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J o u r n a l o f A n a l y t i c a l T o x i c o l o g y , V o l. 2 1 , M a r c h / A p r i l 1 9 9 7

A p p l i c a t i o n s o f L i q u i d C h r o m a t o g r a p h y -M a s s S p e c t r o m e t r y n A n a l y t ic a l T o x i c o l o g y :A R e v ie wH a r a i d H o j a 1 , P i e r r e M a r q u e t 1 , ' , B e r n a r d V e r n e u i l 1 , H a y a t L o f f i 1 ,z , B e r n a r d P ~ n i c a u t 1 , a n d G ~ r a r d t a c b ~ t r e 1,~I Depa r tmen t o f Pha rm aco logy and Tox i co logy , Un i ve rs it y Hosp i ta l, 87042 L imoges , F rance and 2Labo ra to ry o f Tox i co logy ,Facu l t y o f Pha rmacy , 87025 L imoges , F rance

A b s t r a c tL i q u i d c h r o m a t o g r a p h y - m a s s s p e c t ro m e t r y ( L C - M S ) , a f te r lo n g -t e r m d e v e l o p m e n t th a t h a s in t r o d u c e d s e v e n m a j o r in t e r fa c i n gt e c h n i q u e s , is fi n a l ly s u i t a b l e f o r a p p l i c a t io n i n th e f ie l d o fa n a l y t ic a l t o x i c o l o g y . V a r i o u s c o m p o u n d c la s s e s c a n b e a n a l y z e d ,a n d s e n s i t i v it ie s f o r m o r e o r le s s p o l a r a n a l y t e s t h a t a r e a s g o o d a so r b e t te r t h a n t h o s e o f g a s c h r o m a t o g r a p h y - m a s s s p e c t ro m e t ryc a n b e o b t a i n e d w i th m o d e r n i n te r f a c e s . I n a d d i t io n , b e c a u s ei o n i za t io n is o f te n s o f t e r t h a n c l a s s ic a l e l e c t ro n i m p a c t , s o m eL C - M S i n te r f a c e s a r e a b l e t o h a n d l e f r a g i l e s p e c i e s t h a t a r eo t h e r w i s e n o t a m e n a b l e t o M S . T h i s r e v ie w is in t e n d e d t o p r e s e n tL C - M S t o le s s a m i li a r i z e d r e a d e r s a n d t o g i v e a n e x t e n s iv eo v e r v i e w o f t h e a p p l ic a t io n o f t h e d i f fe r e n t c o u p l in g t e c h n i q u e s t od o p i n g a g e n t s , d r u g s o f a b u s e , f o r e n s i c a n a l y s is , t o x i c c o m p o u n d so f v a r io u s n a t u r e , a n d s e v e r a l to x i c o l o g i c a l l y r e le v a n t t h e r a p e u t i cd r u g s . E x p e r i m e n t a l p a r a m e t e r s s u c h a s t h e i n te r f a c e s u s e d ,i o n i za t io n m e t h o d s , d e t e c t io n l im i ts , a n d e x p e r i m e n t a l d e t a il s f o re x e m p l a r y a p p l ic a t io n s a r e g i v e n .

I n t r o d u c t i o nT h e c o u p l i n g o f m a s s s p e c t r o m e t r y ( M S ) a s a d e t e c t o r t o

c h r o m a t o g r a p h i c s e p a r a t i o n s y s t e m s m a y b e t h e r e s p o n s e t oi d e n t i f i c a t i o n a n d q u a n t i t a t i o n p r o b l e m s o f t e n e n c o u n t e r e db y a n a l y ti c a l t o x i c o l o g i s t s . I f t h e s a m p l e m a t r i x i s c o m p l e x( p l a s m a , u r i n e ) , m u l t i p l e e x t r a c t i o n s t e p s a r e u s u a l l y r e q u i r e db e f o r e a n a l y s i s . I n s p i t e o f t h e q u a l i t y o f p u r i f i c a t i o n , i n t e r f e r -e n c e s t h a t m a k e p r o p e r i d e n t i f i c a t i o n a n d q u a n t i t a t i o n i m -p o s s i b l e m a y o c c u r e v e n w i t h d e t e c t o r s s u c h a s u l t r a v i o l e t ,f l u o r i m e t r ic , a n d e l e c t r o c h e m i c a l d e t e c t o r s w i t h l iq u i d c h r o -m a t o g r a p h y ( L C ) a n d e l e c t r o n c a p t u r e a n d n i t ro g e n - p h o s -p h o r u s d e t e c t o r s w i t h g a s c h r o m a t o g r a p h y ( G C ). M S d e t e c t o rsu s i n g e l e c t r o n i m p a c t ( E I ) i o n i z a t i o n o p e r a t e d i n t h e s c a nm o d e c a n p r o v i d e n e a r l y u n a m b i g u o u s s p e c t r a l i n f o r m a t i o na b o u t c o m p o u n d i d e n ti ty , a n d , w h e n o p e r a t e d i n t h e s e le c t e d* Address o which co rrespondence ho uld be sent: Dr. P. Marquet, Servicede Pharm acologieet Toxico log ie , CHRU Oupuytren, 2 Avenue du PasteurMartin-Luther-King, 87042 LimogesCedex, France.

i o n m o n i t o r i n g ( S I M ) m o d e , c a n a ll y e x c e l l e n t s e n s i t i v i ty w i t hh ig h sp ec i fi c it y .I n G C , t h e i n t e r f a c in g p r o b l e m s w i t h M S w e r e o v e r c o m e al o n g t i m e a g o . R e li ab le i n s t r u m e n t s t h a t a r e c u r r e n t l y p a r t o ft h e s t a n d a r d e q u i p m e n t o f t h e m o d e r n t o x ic o lo g i ca l l a b o r a t o r yare avai lab le .

I t i s w e l l - k n o w n t h a t n e a r l y 7 0 % o f e v e ry d a y s a m p l e s b e i n gt r e a t e d i n t o x i co l o g i c a l la b o r a t o r i e s c a n b e h a n d l e d b y L C .U n f o r t u n a t e l y , t h e s i t u a t i o n f o r L C c o u p l i n g i s d i f f e r e n tb e c a u s e L C - M S i n s t r u m e n t s a re f ar m o r e e x p e ns iv e t h a n t h e i rG C c o u n t e r p a r t s , w h i c h p a r t ia l ly e x p l a i n s w h y t h e y a r e u s e dl e s s f r e q u e n t l y . V a r i o u s i n t e r f a c e s h a v e b e e n c o n s t r u c t e d s i n c et h e e a r l y 1 9 7 0 s . E a c h i n t e r f a c e a p p l ie s a d i f f e r e n t t e c h n i q u e t oe l i m i n a t e t h e c h r o m a t o g r a p h i c s o l v e n t , w h i c h , i f i n t r o d u c e dd i r e c tl y to t h e h i g h - v a c u u m r e g io n o f a m a s s s p e c t r o m e t e r(1 0 5 T o rt ) , w o u ld su p p ly an ad d i t i o n a l g as l o ad o f u p t o s ev e ra lh u n d r e d l i te r s p e r r a i n . S e n s i ti v i t y o f LC - M S d e p e n d s b o t h o nt h e c o m p o u n d a n d o n t h e i n te r fa c e u s e d . In a w a y , L C - M S c a nb e s e e n a s a s t r a i g h t f o r w a r d a p p r o a c h f o r t h e a n a l y s i s o f p o l a ro r t h e r m o l a b i l e c o m p o u n d s b e c a u s e n o d e r i v a ti z a ti o n s t e p isr e q u i r e d a s i n G C - M S . H e a t , i f a p p l i e d , is h a r d l y s u f f i c i e n t f o ra n a l y t e d e g r a d a t io n . I t h a s a l so b e e n s h o w n t h a t r a p id h e a t i n gd o e s n o t n e c e s s a r il y l ea d t o c o m p o u n d d e c o m p o s i t i o n b e c a u s et h e k i n e t ic s f o r t h e c o m p e t i n g d e c o m p o s i t io n a n d i o n i z a t io np r o c e s s e s a r e o f t e n i n f a v o r o f t h e l a t t e r ( 1 ). T h e a b i l i t y o fs o m e L C - M S t e c h n i q u e s t o a s s e ss i n t a c t d r u g c o n j u g a t e s ( i .e .,g l u t a t h i o n e , g l u c u r o n o - , a n d su l f o - c o n j u g a t e s ), w h i c h isi m p o s s i b l e i n G C - M S , g r e a t l y f a c i l i t a t e s i d e n t i f i c a t i o n a n dq u a n t i t a t io n o f s u c h c o m p o u n d s , a v o i d i n g h y d r o l y s is s te p so f t e n a p p l i e d i n a n a l y t i c a l t o x ic o lo g y . S o m e L C - M S i n t e r f a c e sa r e e v e n s u i t a b l e f o r t h e a n a l y s i s o f p e p t i d e s a n d p r o t e i n s ,s o m e o f w h i c h a r e d r u g s , s o m e o f w h i c h a r e t o x ic .

T h e a i m s o f t h e p r e s e n t r e v ie w a r e a s f o ll o w s: t o c la r if y t h ei d e a s o f t h e p o t e n t i a l u s e r c o n c e r n i n g L C - M S i n te r f a c e s , t h e i rp o w e r s , a n d w e a k n e s s e s a n d t o c o m p r e h e n s i v e l y r ev ie w t h el i t e r a t u r e o f i ts a p p l i c a t i o n i n a n a l y t i c a l t o xi c ol o g y.

D e s c r i p t i o n o f i o n i z a t i o n m e th o d s a n d L C - M S i n t e r f a c e sI t i s b e y o n d t h e s c o p e o f t h i s a r ti c l e t o 'g i v e a n e x t e n s i v eo v e rv i ew o f t h e p ro ce s ses i n v o lv ed i n an a ly t e i o n i za t i o n . T h e re -

1 1 6 Reproduc t ion (photocopying) o f ed i tor ia l content o f th is journa l is proh ib i ted wi tho ut pub l isher 's permission.

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fore, the principle of eac h m eth od used in LC coupling wil lbriefly be ci ted, and m ore detailed inform ation may be foundelsewhere (2). A succ inc t description of the interfaces will alsobe ma de and m ore pro found information m ay be found ci ted inthe l i terature. The diffe rent types of mass analysis , however,wil l be presented in a m or e detai led m ann er because the ion-izat ion methods used in LC-M S and the chemical backg roundno ise o f the so lven t in the low-mass reg ion som et imes makethe use o f t andem mass spec t rometers (MS-MS) necessary .Ion iza tion occurs up on bom bardm ent wi th e lec t rons (E I),u p o n e x po su re t o p r i m a ry i o n s a t m e d i u m o r a t m o s p h e r i cp ressu re (chemica l ion iza t ion , CI) , upon b om bardm ent wi thaccelerated atom s (fast-atom bom bardm ent, FAB) or ions (sec-ondary ion mass spec t romet ry , SIMS), o r u pon desorp t ion o fions f rom charged d rop le t s ob ta ined by thermospray (TS) o relectrospray (ES) nebulization.T h e d e s o r p t i o n m e t h o d s l i k e E S , T S , a n d F A B a n datmo spheric-p ressure chem ical ionizat ion (APCI) are " so f t "ionizat ion modes, wh ich me ans tha t , in favorable cases , fragi lespec ies ( i. e ., thermo lab i le m olecu les and som e d rug -con ju -ga tes ) can be t rans fo rm ed in to ions wi thou t decom pos i t ionby these techniques. In addition, ES offers the possibility ofp ro d u c i n g m u l t i p l y c h a rg e d i o n s , wh i c h i s i n t e r e s t i n g i fbiomolecules are assessed.M o v i n g b e l t ( M B )The LC f low (1 -2 m L/m in) i s deposi ted on a be l t tha t t rans -p o r t s a s a m p l e s u c ce s s iv e l y th ro u g h a h e a t e d d e s o l v a ti o ncha m ber and a two-stage differential ly pum ped region intothe low-pressure ion source. Ionizat ion may be obtained bysevera l co mp lemen tary ion iza t ion modes : E I , CI , SIMS , o rFAB. If the identification of un kno wn c om poun ds is desired, EIis used. For high ly sensitive analyses, CI is preferable, whereasfor biomolecules , FAB is the m eth od of choice. The power o fMB lies in i ts abi l ity to ac com mo date EI as well as oth er typesof ionization. I ts inconveniences are bel t m em ory effects wh enh a n d l i n g h i g h ly a q u e o u s s o l ve n t s a n d d i s c r i m i n a t i o n o rdecom posit ion of low-volati li ty samples by application of hea tdu rin g the desolvation s tep (3-7).P art i c l e beam (P B )Th e ch romatograph ic so lven t (0 .6 -2 mL, depend ing on i tsvolatility) is nebulized at at m osp her ic pressure into a slightlyhea ted desolvatat ion ch am be r and is t ransferred into the low-pressure ion source thr ou gh a two-stage jet separator. Ioniza-t ion is obtained by EI, C I, or FAB. PB has the advantage ofaccom mod at ing EI , whe reas i t s m ajo r weakness i s the pos -sible discrimination of low-volati le samples in the jet-sepa-rat in g region (8 ,9).D i r e c t l iq u i d in t r o d u c t i o n ( D L I )Direct l iquid introdu ction rel ies on the direct nebulizat ion ofthe chromatographic effluent in to the source of the m ass spec-trom eter where solvent-induced CI occurs . The solvent is evac-ua ted by the vac uum pum ps . The h ighe s t so lven t f low ra teusable w ith MS is approximately 50 laL/min; herefore, the tech-niqu e needs ei ther m icrobore LC or p ostcolum n spl it t ing of thechrom atograp hic flow. Good sensi tiv i ties can be obtained, bu t

solvent spl i t t ing yields a c onc om itant loss of sensi t iv i ty due tothe mass-flow sensitivity of MS detectors (10-12).C o n t i n u o u s - f l o w F A B (C F - F A B ) , r i t- F A B , a n d S I M SA glycerol-containing solvent (5 laL/min) is in trod uced w ithan open-ended (CF-FAB)or frit-te rm inate d (frit-FAB) capillaryin to the low-pressu re ion source o f the m ass spec t rometer ,where i t is bombarded with accelerated atoms. The ions formedare extracted by an electrode and tran sferred to th e analyzer.The d i sadvan tages o f th i s t ech n ique are background- ion con-tributio ns of the matrix , whic h can interfere occasionally , andvery l imited solvent f low rates, w hich im pose a solven t spl i tbefore i ts in t roduc t ion to the ins t rum en t .SIMS can be performed with the same type of in terface w henusing ions instead o f atom s for analyte bom bar dm ent (13-15).T h e r m o s p r a yT h e s o l v e n t (u p t o 2 m L / m i n ) p a s s e s t h ro u g h a h e a t e dcapillary in whic h i t is almo st completely volat il ized. The n i t isnebulized into an expansion cham ber where ionizat ion takesplace at low pressure, induce d by the solv ent buffer, by a fi la-m en t , or by a discharge electrode. After com plete desolvat ion,the analyte ions are t ransferred to MS by mea ns of a repellerelectrode. Ioniza t ion corresponds to solvent-induced CI in theth ree mode s tha t d i f fe r only wi th respec t to in tens i ty and tofragmentation pat tern . F ragm entat ion can also be enha nced byapplying hig her voltages to the repel ler electrode an d acceler-a t ing the desolvated ions , wh ich can g ive f ragmen ta t ion uponcoll is ion with residual so lvent molec ules (16-19).E lec trospray

A f low ra te o f 50 laL /m in o f so lven t i s nebu l ized bo th byhigh voltage applied to a s i l ica capil lary and by pneumaticassis tance. The ionizat ion process takes place under atmo-spheric pressure in the l iquid phase. After desolvat ion, ion-solvent molec ule clusters are destroyed thro ug h accelerat ionin the t rans i t ion reg ion be tween a tmospher ic p ressu re andvacuu m and ass is ted in some ins t rum en ts by a coun tercu rren ts tream of ni trogen . The accelerat ion of ions in th is t ran si t ionreg ion m ay be su ff ic ien t ly energe t ic to induce f ragm enta t ionu p o n i o n - m o l e c u l e c o l li s io n s . T h e n e e d f o r p o s t c o l u m nspli t t ing does not decrease sensi t iv i ty because the responseof the system is concentrat ion-sensi t ive and n ot m ass-flow-sensitive (20-23).A t m o s p h e r i c- p r e ss u r e c h e m i c a l io n i z a t i o nS o l v e n t n e b u l iz a t i o n (u p t o 2 m L / m i n ) is p e r fo rm e d a ta tmosph er ic p ressu re by a hea ted nebu l izer wi th pn eum at icassistance. A discharge electrode ionizes solve nt molecules ,wh ich, after several ion m olecule reactions, t ransfer a ch argeto the ana ly te m olecu les . Fragm enta t ion can be induced inthe same m anne r as fo r ES (24) .

M a s s A n a ly s i sThe ions ob ta ined un dergo mass ana lys is in appropr ia temass fi l ters . Quadrupole m ass fi lters are use d m ost frequently ,

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ion t raps were implem ented recen tly , and sec to r in s t rum en tscan be acc om mo dated by some interfaces . In general , i f h ighmass resolut ion is not necessary, quadrupoles are preferredbecause of their ruggedness . Th e fil ters ca n be opera ted byscanning a specified mass range (scan mode) or by scanningselected masses (SIM). A mo re th an 10-fold increase in sensi-t iv i ty i s ob ta ined in SIM exper ime n ts com pared wi th fu l lscann ing . Coup l ing o f two mass ana lyzers (MS-MS) g ivesadditional opera t ion features , w hich are part icularly valuablefor the identification of unknow ns or for screenin g purposes.An iner t t a rge t gas , wh ich i s in t roduced between the tw o massfi lters, e nhan ces fragm entat ion. This operat ion is cal led col l i-s ion-indu ced dissociat ion (CID).

Fou r types of experim ents are possible:9 Daug hter-ion scanning: A parent-ion mass is selected in thefirs t mass fi l ter and CID fragm ents are col lected by scan-ning th e seco nd one. Identification of unk now ns is easierbecause the ions observed can on ly be i s sued f rom thepare nt ion selected.9 Paren t - ion scann ing : A daugh ter - ion mass i s f ixed in thesecond m ass filter while the first one is scanned. Ion s givingf ragments a t masses co rrespond ing to the one se lec ted inthe second m ass fi lter g ive a s ignal, whereas others d o not .In th i s way , paren t ions o r f ragmen ta t ion pa thways can beidentified easily.9 Cons tant neutral-loss scann ing: The two mass fi l ters ares c a n n e d b y m a i n t a i n i n g a g i v e n m a s s d i f fe r e n c e wh i c hcorresponds to the loss o f neu t ra l spec ies f rom the paren tion. This operat ion is used w hen sc anning for specific drugconjugates.9 Selected-reaction m onito ring (SRM), the M S-M S equiva-

lent of SIM: Only ions which c orrespo nd to a specific frag-me n ta t ion rou te a re m oni to red . Th is type o f exper imen tenha nces select ivity and sensi t iv i ty of analysis in the samem ann er as SIM in a single analyzer m ass spectrometer.

T o x i c o l o g i c a l A p p l i c a t i o n sCriteria for choos ing the articles were as follows: he articlesdeal t with 1 . applicat ions of LC-MS to drugs an d toxic com-pounds tha t were found in h um an o r an im al b iological f lu ids

or in biological matrices that are norm ally absorbed by hum ansand 2. a few comparisons of two or more interfaces for theassessm ent of toxicologically relevant dru gs or toxins w hichwere inc luded even i f pu re com pound s were s tud ied .D o p i n g a g e n t s a n d d r u g s o f a b u s e

This sect ion com prises s teroids, anabolics , d iure t ic drugs,c a rb a m a t e d o p i n g a g e n t s, n o n s t e ro i d a l a n t i - i n f la m m a t o rydrug s (NS AIDs), opiates , an d analgesics. Applicat ions w ithdoping agents in biological f lu ids of animals were includedbecause the extraction procedures re m ain the sam e regardlessof the species and can easi ly be adapted for the assess me nt ofthese co mp ounds in b iological f lu ids o f hum ans .Steroids . MB was used to quan t i t a te p roges terone in hu m anserum wi th CI , SIM detec tion, and deu tera ted p roges terone as118

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i n t e r n a l s t a n d a r d . T h e d e t e c t i o n l i m i t w a s r a t h e r h i g h(4 ng/m L or 200 pg injected) (25). PB, also with CI and SIMdetec t ion , a l lowed the quan t i t a t ion o f bec lom ethasone andi ts der iva t ives in hu ma n se rum w i th a quan t i t a t ion l im i t forlong- t ime ro u t ine use vo lun tar i ly f ixed a t 1 ng /mL (120 pginjected) (26).ES and APCI were app l ied fo r methandros teno lone and i t smetaboli tes in eq uine urin e. APCI was more sui table for neu-t ra l compounds , and ES, wh ich works bes t fo r compoundsthat are ionic in s olut ion , was used for the sulfo-conjugatedmetabolite. As both tec hniq ues gave few fragm ent ions, frag-me ntat io n had to be in duc ed by CID (27). Boidenone an d fiverelated s teroid su lfo-conjug ates we re assessed in equine urineby negative ionization ES coupled to MS-M S. Using s implemass analysis only, detec t ion l im its were 3--4 ng and 100-350pg injected for ful l scan a nd SIM of the molec ular ion, respec-t ively . With M S-MS, th e selectivity for confirmation an d quan-t i tat ion increased. Full-scan d aug hter ions could be obtainedfo r 5 -10 ng in jec ted , and SRM of two f ragm ent ions gavedetec t ion l imi t s o f 110-370 pg onco lumn. Wi th th i s experi -m e n t a l s e t u p , t h e s u l fo -c o n j u g a t e o f b o l d e n o n e c o u l d b edetected up to 17 days postdos e (28).

Other autho rs , who use d TS for confirmation of a num be r ofcort icosteroids in eq uine urine, careful ly optim ized solventcom position to en hance ionization efficiency.Approximately10ng of predn isone or de xam ethaso ne injected was sufficient fora ful l-scan thermospray spectrum in the posi t ive ion mode.Negative ionization wa s slightly m ore sensitive but lacked frag-me n ta t ion (29) . The afo re me n t ioned resu l ts a re cons i s ten tw ith those obtained for the assessmen t of 10 corticosteroids inhum an u r ine by TS. Detec t ion l imi t s ranged f rom 10 to 50 nginjected for full scan spec tra and ranged from 1 to 5 ng withSIM (3O).

Other app l ica t ions used DLI fo r severa l co r t i cos te ro ids(31,32), betame thasone (33,34), and diethylstilbestrol (34 ) andused TS (35) and AP CI (36) for t renbolone.~ -agon i s t s . [3-agonists were assayed by TS, ES, and APCI.W hen using SIM dete ct ion, clenbuterol could be assessed inhum an p lasma a t a de tec t ion l imi t o f 0 .1 ng on-co lum n byAPCI (37) . Com par ing GC-MS, TS, and ES fo r the de termina-t ion of five ~-agonists , an equivalent sensi t iv i ty for G C-MSand E S (100 pg and I ng by SIM and scan m ode , respect ively)was obtained. Both m etho ds gave 50-fold lower detection l imitsthan TS (38).Diure t i c s . DLI and TS w ere used fo r the de terminat ion o fdiuret ic drugs. The DLI applicat ion attained a detect ion l imito f approximate ly 1 ng in jec ted fo r t r i ch lo rmeth iaz ide on alabora to ry -bu i l t dev ice w i th microbore LC as a separa t ionsystem (39). TS was used for confirmation of 12 diuret ics andthe u r icosur ic p robenecide in human u r ine , and n ine wereidentified in real-life extracts. Use of gradient elution w ith TSlead to instabil i ty in ioniz at ion efficiency, herefore th ree iso-crat ic systems were applied. Scans w ere obtained for 50 nginjected for al l but th ree com pou nds, which was sufficientlylow for valuable confirmation (40). Benzthiazide, a diuret ictha t was no t am enab le to GC-MS by rou t ine der iva tizat ionmethods , cou ld be conf i rme d by TS af ter op t imiza t ion o f theLC solvent in order to enh anc e ionizat ion (41).

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J o u r n a l o f A n a l y t ic a l T o x i c o l o g y , V o l . 2 1 , M a r c h / A p r il 1 9 9 7

Carbarnate. A screening procedu re for urinary m etabolites ofmesocarb , wh ich i s a n on the rapeu t ic dop ing agen t o f the car-bamate fami ly , u sed PB w i th E I fo r conf i rm at ion o f the in takeof the paren t d rug . An LC-M S method was necessary as thedru g was no t amenable to GC (42).NSAIDs. PB using thre e ionizat ion m odes and ES were com -pare d as confirmation al devices for f lunixine in eq uine urin e.The de tec t ion l imi t fo r PB was 10 ng /mL, whereas ES w asm ore sensi tive (va lue no t g iven) bu t needed CID to enhancefragm entat ion . A lis t of 40 N SAIDs giving the intensi ty of theresponse for the three PB ionization modes and the thre e m ostabunda n t ions was inc luded (43) .Drugs o f abuse. TS was found sui table for the qu anti tat ion ofmorph ine and codeine in hu m an u r ine in a s tudy on s tandardreference m aterial . The resul ts obtained were in good agree-m en t wi th GC-MS and d i rec t-p robe MS-MS. The de tec t ionl imi t fo r TS was es t imated to be 2 ng /mL (44) . Other d rugs o fabuse, inc luding methad one, dextropropoxyphene, and dextro-moram ide, gave excellent sensitivities in whole blood with TSand SRM. Detect ion l im its were 10-100 pg for MS-M S (45).LSD was detected in uri ne w ith a de tect ion l imit of 200 pg forDLI with SIM detect ion using a coupled-column tec hnique andhea rt-cut t ing of the peak of in terest (46). Other applicationsused CF-FABfor dex t romethorphan wi th a de terminat ion l imi tof 110 ng/mL, whereas the plasma conc entrat ions could be aslow as 10 ng /mL (47) and TS fo r metham phetam ine (48).I d e n t if ic a t io n o f u n k n o w n s a n d s c r e e n in g p r o c e d u r e s inf o r e n s i c a n a l y s isMS coupling becomes part icularly valuable whe n unkn ow ncom pounds are to be iden t if ied , p rov ided tha t the spec t rumgives enough informative fragments for in terpretat ion. Theapplicat ions found on the subject u se DLI, MB, PB, frit -FAB,TS, ES , and APC I. Use of EI spectra is advantageou s because ofthe possibility for library searching, alth oug h molecular weightin fo rmat ion may be absen t . Th is i s no t a majo r d rawbackbecause the interfaces using EI are al l able to handle CI, whichmos t o f ten p rov ides molecu lar weigh t in fo rmat ion . In th i scase, quan t i t a t ion i s perfo rm ed wi th CI and SIM detec t ionbecause spe ctra are s impler, w hich gives less rise to in terfer-e n c e f ro m o t h e r c o m p o u n d s .Using MB with EI a nd posi tive CI al lowed the identificationof unknown com pounds , wh ich were found dur ing a screen ingp ro c e d u re fo r b e n z o d i a z e p in e s in p o s t m o r t e m p l a s m a a n durine , as the pest icide diur on and four of its metabolites . C on-firmation of co mp ound identity was real ized by high resolut ionm e a s u re m e n t s on a s e c t o r i n s t ru m e n t i n o rd e r t o m e a s u rethe exac t molecu lar weigh t . The de tec t ion limi t was 50-80 ngfor a scan. LC-M S was, in th is case, superior to GC-M S becauset h e c o m p o u n d was therm olabile (49,50). The sam e group usedMB with CI for the quan ti tat ion of clopenthixol , bromazepam,a n d r e s e r p in e i n s e r u m t a k e n f r o m a n i n d iv i d u a l w h oat tempted su ic ide . D etec t ion l imi t s by SIM ranged f rom 35to 100 pg . The p resence o f the com pounds in serum , excepttha t o f reserp ine w hose co ncen t ra t ion was too low, cou ld bec o n f irm e d wi t h a c c u ra te m a s s m e a s u re m e n t s w i t h a s e c t o rins t rumen t (51) .Electrospray M S-MS was used to identify and to qua nti tate

a toxic conta min ation in a dexamethasone-injectable solution.Analys i s was qu i te demand ing because the compound suc-cinylcholine c ould no t be separated f rom the adjuvants of thedrug fo rmu la t ion because o f i ts hydroph i l i c na tu re . For th i sreason, i t was overlooked in routin e screen ing procedures (52).A routine AP CI screenin g procedure for 21 organophosphor-ous pe sticides in blood was deve loped for cases of intoxicationby agrochemicals . After optimizing mobile phase com posit ionto enhanc e sens it iv i ty , de tec t ion l imi t s ranged f rom 200 to1000 ng fo r scann ing and f rom 2 to 50 ng fo r SIM, which wasonly slightly inferior to GC -MS . Positive or ne gative ionizationwas used , depend ing on the comp ound , and two o r th ree con-f i r m a t o r y f r a g m e n t i o n s c o u l d b e o b t a i n e d f o r e a c hc o m p o u n d (53 ).The pes t ic ide e thy lene th iourea was de tec ted in u r ine downto 0 .1 ng injected with TS and SIM of the protonated molecularion (54) . A thermospra y screen ing me thod fo r 21 benzod i-azepines used the combined information of retent ion t imes andisotopic ratios , which are related to the presence of halogen, toidentify posit ive samples . A lthough peaks were not completelyreso lved when acqu i r ing the to ta l ion cu rre n t (T IC) , he i r res-o lu t ion cou ld be ob ta ined wi th the se lec ted ion channels (55) .F r it -F AB wi t h m i c ro -L C u s i n g a n o n l i n e c o n c e n t r a t i o ncolu m n was applied to several forensic applicat ions. Full scansof benzodiazepines, cephalosporine ant ibiotics , a nd ph eno thi-az ines cou ld be ob ta ined wi th de tec t ion l imi t s o f 4 -10 ng /mL,10-100 ng , and 0 .25-10 ng on c olum n, respect ively (56).A DLI source w as used to d etect metabolites of the explosive2 ,4 ,6 - t rin i t ro to luene in hum an u r ine wi th an SIM detec t ionl imi t o f 0 .1 n g /mL (57) , and exp los ives and the i r mix tu rescould be identified in 1 to 10 I~g of sample (58).

ES was used fo r penic il lin s and cephem s in serum wi th ade tec t ion l imi t o f 2 -15 ng fo r scann ing (59) . TS was used fo rthermolab i le benzod iazep ines in who le b lood wi th on co lum ndetec t ion l imi t s o f 10 -200 pg fo r M S-MS and SRM (60) , fo rcyc lospor ins in who le b lood (61) , and fo r t r imethy l lead inurine (62).P e s t ic id e s a n d t o x i n s in n o n h u m a n s a m p le s

This sec t ion dea l s wi th th e assessm en t o f compou nds suchas mycotoxins, marin e toxins, and pest icides m ostly in anim aland vegetable samples . The interfaces used w ere MB, PB, CF-FAB, ES, an d T S.

A TS m ethod was app lied for the de tec t ion o f th ree myco-toxins and so me of their m etaboli tes , includ ing a glucu ronideconjugate, in excretion sam ples from rats, hens, an d cows. Pos-itive filament-on ionization was preferred to negative ionizationbecause o f a mo re a bundan t f ragmenta t ion pa t te rn . Detect ionl imi t s ranged f rom less than 1 ng to 20 ng and f rom 50 to 1000pg by scan and SIM detect ion, respect ively (63). In a noth ers tudy us ing TS, seven m yco tox ins were de term ined in wh eatsamples . Fragmentat ion could be enhanced or suppressed whenvarying the buffer conce ntrat ion. Very low concen trat ions andfi lament-enhanced ionizat ion yielded fragments for three ofthe com pounds , whereas h igher conce n t ra t ions favored theprotona ted molecular ion a nd low ered detect ion l imits. Overal ldetect ion l imits by SIM were 75-1000 pg, and cal ibrat ion w asl inear in th e ran ge of 3-40 ppb (ng/g) (64).

1 1 9

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Marine toxins cause severe cases of in toxication that can belethal for fragi le subjects . D omoic acid , a neurotox ic aminoacid, was c onfirm ed in shel lfish with a laboratory-modified ESusing SIM detect ion of three characteris tic ions down to 0 .1tJg/g . An optimize d extract ion proce dure from rat p lasma a ndfeces gave de tec t ion l imi t s in th e low p icogram range (65).CF-FAB and SIM detec t ion in con junc t ion wi th an open-borefused-si l ica col um n were use d for the detect ion o f saxi toxin inhum an u r ine . ES, wh ich was a lso tes ted by the au thors , gave a20-fold lower detect ion l imit (66).C o n f i rm a t i o n o f t h e p re s e n c e o f 1 7 o rg a n o p h o s p h o ro u spest icides and m etaboli tes in beef musc le could be obtained byTS discharge-on negative ionizat ion and SIM detect ion. Allbut fo ur could be detected at the i ppm level or lower. Identi-f ica tion was ach ieved by m oni to r ing o f the f ive mos t ab undan tions. In order to increase select ivity , the reten t ion t im es andthe ion in tens i ty rat ios w ere com pared wi th those o f s tandardcom pound s (67) . A num ber o f comp ounds , inc lud ing ant ib i -ot ics , were evaluated for their sui tabi l ity for PB with E I and CIand for TS. PB with C I gave detec t ion l imits of 100 ng for al-m ost al l of the compounds, whereas TS de tect ion limits rangedfrom 10 to 400 ng fo r full-scan m ass spectra. The interfaceswere also com pared for the ir sensi tiv i ty and specifici ty for thedeterm ination of cephalosporins in m ilk and me thylene blue inmu scle extracts. PB w ith CI was preferred to TS because SIMdetect ion of five ions was possible w ith PB and CI (68). APCI,ES, TS, and PB were used for carbamate pest icides in pepper.APCI gav e excel lent de tect ion l imits; ES and T S perform edwell . PB was not sensit ive enou gh (69).Other applications used MB for pesticides and alkaloids (70).ES was used for the shel lfish toxins okadoic acid and dinoph -ysistoxin-1 (71) and for penicillins in bov ine m ilk (72,73), andCF-FAB was use d for tetracycline antibiotics in ho ney (74).T h e r a p e u t i c drugsIn this section, the assessmen t of drugs in biological fluids willbe discussed. In applications dealing with the identification andquantitat ion of known compounds, the use of SIM or SRM willoften be encountered. LC-M S methods are often used during theearly stages of drug developm ent because they are able to providerel iable pharmacological data with minim al m ethod-develop-m ent t im e (75). As mos t of the analyses have been performedroutinely for large sample series, deuterated internal standardswere used frequently in order to minim ize intra-assay varia-tions. For studies in wh ich the elucidation of metabolites was de-sired, M S-MS was used almo st systematically .Drugs for human usage. In a s tudy on the sui tabi l i ty ofAPCI, var ious c om poun d c lasses w ere inves t iga ted by f lowinject ion analysis (FIA).As an exam ple of the sensi tiv i ty of theinterface, serum containing theophyll ine at the level of i tsde tec t ion l imi t (5 pg) was in jec ted on co lumn. Compoundclasses and the min imal de tec tab le quan t i ty ob ta ined wi thAPCI and SIM detect ion were as fol lows: amin es, free am inoa c i d s, p h e n y l h y d a n t o i n a m i n o a c i d s , s t e ro i d s , v i t a m i n s ,alkaloids (10-100 p g), peptides with mo lecular weights greaterthan 1000 Da (100 pg-20 0 ng), nucleosides and nucleotide s(100 pg-50 ng) , b i le ac ids (10 ng) , and sugars and l ip ids(50-200 ng) (76).120

Journal of An aly t i ca l Tox icology , Vol . 21, Ma rch/Apr i l 1997

TS was used fo r the assay o f compoun ds o f neurochem ica linterest com pris ing 19 ind olic acids , t r icycl ic ant idepressants,and catecholamines. T he indolic com poun ds gave a detec t ionlimit of 50-100 pg by FIA and SIM. In addit ion, extract ionf ro m u r i n e w a s p e r fo rm e d s u c c e s s ful ly fo r f iv e o f t h e m .Detect ion l imits were 10 pg for the t r icycl ic ant idepressa ntsimipram ine and c lomipram ine . For the p revious ly men t ionedcom pou nd classes, sensi tiv i ty , except for catecholamines, wasbetter w ith TS than w ith G C-MS, an d no derivatization s tepwas needed for the LC metho d. The authors observed tha t forconcen t ra t ions near the de tec t ion l imi t , add i t iona l samplecleanup would be necessary because coeluting endogenous com-pounds com pete fo r ion iza t ion and are ab le to mask , to someextent, the ionization of the analyte (77). Butyrophen one tra n-quilizers were quanti tated in whole blood with TS and MS-M S.The SRM detect ion l imit for haloperidol was approximately2 5 0 p g / m L ( 4 5 ) . T h e a n t i a s t h m a t i c d r u g s b a m b u t e r o l ,t e rbu ta l ine , and b ude son id e were assayed by TS and SIMdetec t ion us ing deu tera ted in te rna l s tandards , wh ich gaveexcellent l inearity of cal ibrat ion graph s for gradient e lut ions.The quanti tation l imit was 0 .1 ng/mL for plasma extracts of thecorticosteroid budeson ide (78). TS positive ionization was usedto m easure the ena ntiom ers of terbutal ine, a [~-stimulant witho n l y o n e p h a rm a c o l o g i c a l l y a c ti ve e n a n t i o m e r , i n h u m a nplasma usin g the de uterate d analogue as an internal s tandard.Quan t i t a t ion was perfo rmed on the racemic m ix tu re , whereasthe enantiomeric rat io was determ ined using a coupled colu mntechn ique with a chiral colu m n. Because no chiral separationwas performed on the fi rs t column, hear t cut t ing of the peak ofthe racemates provided information about the enantiomericrat io at each m om ent, regardless of the w idth of the fract ion.Using this ins trum ental setu p, l inear cal ibrat ion w as obtaineddown to 4 pm ol (approximately 1 rig) (79). The quaternarya m m o n i u m s t e ro i d s p a n c u r o n i u m a nd v e c u r o n i u m w e redetermined in p lasma and u r ine us ing MB coup led to a sec to rinstr um ent (80). Other au thor s used ES for the sensit ive me a-s u r e m e n t o f s t e r o id s a n d t h e ir g l u c u r o n i d e s i n u r in eand reached a detec t ion l imit of 15 pg, which was bet ter thanGC-MS (81) . Sum at r ip tan , a d rug used fo r the t rea tm en t o fmigra ine headaches, was determ ined in plasma by TS, posi t iveionizat ion, and SIM. The u se of a homo logue of the drug as aninternal s tand ard led to hig h intra- and interassay variations;therefore, a labeled com po und was preferred, w hich s ignifi-cantly improved results for linearity, precision, and accuracy.The rou t ine q uan t i t a t ion l im i t was se t a t 2 ng /mL, a l thoughcalibrat ion graphs were l inear dow n to 500 pg/m L (75). Thesame group assessed the ant ihypertensive drug labetalol inplasma by TS positive ionization an d SIM detection. W ith adeuterated internal s tandard , the detect ion l imit was 5 ng/mL,and th e l inearity ranged f rom 10 to 100 ng/mL (82). For phar-ma cok in e t ic s tud ies , abanoqu i l , a low-dosage % -adre no-recep tor antagonist , was assessed in whole blood by APCI witha deuterated internal s tandard. Sensi t iv i ty with MS-MS andSRM for the p aren t to da ugh ter t ransi t ion was excel lent w itha l inear cal ibration range fro m 10 to 500 pg/m L (83). Anthra-cycline ant ibiot ics used in cancer c hem othera py were investi-gated by PB with EI and CI as well as with TS in order to obtainstructu ral info rma tion and to determ ine the sui tabi l ity o f the

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Journal of Analy t ica l Tox icology , Vol . 21, March/Ap r i l 1997

LC-M S techniques. B oth LC-M S interfaces were foun d fit forthe analysis of the d rugs , bu t PB was preferred because i t pro-v ided mo re abundan t f ragm ent ions , though i t l acked sensi -t ivi ty compared with th e othe r m ethod s (84). Tacrol imus (for-merly FK-506) and som e of i ts metaboli tes were determ ined inwhole blood using PB negative CI. For qu anti tat ion purposes,the mon oacetylated derivative was used as an internal standa rd.Other cl in ically relevant drugs were checked and fou nd not tointerfere w ith the assay. The LC -M S m ethod was also cross-val idated with an enzyme immunoassay for the drug and i tsmetabolites in plasma, b ut n o s ignificant correlat ion could beobtained because of crossreactivi ty that occu rred in the lat terme thod (85). A new derivative of the fentanyl group, m irfen-tanil, whic h is actually un de r clinical investigation, was dete r-min ed in plasma by TS posi t ive ionization and SIM detect ion.Detect ion and quanti tat ion l imits were 0 .1 and 0 .4 ng/mL,respect ively , with a deuterated internal s tandard (86). Anti-malarial Cinchona alkaloids issued from cel l cul tures wereassayed with TS. FIA yielded a detect ion l imit of 10 pg forquinidine when m onito ring the protonated molecular ion. Theauthors concluded that for the analysis of complex mixturesand accurate quan t i t a t ion , the use o f MS-MS and deu tera tedinternal s tandards would be necessary (87). The an tihis tam inedrug triprol idine a nd one of i ts m etaboli tes were investigatedby TS. The est imated detect ion l im it for the paren t drug w as 10pg when us ing SIM detec t ion (88) .A synthet ic hetraze pine derivative with platelet-act ivat ingfactor (PAF) antag onizin g propert ies w as determ ined by PBnegative ion CI with m etha ne as a reagent gas yielding a quan-t i tat ion l im it of 0 .1 ng/m L (89). The performances of ES andAPCI were i l lustrated for the detect ion of a PAF antag onistand a m etalloproteinase inhibi tor with de tect ion l imits of 100pg/m L (90). In othe r applicat ions, TS was used for the deter-minat ion o f m or ic iz ine in p lasma w i th a quan t i t a t ion l imi t o f10 ng /mL (91) . TS and PB were used fo r coumarin an t icoagu-lant drug s in plasma (92 ), and ES w as used for the pentapep-t ide d rug IRI-514 wi th a quan t i t a t ion l imi t o f 2 ng /mL (93).Drugs for veterinary usage. A TS in ter face was used toconf i rm t r imethop r im in bov ine serum by SIM of the p ro to -nated molecular ion (9 4). The benzimidazole ant ihe lmin ticsoxbendazole and fenbendazole w ere qu anti tated in l iver andmu scle of sheep usin g TS and SIM detect ion. Detect ion l imitsfo r oxbendazo le and fenbend azo le were 50 and 100 ng /g ,respectively (95). An othe r application used APC I for m edeto -midin e, an analgesic sedat ive (96).M e t a b o l ic s t u d ie sFor th is sect ion, l i terature was mos t abunda nt because MSanalysis can easi ly reveal impor tant informat ion wi th regard tome tabol ic pathways. Fo r the sake of completeness and becausethe nu mb er of appl icat ions is imp or tant , on ly c i tat ions wi l l bemade.The ionizat ion ef f ic iencies of APC I , ES, and TS as a funct ionof hydropho bic i ty were com pared for the drugs pravastat inand i ts m etabol ites and for two other d rugs using posi t ive andnegat ive ionizat ion. R esul ts showed that AP CI w i th posi t iveionizat ion was the mo st sensi t ive me thod for nonp olar com-pound s whereas polar com poun ds were less sui table; the b est

sensit iv ity with TS for hydrophobic com pou nds was obtaine dw ith negative ionization; an d ES was very sensitive reg ardlesso f compo und po lar ity and ion iza tion mode and w as the mo s tsensit ive interface for hydrophil ic com pou nds (97).Thermospray was used fo r the iden t if ica tion o f an u nkno wnurinary metaboli te of heptabarbi tal (98), for the ant iepi lept icdru g lam otrigine (99), for glucuron ide metaboli tes of doxyl-am ine (100), for SK &F 101468, a low-dosage dopamine-d2 re-cepto r agonist (101), for haloperidol (102), for the u ricosur icd rug be nzbrom arone (103) , fo r n ico t ine and i ts g lucuro -con -jugates (104) , fo r metabo l i t es o f t em elas t ine S K& F 93944(105), for chlorpromazineoN-oxide, an ant ipsyc hotic agen t ofthe phen othiaz ine family (106), for the DNA addu cts of thean t icancer d rugs mi tomycin C, po rf i romycin , and th io tepa(107) , fo r ace taminophen con jugates (108) , fo r coumarin icanticoa gulan ts (109), for terfenadine (110), and for ampici l l inantibiotics (111).ES was used for N-0923, a hydroxytetral in ena ntiom er withpotent dopamine-d2 receptor agonizing act ivi ty (112); for S12813, a drug with analgesic activity (113); for clozapine N glucu ronid e (114); and for vi tamin D3 metaboli tes (115).CF-FAB was u sed for vi tam in D3 metabolites (116) and foramp icillin antibiotics (111 ); PB with EI was used fo r oxodipine,a new calcium agonist (117); DLI was used for a glucurono-con jugate o f f lu razepam (118) ; and MB was used fo r thesyn theti c corticosteroid budesonide (119).

D i s c u s s i o n

The principle functions o f the seven LC-M S interfaces whichwere o r a re co mm only used have b r ie f ly been p resen ted , andt h e i r r e s p e c t i v e a d v a n t a g e s a n d d i s a d v a n t a g e s h a v e b e e npo in ted ou t . The app l ica t ions o f these in te r faces cover ingvarious fields of toxicological analysis have been extensivelyreviewed.Because o f the i r m ore o r l es s po lar na tu re s , a l l o f thecom pou nds were well-suited l iquid chrom atogra phy analytes .T h e a p p l i c a t i o n s h a v e b e e n g ro u p e d fo l lo wi n g p re v io u s l ydefined toxicological f ields; a discussion ab out the intere st ofthe interfaces with respect to compound classes may give aclearer un ders tandin g of their respective performance.

Direct l iquid intro duc tion (DLI), with w hich MS coup lingwas real ized for the fi rs t t ime, was rarely applied al thoughgood l imits of detect ion could be reached for the analysis ofe x p l o s i v e s a n d , w i t h a n a p p ro p r i a t e e x p e r i m e n t a l s e t u p ,for LSD.Applications with the mo ving bel t (MB) were also rare. I tssensitivity for steroids was mediocre, wherea s benzo diazepineswere detected more sensi t ively . The major advantage of theinterface, which is its potential to obtain library-searchableEI spectra, was only used in confirmational s tudies for rela-t ively high conc entrat io ns because i ts sensi tiv i ty w as poore rthan th a t o f CI.App l ica t ions us ing par t i c le beam (PB) were foun d m oreoften. Sensi tiv i ties for s teroids and NSAID s were good, andthose for antibiot ics were mediocre. The ins tru m en t was not

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con ven ient for the analysis of pesticides . Just as with MB, EIionizat ion w as not used wh en sensi t iv i ty was desired.Fas t a tom bom bardm ent was used fo r the ana lys is o f ben-zodiazepines, ant ibiot ics , and p heno thiazine s w ith very goodperform ance because of an experimental setup tha t used on-l ine co lumn p reconcen t ra t ion . However , dex t romethorphancou ld no t be quan t i t a ted in p lasma us ing FAB because i t squan t i t a t ion l imi t was too h igh .Thermospray was the in te r face tha t w as app lied mo s t o f tenand th at w as used in nearly al l f ields . I ts perform ances wereequivalent to or bet ter than the previously discussed inter-faces. High sen sitivities could be obtained for steroids, drug s ofabuse, th iourea pest icides , mycotoxins, indolic compounds,and tricyclic antidepressants. Its application range includedma ny therape utic drugs and metabolic s tudies .E lec t rosp ray i s a re la t ive ly new in ter face tha t has beenapplied for steroids, diuretics, NSA IDs, carbam ate pesticides,and ma rine toxins . Limits of detection w ere at least of the sameorder as those o f TS. Further, ES was som etim es used for thedeterm ination of therap eutic drugs and for m etabolic s tudies.Atm ospheric pressure chemical ion izat ion gave low detec-tion limits for steroids, 13-agonists,carbamate pesticides, alka-lo ids , and v i tamins . A com par i son wi th ES and TS showedtha t APCI was the m ost sensi tive interface for pesticides and13-agonists.This overview shows th at DLI, MB, PB, and FAB have fo undrestricted applicat ion in analyt ical toxicology, wh ereas APC Iand E S were used m ore often and TS was used very frequently .Good sensit ivi t ies could be obtained w ith al l of them , providedthe com poun d chosen ionizes wel l . Valuab le conclus ions wi thregard to th e sui tabi l ity of each interface can not be drawn ifone compares only their respect ive number of applicat ionsbecause some o f them have been used fo r near ly 20 years(MB, DLI), whereas other s are comparat ively new (APCI, ES).For the three m ost prom ising interfaces , APCI, ES, and TS,a comparat ive s tudy of instru m en t sensit iv ity as a functio n ofcompound polari ty has shown that ES was applicable for awide ran ge of analyte polari ty , wh ereas AP CI and T S werebetter su i ted for hydrophobic com pou nds (97). These tech-niques have a relat ively soft ionizat ion in common, so themass spec t ra somet imes lack f ragment ions. Frag me nta t ioncan be enhanced fo r a l l th ree by acce lera t ing the ions inregions of in term ediate pressure w here col l is ion with neutr almolecules occurs . Nevertheless, confirmational fragm ents areusually less abu nda nt tha n w ith classical EI ionization. If th isfirs t m etho d is not efficient enou gh, col l is ion-induced disso-c iat ion in t andem mass spec t rom et ry prov ides f ragmenta t ionin a more co n t ro l led manner .W ith regard to the frequency of their use and bearing in min dthat ES and APC I have certainly not been developed o the ir ful lpotential, one can think th at these two interfaces and TS areactually the m ost in teresting techniqu es for toxicological anal-ysis . If genetic therapy is developed, ES could play a moreimp ortan t role because the analysis of proteins can easi ly beperformed, taking advantage of i ts abi l i ty to form mult iplycharged ions tha t can be de termined on co mm on quadrupo lemass fi l ters . Moreover, ES, which handled the w idest range o fcom pou nd polari ty w ith a g ood sensi tiv i ty , and APC I, wh ich12 2

J o u r n a l o f A n a l y t i c a l T o x i c o l o g y , V o l . 2 1 , M a r c h / A p r i l 1 9 9 7

showe d the highest sensitivity for lipophilicanalytes, can be per-formed on the same instru me nt by s implychanging he ion source.Such a combination may replace thermospray in the near future.

ConclusionL C -M S h a s b e e n s h o wn t o b e a p p l i c a b l e t o s a m p l e s o fvarious natures that may have a wide range of polari ty . Theinterfacing problem that ham pered i ts rapid applicabili ty seemsto have been overcome in m ore th an 20 years of development.S o m e o f t h e i n t e rf a c e s , wh i c h e i t h e r h a d t h e m e r i t t o b epioneers in the field of LC-MS or showed new perspect iveswh en in t roduced , l ack sens it iv i ty whe n com pared wi th mo rerece nt instru me nts . This is why i t may be just if ied to classifythe m according to th eir sui tabil i ty for trace analysis and theirpotential interest for toxicological applications.Instruments of historical interest. Direct l iquid introdu ctionand mo ving bel t have virtual ly disappeared because they are

t e c h n i c a l l y d e m a n d i n g a n d a r e n o t s e n s i t i v e e n o u g h fo rtrace analysis .Instruments of secondary interest . Part icle beam and con-t inuous-flow or fri t fas t ato m bom bard me nt have proved to bevaluable tools for ide ntificat ion and quan ti tat ion: PB becauseof the l ibrary search ing possibi li ty for spectra acquired w ithelectron im pact ionizat ion and CF-FAB because ionization isrelatively soft and b ecause i t is able to ass ess com pou nds ofhigh mo lecular mass. Nevertheless , these may also eventual lydisappear. PB lacks sensi tiv i ty wh en used w ith EI, and there arebet ter me thods avai lable for use w ith CI. FAB can only achievelow de tec t ion l imi t s wi th coup led c o lumn me thods tha t a retechnical ly demanding.Instruments of primary interest . Thermospray, electrospray,and atmospheric pressure chemical ionization are the methodsthat w ere mo st often applied in the past years . In the mid-1980sand the beg inn ing o f the 1990s, LC-MS coup l ing and TS werenearly indiscernable, as can be seen by the t rem endo us num berof applicat ions published using this in terface. The l imits ofdetect ion and the rel iabi li ty o f the in stru m ent are sat isfying,and variat ions in io nizat ion efficiency often require the u se oflabeled internal s tandards. ES and APCI have found wider usein the las t seven years . These methods, which are general lyu s e d o n t h e s a m e t y p e o f i n s t ru m e n t b y c h a n g i n g th e i o nsource , a re com plem enta ry wi th regard to sens i t iv i ty as afunction o f the polari ties of the analytes . The excel lent sensi-t iv ity at tained for hyd ropho bic comp ound s by APC I, the w iderange of solute polari ty , a nd th e abi li ty to assess m ult iplycharged ions by ES mak es this coupling a highly perform ingins t rumen t tha t shou ld ga in more impor tance in the fu tu rewh en pep t ide and p ro te in d ru gs wi l l be used m ore f requen tly .

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