC-01 column.
The T I C shown i n Figure 88 demonstrates the
q u a l i t y o f i o n , c u r r e n t and mass spectrometer s t a b i l i t y during the course o f a low nanogram micro LC/MS experiment.
The o v e r a l l peak shapes o f t h e TIC
are e s s e n t i a l l y as good as the UV chromatogram o f the three promazines. The baseline o f the T I C i s r e l a t i v e l y constant w i t h no i n d i c a t i o n o f noise o r "spiking." The C I micro LC/MS mass spectra shown i n Figures 9A-C were obtained from the corresponding scan numbers 23, 44 and 67 shown i n t h e T I C o f Figure 88.
The [ M t l f
i n t h e i r mass spectra.
ions f o r each o f the promazines are t h e base peaks Thus, 2-OH-Pro (MW 300), Ace Pro (MW 326) and 2-C1-Pro
(MW 318) are e a s i l y d i s t i n g u i s h e d by t h e i r [ M t l ] '
ions o f
fn/r 301, 327 and
319 i n Figures 9A, 9B and 9C, respectively. As o t h e r researchers came t o r e a l i z e the p o t e n t i a l advantages o f s e n s i t i v i t y and s p e c i f i c i t y provided by micro LC/MS, new ideas emerged i n the l i t e r a t u r e u t i l i z i n g the JASCO micro LC and i t s PTFE packed microbore columns.
Schafer
and Levsen ( r e f . 29) have reported t h e micro LC/MS determination o f p o l y c y c l i c aromatic hydrocarbons u t i l i z i n g a homemade s t a i n l e s s steel c a p i l l a r y (0.1 mm i.d., 0.2 mm o.d., 30 cm long) connected d i r e c t l y t o a packed PTFE microbore column.
The s t a i n l e s s c a p i l l a r y was contained i n s i d e a l a r g e r s t a i n l e s s o r
brass tube whose o u t s i d e dimensions a1 lowed d i r e c t i n t r o d u c t i o n v i a t h e conventional GC vacuum lock.
Although the o r i g i n a l r e p o r t was n o t very
encouraging, recent work ( r e f . 30) has provided impressive micro LC/MS r e s u l t s from these workers on more d i f f i c u l t compounds. Although Bruins ( r e f . 31) had experimented w i t h micro LC/MS u t i l i z i n g the glass c a p i l l a r y DLI i n t e r f a c e , a r e v i s e d i n t e r f a c e u t i l i z i n g fused s i l i c a c a p i l l a r y tubing has afforded very encouraging micro LC/MS r e s u l t s from the JASCO FAMILIC l O O N micro LC on an unchanged Finnigan 3300 quadrupole MS ( r e f . 32).
A 70 cm long x 50 mm i.d.
fused s i l i c a c a p i l l a r y t r a n s f e r r e d micro
LC e f f l u e n t from the micro LC UV c e l l t o the chemical i o n i z a t i o n i o n source. Figure 10 shows how a copper block was a f f i x e d t o t h e t i p o f the fused s i l i c a t r a n s f e r l i n e t o provide heat t r a n s f e r from t h e i o n source t o the capillary.
This arrangement precluded f r e e z i n g o f aqueous eluents a t the
tip,which had caused problems e a r l i e r .
266
L........... 140
60
I10
1I
M I so0
. . , . . . . . . . . . . . . . . . . . . ~ . . .. -. . 200
220
240
260
210
300
320
. ' i n ' LCMSl
SCAN
- . . I
340
WI
z ; i l s 40
MI
am
I
20
140
160
IW
1 0 ~
no
240
250
200
900
am
340
I
40
uw u a
20
uo
m
w
zoo
zm
240
2c4
zw
aoo
a20
a40
Fig. 9. Micro LC/MS C I mass spectra f o r (a) 2-hydroxypromazine; (b) acepromazine; and ( c ) chlorpromazine taken from the corresponding scan numbers o f Figure 88. The micro LC eluent C I reagent gas was 90/10 CH3CN/H20 containing 0.1% TIIA. Reproduced w i t h permission from Ref. 20. Copyright 1980 John Wiley and Sons, Ltd.
266
I
2
3
1
5
F i g. 10. Schematic representation o f t h e i n t e r f a c e probe ( n o t drawn t o scale). 1. copper, 4.9 nun 0.d.; 2. s t a i n l e s s s t e e l 0.5 mm 0.d. x 0.25 mn i.d.; 3. Teflon i n s u l a t o r ; 4. s t a i n l e s s s t e e l 6.4 mn 0.d. x 4.6 mm i.d.; 5. fused s i l i c a c a p i l l a r y . Reproduced from Ref. 32.
The Finnigan methane.
CI source used i n t h i s work can accept 20 atm mL/min o f
A f l o w o f 10 pL/min o f water produces 12 atm mL/min water vapor.
A mixture o f water w i t h a c e t o n i t r i l e o r methanol presents a lower gas l o a d t o the vacuum system, so the CI source can e a s i l y accept the t o t a l e f f l u e n t from t h e micro LC.
A t y p i c a l l i q u i d chromatogram i s shown i n Figure 11.
The column was a 150 nim x 0.5 mm i.d.
PTFE tube, packed w i t h Nucleosil 5 um C18,
the solvent system was acetoni t r i l e / w a t e r 70:30 ( r e f . 32). Figure 12 gives the l i q u i d chromatogram o f t h e same mixture, recorded by the mass spectrometer a t 250'
C source temperature; ammonia gas was added,
but t h i s had no i n f l u e n c e on t h e mass spectra o f compounds under i n v e s t i g a t i o n . The sample components a r e i o n i z e d by t r a n s f e r o f a proton from a r e a c t a n t ion.
Thus, a simple i n t e r f a c e was constructed using inexpensive components.
The commercially a v a i l a b l e fused s i l i c a c a p i l l a r y can e a s i l y be replaced i f necessary, because no r e s t r i c t i o n or o t h e r m o d i f i c a t i o n has been made t o it. Adequate s e n s i t i v i t y i s obtained w i t h t h i s system, b u t a n a l y s i s o f so-called "non v o l a t i l e " samples i s n o t possible ( r e f . 32). As noted above, one of the shortcomings o f t h e JASCO FAMILIC l O O N and i t s associated packed PTFE microbore columns i s the r a t h e r low column ( r e f . 33) have reported a simple, improved e f f i c i e n c i e s . Games
et.
technique f o r packing t h e PTFE columns so they provide up t o a 1 0 - f o l d improvement i n e f f i c i e n c y .
267
A.U.
M W * 265
0.05
M W e 265
CI
MW. 2'19
ir 0 0
I
M W * 2991301
L 10 M l l .
Fig. 11. L i q u i d chromatogram o f a mixture o f f o u r components (10 ng each); UV d e t e c t o r 390 nm, a c e t o n i t r i l e / w a t e r 70:30, 8 pL/min. Reproduced from Ref. 32.
268
a; llCP
EICP 268
Fig. 12. Total i o n c u r r e n t p r o f i l e (m/z 150-350) and e x t r a c t e d i o n c u r r e n t p r o f i l e o f m/z 266; a c e t o n i t r i l e / w a t e r 70:30; 8 pl/min; NH3 gas added; same mixture as i n Figure 15, 50 ng per component.
Reproduced from Ref. 32.
One goal o f any LC/MS i n t e r f a c e i s t o maintain h i g h chromatographic e f f i c i e n c i e s throughout the system.
An advantage o f t h e moving b e l t system
f o r LC/MS over o t h e r types o f i n t e r f a c e s i s t h a t t h e microbore l i q u i d chromatographic column can be connected such t h a t i t feeds i t s e f f l u e n t d i r e c t l y onto the b e l t , thus ensuring a minimum dead volume connection. Thus, one can o b t a i n s i m i l a r e f f i c i e n c i e s by LC/MS t o those obtained using the UV detector.
This i s i l l u s t r a t e d i n Figure 13 where 7450 and 8250
t h e o r e t i c a l p l a t e s were obtained f o r naphthalene and biphenyl, r e s p e c t i v e l y ( r e f . 33).
Use o f the microbore l i q u i d chromatograph f o r LC/MS w i t h a
moving be1 t i n t e r f a c e leads t o improved s e n s i t i v i t y w i t h t h i s i n t e r f a c e , since there i s a lower background due t o solvent i m p u r i t i e s and h i g h e r mass s e n s i t i v i t y from the microbore l i q u i d chromatograph.
This improvement i s
not as dramatic as t h a t observed w i t h the i n t e r f a c e of the d i r e c t l i q u i d i n t r o d u c t i o n type ( r e f . 33).
269
5
0
10
min
15
25
20
Fig. 13. Computer reconstructed t o t a l i o n c u r r e n t t r a c e o b t a i n e d d u r i n g microbore LC/MS from a m i x t u r e o f naphthalene (A) and biphenyl (B) u s i n g a moving b e l t i n t e r f a c e , A 300 x 0.5 mm column packed w i t h 5 pm ODS was used w i t h a c e t o n i t r i l e / w a t e r 70:30 a t 5 p 1 min-’ as mobile phase. The mass spectrometer was used i n t h e C I mode and t h e o r e t i c a l p l a t e s were c a l c u l a t e d from t h e mass chromatograms o f m/z 128 f o r naphthalene and m/z 154 f o r biphenyl. Reproduced w i t h permission from Ref. 33. Copyright 1982 John Wiley and Sons, Ltd. PACKED METAL MICROBORE COLUMNS
When t h e a v a i l a b l e sample f o r a n a l y s i s i s l i m i t e d , a s w i t h blood o r t i s s u e extracts, i t i s v e r y important t o be a b l e t o i n t r o d u c e t h e sample e x t r a c t on-column i n a concentrated form and f o r t h a t column t o p r o v i d e h i g h e f f i c i e n c y Good examples o f these problems a r e found i n t h e d e t e r m i n a t i o n
separations.
o f d i e t h y l s i l b e s t e r o l i n t i s s u e s ( r e f . 34) and drug r e s i d u e s i n plasma ( r e f . 35).
The use o f packed m i c r o b w e columns (1 mm i.d.)
developed d u r i n g t h e r e c e n t p a s t ( r e f s . 36,37)
which have been
a r e considered an a t t r a c t i v e
s o l u t i o n t o these problems. Packed microbore columns w i t h one m i l l i m e t e r i n s i d e diameters have r e c e n t l y become commercially a v a i l a b l e as f l e x i b l e 1/16 i n c h 0.d. columns (refs.
38A-C) o r g l a s s l i n e d 1/8 0.d.
stainless steel
columns ( r e f . 38D).
The
demonstrated e f f i c i e n c i e s f o r these columns w i t h t e s t standard compounds
260
approach 50,000 p l a t e s per meter, and are therefore, s i g n i f i c a n t l y b e t t e r choices over the packed PTFE columns described above. I n addition, s t a t e o f - t h e - a r t s t a i n l e s s s t e e l compression f i t t i n g s may be u t i l i z e d w i t h t h e new metal microbore columns which f a c i l i t a t e l e a k - t i g h t , zero dead volume connections t o t h e i n j e c t o r and d e t e c t o r systems. Packed metal microbore LC columns a l s o provide o t h e r d e s i r a b l e features t h a t a r e r o u t i n e l y a v a i l a b l e from conventional HPLC equipment.
I n particular,
dual pump systems provide the c a p a b i l i t y f o r f a c i l e changes i n e l u e n t composit i o n without t h e need t o prepare a new solvent mixture each time t h a t a d i f f e r e n t solvent strength i s needed. Also, t h e a b i l i t y t o u t i l i z e convenient gradient e l u t i o n , o r solvent programming, i s extremely useful f o r a c c e l e r a t i n g the e l u t i o n o f more h i g h l y r e t a i n e d solutes from t h e column. O f course, the most important aspect o f micro LC f o r LC/MS purposes i s
the s i g n i f i c a n t l y reduced e l u e n t f l o w rates.
Favorable micro LC separations
may be accomplished a t eluent f l o w r a t e s ranging from 10
-
60 pL/min-’.
Most C I mass spectrometer vacuum pumping systems are capable o f maintaining the required instrument vacuum w i t h the d i r e c t i n t r o d u c t i o n o f between 10
- 20 p 1 h i n - l
o f aqueous organic e l u e n t mixtures i n t o t h e i o n source.
Thus, micro LC/MS a t these f l o w r a t e s may be accomplished on unchanged commercially a v a i l a b l e C I MS systems. However, i f one increases t h e pumping capacity o f t h e MS by i n s t a l l i n g a l i q u i d n i t r o g e n (LN) cryopump, micro LC may be accommodated by t h e MS. These increased
f l o w r a t e s up t o 60 UL/min-’
micro LC f l o w r a t e s have the advantage o f decreasing a n a l y s i s time b u t the disadvantages include reduced column e f f i c i e n c y and increased pressure on the associated plumbing system.
These l a s t two features cause problems
w i t h the above described JASCO micro LC system, but a r e more e a s i l y d e a l t w i t h by the metal microbore LC columns,
F i r s t , the e f f i c i e n c i e s o f t h e
metal microbore columns are considerably greater than those o f t h e PTFE microbore columns, so even a t increased f l o w rates, one obtains chromatographic e f f i c i e n c i e s adequate f o r most analyses. Second, the s t a i n l e s s compression f i t t i n g s on metal microbore columns have no d i f f i c u l t y coping w i t h higher system pressures r e s u l t i n g from increased f l o w rates. The PTFE columns and associated plumbing f r e q u e n t l y s u f f e r from leaks and various system f a i l u r e s i f the pressure exceeds 1500 psi.
Thus, the new a l l - m e t a l micro LC columns
and t h e i r associated hardware o f f e r some d i s t i n c t advantages f o r both micro LC i t s e l f and e s p e c i a l l y micro LC/MS. The major underlying advantage f o r LC/MS i s t h a t most o r a l l o f t h e micro LC e l u t e d solutes may be introduced d i r e c t l y i n t o t h e MS. Due t o the r e l a t i v e l y recent commercial i n t r o d u c t i o n o f metal microbore However, columns there a r e o n l y a few r e p o r t s o f t h e i r use i n micro LC/MS.
261 the i n i t i a l f i n d i n g s are very encouraging,and i t i s l i k e l y t h a t many applicat i o n s w i l l be seen i n the near future,
One o f the f i r s t reported examples
o f metal microbore LC/MS involved the c o n s t r u c t i o n and implementation o f a special d i r e c t i n s e r t i o n probe f o r a double-focusing A E I MS-902 h i g h r e s o l u t i o n MS f i t t e d w i t h an unmodified S I R C I S I 1 chemical i o n i z a t i o n source ( r e f . 39). These workers succeeded i n coping w i t h r a t h e r d i f f i c u l t instrumental problems, which included an 8000 v o l t a c c e l e r a t i n g voltage, low pumping speed a t t h e i o n source, and a r a t h e r small 1/4-inch 0.d. a c t u a l l y contained the 1 mm i.d.
d i r e c t i n s e r t i o n probe, which
x 75 cm microbore column.
The unique f e a t u r e o f t h i s work i s t h a t t h e r e was e s s e n t i a l l y no LC/MS The e x i t o f the microbore column a c t u a l l y entered t h e
i n t e r f a c e involved.
i o n source chamber where column e f f l u e n t passed through a 2 micron metal frit Solvent d e l i v e r y was n o t by a conventional LC
d i r e c t l y i n t o the C I source.
pumping system, b u t r a t h e r a compressed argon gas c y l i n d e r connected t o
5 m o f 1/4-inch 0.d.
copper t u b i n g which acted as the solvent r e s e r v o i r .
This was j o i n e d t o the microbore LC column by 1 m o f 1/4-inch Teflon t u b i n g which provided f l e x i b i l i t y and e l e c t r i c a l i n s u l a t i o n from t h e 8000 v o l t accelerating voltage. This micro LC/MS system accommodated e l u e n t f l o w r a t e o f 10 uL/min-’ w i t h normal phase eluents.
Separations and S I M traces o f f a t t y a c i d e s t e r
protonated molecular ions were obtained w i t h 4000 t h e o r e t i c a l p l a t e s f o r these compounds on the homemade microbore columns.
A1 though some reversed
phase separations were attempted, t h e methanol/water mixtures caused e a r l y f a i l u r e o f the MS filament.
These workers t h e r e f o r e l i m i t e d t h e i r s t u d i e s
t o silica-packed microbore columns.
These d i f f i c u l t i e s combined w i t h t h e
f i n d i n g t h a t l e s s v o l a t i l e compounds could f r e q u e n t l y n o t be detected may be responsible f o r t h e f a c t t h a t l a t e r developments i n t h i s approach t o micro LC/MS have n o t been reported.
Another r e p o r t o f metal column micro LC/MS described an adaptation o f the Hewlett-Packard DLI diaphragm i n t e r f a c e t o micro LC c o n d i t i o n s ( r e f . 21). The s i m p l i f i e d version o f the commercially a v a i l a b l e s p l i t e f f l u e n t i n t e r f a c e i s shown i n Figure 14.
The s a l i e n t feature o f t h i s new micro LC/MS probe
i s the narrowbore (0.004 i n c h i.d.)
c e n t r a l throughput tube which t r a n s f e r s
t o t a l e f f l u e n t from the micro LC column t o t h e C I mass spectrometer i o n source o f an unchanged commercially a v a i l a b l e quadrupole MS.
The water-
cooled probe t i p features a removable s t a i n l e s s s t e e l diaphragm c o n t a i n i n g a p r e c i s e l y centered lasebgenerated f i v e micron pinhole.
The device niay be
i n s e r t e d i n t o a standard 1/2-inch d i r e c t i n s e r t i o n i n l e t w i t h o u t any a l t e r a t i o n o f t h e MS system.
This system does n o t s u f f e r from t h e hazards o f high
acceleration voltage, long run times, d i f f i c u l t y w i t h l e s s v o l a t i l e solutes,
262
o r r e s t r i c t i o n t o normal phase LC conditions reported i n e a r l i e r micro LC/MS work ( r e f . 39).
A
J l i
6'
E
F
G
Fig. 14. Micro LC/MS probe: (A) micro LC e f f l u e n t i n l e t l i n e ; ( B ) water c o o l i n g i n l e t tube; (C) Teflon washer f o r maintaining vacuum seal between probe t i p / c o o l i n g chamber and probe shaft; (D) throughput tube c o l l e t ; ( E ) 0.004 inch (i.d.) x 0.062 i n c h (0.d.) s t a i n l e s s s t e e l throughput tube; (F) water cooling chamber; (G) Kalrez O-ring; (H) diaphragm containing 5 urn pinhole i n center; ( I ) removable endcap. Reproduced w i t h permission from Ref. 40. Copyright 1982 American Association o f C l i n i c a l Chemistry. When a stable, s h o r t " j e t " o f micro LC e f f l u e n t has been established through the diaphragm pinhole, t h e micro LC/MS probe i n t e r f a c e may be i n s e r t e d through the d i r e c t probe i n l e t t o t h e cryogenically-pumped C I source.
The Hewlett-Packard 59858 quadrupole MS u t i l i z e d i n t h i s work
( r e f . 17) operates through an e l u e n t f l o w range from 10
-
60 pL/min-'.
Optimum performance, however, occurs i n the neighborhood o f 40 p 1 h i n - l w i t h any combination o f aqueous methanol o r a c e t o n i t r i l e eluents. V o l a t i l e b u f f e r s such as ammoni um hydroxide, t r i m e t h y l amine, t r i e t h y l ami ne, ammoni um acetate, formic acid, a c e t i c acid.and t r i f l u o r o a c e t i c a c i d o f f e r no d i f f i c u l t y because they produce low molecular weight organic compounds t h a t are r e a d i l y pumped away by the MS vacuum system. Figure 15 shows t y p i c a l i o n c u r r e n t chromatograms obtained from t h e DLI micro LC/MS diaphragm probe i n t e r f a c e under negative i o n chemical i o n i z a t i o n (NCI) conditions ( r e f . 21).
I t should be noted t h a t these data
are acquired as f u l l scan mass spectra (e.g.
z/r 80-500)
and t h a t t h e i o n c u r r e n t s t a b i l i t y and micro LC/MS s e n s i t i v i t y appear comparable t o t y p i c a l GC/MS data a t these level's.
The r e s o l u t i o n o f t h e components o f dexamethasone
263
97
6
39.
n
A
&"..
33 NG DEXAl'lETHRSONE
38 NG 6-B-OH-PREDNSSOLONL
I
W$OH
rI
Fig. 15. N C I micro LC/MS T I C P and E I C P f o r 30 ng l e v e l s o f dexamethasone and 6-B-hydroxyprednisolone using 50% CH3CN/H20 a t 34 pL/min as micro. LC/MS eluent/CI reactant gas.
The micro LC column was a C18 HRSM connected t o
an unchanged Waters ALC-202 pump and solvent programmer. Reproduced w i t h permission from Ref. 21. Copyright 1981 American Chemical Society. and 6-f3-hydroxyprednisolone was accomplished on a 50 cm C18 HRSM microbore column ( r e f . 386) using a f l o w r a t e o f 34 UL/min-'
50% CH3CN/H20 as micro
LC/MS eluent/CI reactant gas ( r e f . 21).
An a p p l i c a t i o n o f micro LC/MS t o actual problem s o l v i n g i s shown i n Figure 16. The upper panel shows the micro LC UV t r a c e from a TLC scrape of an unknown powder sample confiscated from a race track. The f l o w r a t e was 34 VL/min-'
50% CH3CN/H20 on a 50 cm C18 HRSM micro LC column and UV
d e t e c t i o n (15 pL f l o w c e l l 239 nm.
, Perkin
Elmer LC-55, Norwalk, Connecticut) was
In the lower panel o f Figure 16, t h e corresponding micro LC/MS
i o n c u r r e n t traces f o r t h i s unknown sample a r e shown.
The major component
observed a t 2.8 min r e t e n t i o n time had an N C I micro LC/MS mass spectrum i d e n t i c a l w i t h t h a t o f authentic dexamethasone.
The minor component
observed a t 4.4 min r e t e n t i o n time had an abundant n j ~ 1 2 7i o n and an apparent molecular weight o f 366.
Its i d e n t i t y i s unknown, although i t had gone
undetected by the UV detector.
These data demonstrate both t h e f e a s i b i l i t y
264
Fig. 16. (A, upper) Micro LC UV t r a c e f o r a TLC scrape of an unknown sample confiscated from a race track. The f l o w r a t e was 34 pL/min 50% CH3CN/H20 on a C18 HRSM micro LC column and UV d e t e c t i o n was 239 nm. (B, lower) N C I micro LC/MS T I C P and EICP f o r the sample described i n 4A. The major component observed a t 2.8 min r e t e n t i o n time was shown t o be dexamethasone. Reproduced w i t h permission from Ref, 21. Copyright 1981 American Chemical Society.
266
and v e r s a t i l i t y o f micro LC/MS.
The analysis times can be l e s s than 20 minutes
and s e n s i t i v i t y s u i t a b l e f o r t r a c e analysis i s possible by micro LC/MS. The CLI micro LC/MS diaphragm i n t e r f a c e described above has been improved and a d d i t i o n a l a p p l i c a t i o n s reported ( r e f . 40).
The c o n s t r u c t i o n o f t h e
i n t e r f a c e was simp1 i f i e d by r e p l a c i n g t h e two concentric narrow-bore t r a n s f e r tubes w i t h one c e n t r a l throughput tube whose dimensions a r e 0.004-inch 1/16-inch 0.d.
i.d.
x
Experimental d e t a i l s f o r accomplishing micro LC/MS were
described i n a d d i t i o n t o s p e c i f i c information concerning m o d i f i c a t i o n o f conventional Waters HPLC equipment f o r micro LC work.
Examples o f micro LC
UV chromatograms f o r three t h i a z i d e d i u r e t i c s and three c o r t i c o s t e r o i d s were
shown i n a d d i t i o n t o p r a c t i c a l NCI micro LC/MS d e t e c t i o n l i m i t s using t h e thermally 1abi 1e, i n v o l a t i l e compound, t r i c h l ormethiazide (TCM)
.
The d e t e c t i o n
l i m i t f o r t h i s compound was 1.25 ng i n j e c t e d onto the micro LC column which
provided an acceptable f u l l scan N C I mass spectrum of t h e r a t h e r i n v o l a t i l e molecule ( r e f . 40). Figure 17 shows the N C I micro LC/MS t o t a l i o n c u r r e n t chromatograms o f a TLC scrape from a zero-hour equine u r i n e e x t r a c t (lower) and an equine u r i n e c o l l e c t e d 2h post-oral a d m i n i s t r a t i o n o f TCM (upper). The micro LC/MS eluent/CI reactant gas was CH3CN/H20 (70:30 by v o l ) maintained a t 40 pL/min- 1 through a Chrompack 1 mm i.d.
x 50 cm microbore column.
Sample clean-up
by preparative TLC g r e a t l y f a c i l i t a t e s the a n a l y s i s by precluding t h e i n t r o duction o f high l e v e l s o f endogenous compounds onto the microbore LC column and shortens the micro LC/MS a n a l y s i s time t o l e s s than 10 minutes.
The
determination o f TCM by p o s i t i v e i o n chemical i o n i z a t i o n (PCI) i s complicated by a 100-fold decrease i n s e n s i t i v i t y f o r TCM, and i n t e r f e r e n c e by numerous o t h e r components t h a t are n o t even observed i n the NCI data o f Figure 17. Thus, the f a c i l i t y o f s e l e c t i n g e i t h e r P C I o r N C I modes can improve the performance o f micro LC/MS i n c e r t a i n instances.
The above described method
allows d e t e c t i o n o f TCM i n racehorse u r i n e through 24 hours post-oral admi n i s t r a t i on. The a p p l i c a t i o n o f micro LC/MS determinations o f betamethasone and i t s metabolites i n equine urine, a n t i b i o t i c s i n t h e crude e x t r a c t s o f a fermentat i o n b r o t h and i m p u r i t i e s i n a preparative HPLC sample obtained from a s y n t h e t i c mixture of Felodipine have been reported ( r e f . 41).
Figure 18,
f o r example, shows both the UV chromatogram and the i o n c u r r e n t chromatograms from the micro LC and micro LC/MS analysis, r e s p e c t i v e l y , o f an equine u r i n e extract.
The u r i n e had been c o l l e c t e d 6 hours a f t e r the a d m i n i s t r a t i o n
o f betamethasone.
Both the major betamethasone metabolite and a p r p v i o u s l y
undetected minor metabolite are observed i n these data, i n a d d i t i o n t o t h e parent c o r t i c o s t e r o i d , betamethasone, which i s e a s i l y observed a t a r e t e n t i o n
266
Fig. 17. Negative ion CI micro LC/MS total ion current profiles of a TLC scrape from a zero-hour equine urine extract (lower) and an equine urine collected 2 h post oral administration o f trichlormethiazide (upper). The micro LC/MS eluent/CI reactant gas was CH3CN/H20 (70:30 by vol) maintained at 40 pL/min through a C,8 Chrompack 1 mm i.d. x 50 cm microbore column. Reproduced with permission from Ref. 40. Copyright 1982 American Association of Clinical Chemistry.
267
time o f 15.5 min.
F u l l scan N C I mass spectra are provided by these experiments.
The micro LC conditions f o r these experiments were 40 pL/min-l 60:40 CH30H/H20 u t i l i z i n g an A l l t e c h 1 mm i.d.
x 1/16-inch 0.d.
x 50 cm C18
reversed phase micro LC column ( r e f . 38A).
L
b
5
12
Fig. 18. (A) Micro LC UV chromatogram o f an equine u r i n e e x t r a c t . The u r i n e was c o l l e c t e d 6 hours a f t e r an a d m i n i s t r a t i o n o f betamethasone t o a horse. This micro LC separation was accomplished on an A l l t e c h 1 mm i.d. x 1/16-inch 0.d. x 50 cm C18 reversed phase column w i t h an e l u e n t o f 65:35 CH30H/H20 a t a f l o w r a t e o f 40 pL/min and UV d e t e c t i o n a t 254 nm. ( 6 ) Total i o n c u r r e n t and e x t r a c t e d i o n c u r r e n t p r o f i l e s obtained from the micro LC/MS a n a l y s i s o f t h e sample described i n (A). Reproduced from Ref. 41.
The use o f g l a s s - l i n e d s t a i n l e s s s t e e l microbore columns f o r micro LC/MS w i t h a moving b e l t t r a n s p o r t i n t e r f a c e has been reported r e c e n t l y ( r e f . 42). The goal o f t h i s work was t o introduce t o t a l micro LC e f f l u e n t t o t h e i n t e r f a c e and maintain good chromatographic e f f i c i e n c y from t h e i o n c u r r e n t chromatograms.
These goals were n i c e l y met by u t i l i z i n g a Finnigan 4000 MS
equipped w i t h a Finnigan moving b e l t LC/MS i n t e r f a c e ( r e f . 15). and C I micro LC/MA mass spectral data were obtained.
Both E I
268
Representative micro LC/MS data from t h i s work are shown i n Figures 19 and 20.
The computer reconstructed t o t a l i o n c u r r e n t chromatogram (amnonia
C I ) obtained from an e x t r a c t o f the r o o t s o f Imperatoria ostruthium i s
shown i n Figure 19.
This micro LC separation was accomplished on a Whatman
P a r t i s i l 10 ODs-3 250 x 1 mm microbore LC column (Ref. 38D) u t i l i z i n g a mobile phase o f 9O:lO CH30H/H20 a t a f l o w r a t e o f 40 pL/min-’.
’
The u t i l i t y
o f the high e f f i c i e n c y o f the microbore LC columns i s shown i n Figure 20. where t h e computer reconstructed t o t a l i o n c u r r e n t chromatogram, obtained from t h e same mixture under d i f f e r e n t chromatographic conditions, i s shown.
5
10
min.
15
20
Fig. 19. Computer reconstructed t o t a l i o n c u r r e n t t r a c e (ammonia e x t r a c t o f t h e r o o t s o f Imperatoria ostruthium, obtained by LC/MS P a r t i s i l 10 .ODS-3 250 x 1 m Whatman microcore LC column, Mobile Reprinted w i t h permission Methanol/water (9O:lO) a t 40 pL/min-’ Copyright 1982 John Wiley and Sons, Lid.
CI) o f an using a phase: from Ref. 42.
This improved separation allowed t h e compounds present between t h e f i r s t two major components i n Figure 19 t o be studied and t h e s t r u c t u r a l assignments made. The mobile phase i n t h i s case was CH30H/H20/CH3C02H (58:40:1) i n i t i a l l y flowing a t 40 p1 min-’, increased t o 80 p1 min-’ i n the l a t t e r p a r t o f the chromatogram.
269
B
T 10
Fig. 20. Computer reconstructed t o t a l i o n c u r r e n t t r a c e (ammonia C I ) o f an e x t r a c t of the r o o t s of Imperatoria ostruthium, obtained by LC/MS using a P a r t i s i l 10 ODS-3 250 x 1 mm Whatman microbore LC column. Mobile phase: Methanol/water/acetic a c i d (58:40:1)
i n i t i a l l y a t 40 pL/min-’
increased t o
80 pL/min-l i n l a t t e r p a r t o f chromatogram. Reprinted w i t h permission from Ref. 42. Copyright 1982 John Wiley and Sons, Ltd. These authors conclude t h a t comnercially a v a i l a b l e g l a s s - l i n e d s t a i n l e s s steel reversed phase microbore LC columns provide chromatographic performance comparable t o t h a t obtainable from conventional HPLC columns.
Thus, use
f o r combined LC/MS w i t h i n t e r f a c e s o f t h e moving b e l t type i s advocated since improved mass spectral s e n s i t i v i t y i s obtained i n the absence o f a s p l i t t e r . The problems associated w i t h reversed phases and background solvent i m p u r i t i e s on t h e moving b e l t i n t e r f a c e a r e a l s o s i g n i f i c a n t l y reduced ( r e f s . 42-44). O f course, the o t h e r LC/MS i n t e r f a c e s should enjoy some o f these advantages
from microbore LC, too.
270
OPEN TUBULAR MICRO LC/MS
The d i r e c t coupling o f open t u b u l a r m i c r o c a p i l l a r y LC columns would appear t o be a very d e s i r a b l e goal.
There have been several inferences toward t h i s
end b u t r e l a t i v e l y few published reports.
I s h i i and Takeuchi made b r i e f
mention o f d i r e c t coupling o f an open t u b u l a r column v i a the GC i n l e t o f a Finnigan 3300E quadrupole G U M S ( r e f . 45). connected t o a 0.13 nun i.d.
The open t u b u l a r LC column was
s t a i n l e s s s t e e l c a p i l l a r y tube which penetrated
the system v i a the GC i n l e t h e l d a t 150' C.
They monitored the (M+1)+ ions
o f 50 ng l e v e l s f o r several isomeric xylenols.
The authors admit t h a t d i r e c t
i n t r o d u c t i o n o f micro LC e f f l u e n t i n t o the i o n source would be preferable and w i l l r e p o r t these r e s u l t s a t a l a t e r date. T i j s s e n and co-workers have published some o f the more impressive micro LC/MS r e s u l t s using d i r e c t l y coupled open t u b u l a r LC columns t o the i o n source o f the MS ( r e f . 46).
For samples o f low molecular weight (MW 250),
the d i r e c t i n l e t o f column e f f l u e n t from unmodified m i c r o c a p i l l a r y columns i s w e l l - s u i t e d f o r p r a c t i c a l q u a l i t a t i v e as w e l l as q u a n t i t a t i v e analysis. An i l l u s t r a t i v e example i s the reversed phase separation o f polynuclear aromatics (PNAs).
This separation was performed on a32 vm x 4.5 m column
w i t h i n 3 min as shown i n Figure 21.
The peak shape q u a l i t y i s comparable
t o those obtained from the UV detector and t h e d e t e c t i o n l i m i t o f 100 pg was f a r b e t t e r than t h a t obtained from t h e UV detector. T i j s s e n reports t h a t l i q u i d j e t formation may be r e a l i z e d by passing the open t u b u l a r LC e f f l u e n t through a conical t i p w i t h rjet = 2.5 pm a t the end o f a microcapillary.
I n t h i s case, samples o f higher molecular weight can
be introduced i n t o the MS i o n source w i t h o u t d i f f i c u l t y .
Figure 22 shows
the i o n c u r r e n t chromatogram (upper) obtained by m i c r o c a p i l l a r y LC/MS w i t h j e t formation.
The f o u r resolved components were toluene (scan 22), Sudan
ye1 low (scan 25) , 2-ethyl anthraquinone (scan 29) and 1,4 naphthoquinone (scan 33).
The column was a 32 cm x 322 pm and isooctane was used as eluent.
The lower p o r t i o n o f Figure 22 shows t h e C I mass spectrum o f Sudan y e l l o w which e l u t e d a t scan 25. These r e s u l t s , too, are encouraging and i n t e r e s t i n g . However, open t u b u l a r micro LC/MS r e s u l t s from r a p i d separations o f p o l a r , l a b i l e compounds occurring i n complex matrices are s t i l l needed t o demonstrate the r e a l i s t i c u t i l i t y o f t h i s technique.
271
MASS SPECTROMETRIC SIGNAL TUNED TO SPECIFIC MASSES
RETENTION TIME, sec
Fig. 21. Chromatogram showing t h e superimposed s i g n a l s of four s p e c i f i c masses f o r p o l y n u c l e a r aromatics separated by reversed-phase m i c r o c a p i l l a r y LC. (Benzene i n CI/MS behaves d i f f e r e n t l y from o t h e r PNAs and i s d e t e c t e d Peak h e i g h t s ( i n counts): benzene 383, naphthalene 2939, a t m/z = M W - 1.) anthracene 158, pyrene 539. Sample c o n c e n t r a t i o n s : ug/ml. Flow r a t e : 2 uL/min. S p l i t t i n g r a t i o : 1:5000. Each peak c o n t a i n s 10 ng o f t h e compound. Reproduced from Ref. 46.
272
K INTENSITY
loo-
0
00-
0
0:o
0:24
0.49
20 100
1.13
1.37
0 140
I80
24 TIME
0 220
260
300
Fig. 22. Chromatogram o b t a i n e d by m i c r o c a p i l l a r y LC/MS w i t h j e t f o r m a t i o n . A. S o l u t e s : t o l u e n e (scan 22); Sudan y e l l o w (scan 25); 2 - e t h y l a n t h r a q u i n o n e (scan 29) , l Y 4 - n a p h t h o q u i n o n e (scan 33) and p-aminoazobenzene (scan 87). Column 322 pm x 32 cm, rjet = 2.5 urn, e t c h e d g l a s s . M o b i l e phase: i s o o c t a n e .
B.
Mass spectrum accompanying t h e Sudan y e l l o w peak. Ref. 46.
Reproduced f r o i n
273
SUMMARY AND CONCLUSIONS I t i s c l e a r t h a t a r o u t i n e LC/MS system p r o v i d i n g broad a p p l i c a t i o n
c a p a b i l i t y would be welcomed by many a n a l y t i c a l chemists.
Unfortunately,
LC/MS i s n o t y e t q u i t e as easy t o perform as HPLC by i t s e l f .
I n addition,
many f e e l t h a t LC/MS i s n o t y e t s u f f i c i e n t l y r o u t i n e t o m e r i t t h e e f f o r t necessary t o g e t r e s u l t s .
T h i s a u t h o r knows o f several researchers and
s e r v i c e o r a p p l i c a t i o n l a b o r a t o r i e s t h a t r o u t i n e l y u t i l i z e o n - l i n e LC/MS when t h e s o l u t i o n t o a problem r e q u i r e s t h i s technique.
The technique o f
LC/MS can be r o u t i n e i f t h e chemist decides t h e r e s u l t s a r e i m p o r t a n t enough t o m e r i t t h e e f f o r t . M i c r o LC/MS r e a d i l y provides LC/MS d e t e c t i o n l i m i t s comparab1.e t o todays GC/MS c a p a b i l i t i e s .
I f t h e a n a l y s t can r e l i a b l y achieve m i c r o LC s e p a r a t i o n
and d e t e c t i o n o f compounds o f i n t e r e s t i n drug metabolism o r t o x i c o l o g y s t u d i e s , m i c r o LC/MS can p r o v i d e d e t e c t i o n and mass s p e c t r a l i n f o r m a t i o n on t h e components o f i n t e r e s t .
I n f a c t , a t t h e p r e s e n t time, each o f t h e
v a r i o u s approaches t o LC/MS b e n e f i t from t h e reduced e l u e n t f l o w r a t e s f r o m micro LC and b e t t e r p r o v i d e low d e t e c t i o n l i m i t s from m i c r o LC/MS than any form o f s p l i t e f f l u e n t LC/MS. The major l i m i t a t i o n t o r o u t i n e o r f a c i l e m i c r o LC/MS so f a r has been t h e l a c k o f a commercially a v a i l a b l e m i c r o LC/MS i n t e r f a c e .
The s i t u a t i o n
i s changing now t h a t several companies a r e marketing t h e necessary HPLC hardware and a t l e a s t one mass spectrometer manufacturer ( r e f . 17) now provides a micro LC/MS diaphragm probe i n t e r f a c e .
Perhaps, as more chromato-
graphers become f r u s t r a t e d w i t h n o t r e a l l y knowing what t h e i r HPLC cliroinatographic peaks are, they w i l l implement m i c r o HPLC techniques i n t o t h e i r l a b o r a t o r i e s and g e t t o g e t h e r w i t h t h e i r f r i e n d l y mass s p e c t r o s c o p i s t . When these areas o f e x p e r t i s e combine toward a productive, p o s i t i v e goal, one can have m i c r o LC/MS as a unique and very powerful a n a l y t i c a l technique. REFERENCES P.J. Arpino and G. Guiochon, Anal. Chem., 51 (1979) 682A-701A. W.H. McFadden, J. Chromatogr. Sci., 18 (1980) 97-115. P.J. Arpino, TRAC, 1 (1982) 154-158. J.D. Henion, J.S. Nosanchuk and B.M. B i l d e r , J. Chromatogr., 213 (1981) 475-4813 . . - .- - R.E. Lovins, S.R. E l l i s , G.D. T o l b e r t and G.P. McKinney, Anal. Chem., 45 (1973) 1553-1556. R.P;W. Scott, C.G. Scott, M. Munroe and J. Hass, Jr., J. Chromatogr., 99 (1974) 395-405. W.H. McFadden and J.L. Schwartz, J. Chromatogr., 122 (1976) 389-396. R.P. Jones and S.K. Yang, Anal. Chem., 47 (1975) 1000-1003. E.C. Horning, D.I. C a r r o l l , I . D z i d i c , K.D. Haegele, M.S. Horning and R.N. S t i l l w e l l , J. Chromatogr., 99 (1974) 13-21. ~
274
10 11 12 13 14 15 1G 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
39 40 41 42 43 44 45 46
P.J. Arpino, M.A. Baldwin and F.W. M c L a f f e r t y , Biomed. Mass Spectrom., 1 (1974) 80-82. J.D. Henion, Anal. Chem., 50 (1978) 1687-1693. R.G. Christensen, H.S. Hertz, S. Meiselman and E. White, Anal. Chem., 53 (1981) 171-174. C.R. Blakely, J.J. Carmody and M.L. Vestal, J. h e r . Chem. SOC., 102 ( 1 980) 5931 -5933. J.L. DiCesare, M.W. Doug and J.G. Atwood, J. Chromatogr., 217 (1981) 369-386. Finnigan Mat, Sunnyvale, C a l i f o r n i a 94086. V.G. A n a l y t i c a l , A1 trincham, Cheshire, Great B r i t a i n . Hewlett-Packard Co., Palo A l t o , C a l i f o r n i a 94304. Nermag Co., Houston, Texas 77004. D.E. Games, P. H i r t e r , W. Kuhnz, E. Lewis, N.C.A. Weerasinghe and S.A. Westwood, J. Chromatogr., 203 (1981) 131-138. J.0. Henion and G.A. Maylin, Biomed. Mass Spectrom., 7 (1980) 115-121. J.D. Henion and T. Wachs, Anal. Chem., 53 (1981) 1963-1965. P.J. Arpino, B.F. Dawkins and F.W. McLafferty, J. Chromatogr. Sci., 12 (1974) 574-578. F.W. McLafferty, R. Knutt, R. Vankataraghavan, P. Arpino and B.F. Dawkins, Anal. Chem., 47 (1975) 1503-1505. JASCO, Inc., Easton, Maryland 21601, J.O. Henion, J. Chromatogr. Sci., 19 (1981) 57-64. Y . Y . L i n and L.L. Smith, Biomed. Mass Spectrom., 6 (1979) 15-18. E.A. Dewey, G.A. Maylin, J.G. Ebel and J.D. Henion, Drug Metab. Dispos., 9 (1981) 30-36. J.D. Henion, Proc. Int. Symp. Equine Med. C o n t r o l , 3 (1980) 133-140. K.H. Schafer and K. Levsen, J. Chromatogr., 206 (1981) 245-252. K. Levsen and K.H. Schafer, I n t . J. Mass Spectrom. I o n Phys., 46 (1983) 209-21 2. A. P. Bruins , personal communication. A.P. Bruins and B.F.H. Drenth, I n t . J. Mass Spectrom. I o n Phys., 46 (1983) 213-216. D.E. Games, M.S. Lant, S.A. Westwood and M.J. Cocksedge, Biomed. Mass Spectrom. , 9 ( 1 982) 21 5-224. M. Metzler, CRC C r i t . Rev. Biochem., 10 (1981) 171-212. J.D. Henion and G.A. Maylin, J. Anal. Toxicol., 4 (1980) 185-191. R.P.W. S c o t t and P. Kucera, J. Chromatogr., 169 (1979) 51-72. P. Kucera, J. Chromatogr., 198 (1980) 93-109. A. A l l t e c h Associates, Deerfield,, I l l i n o i s 60015. B. Chrompack, The Netherlands. C. CM Laboratories, Nutley, New Jersey 07110. D. Whatman, C l i f t o n , New Jersey 07014. J.J. Brophy, D. Nelson and M.K. Withers, I n t . J. Mass Spectrom. I o n Phys., 36 (1980) 205-212. C.E. Eckers, D.S. Skrabalak and J.D. Henion, C l i n . Chem., 28 (1982) 1882-1 886. C. Eckers, J.D. Henion, G.A. Maylin, D.S. Skrabalak, J. Vessman, A.M. T i v e r t and J.C. Greenfield, I n t . J. Mass Spectrom. I o n Phys., 46 (1983) 205-208. N.J. Alcock, L. C o r b e l l i , D.E. Games, M.S. Lant and S.A. Westwood, Biomed. Mass Soectrom.. 9 (1982) 499-506. S.A. Westwood,' D.E. Games,'M.S.'Lant and B.J. Woodhall, HPLC i n Pharmaceut. Anal., (1982) 121-123. D.E. Games, C. Eckers, M.S. Lant, E. Lewis, N.C.A. Weerlasinghe and S.A. Westwood, Recent Adv. Chromatogr. Mass Spectrom., (1982) 253-256. T. Takeuchi, 0. I s h i i , A. S a i t o and T. Ohki, J. High Resolut. Chromatogr. Chromatogr. Commun., 2 (1982) 91-92. R. Tijssen, J.P.A. Bleumen, A.L.C. S m i t and M.E. Van Kreveld, J. Chromatogr. , 218 (1983) 137-165.