Mass Spectrometry

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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

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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

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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

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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. ~

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39 40 41 42 43 44 45 46

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