Tandem Mass Spectrometry Applied to the Characterization of

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Characterization and Measurement of Flavor Compounds Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 11/06/18. For personal use only.

Tandem Mass Spectrometry Applied to the Characterization of Flavor Compounds Kenneth L . Busch and Kyle J. Kroha Department of Chemistry, Indiana University, Bloomington, IN 47405

Tandem mass spectrometry (MS/MS) i s a new analytical technique applied to problems in food and flavor analyses. Rapidity of analysis, a high discrimination against chemical noise, and the ability to analyze mixtures for functional groups are attributes of MS/MS that make it attractive for such problems. Sample collection and pretreatment differ from methods used in GC/MS. Correct choice of an ionization method i s paramount. Daughter ion MS/MS spectra are used for compound identification via comparison with those of authentic compounds, and parent and neutral loss spectra are useful in functional group analysis. Applications to direct analysis of volatiles emitted from fruits and to spice analyses are considered. Why use MS/MS a n a l y s i s o f v o l a t i l e components from food and f l a v o r components? Figure 1 provides the answer. The top trace i s the c a p i l l a r y column gas chromatographic p r o f i l e o f the concentrated v o l a t i l e s from a knockwurst sausage sample. The temperature program o f 55°C t o 180°£ a t 5° per minute establishes the time scale from beginning t o end o f run as 25 minutes. Coupled t o a mass spectrometer f o r i d e n t i f i c a t i o n , each o f the many compounds can be examined by the mass spectrometer f o r only a few seconds. The bottom s e r i e s o f figures i l l u s t r a t e s the d i r e c t MS/MS a n a l y s i s of the v o l a t i l e s from the sausage sample. A stream o f a i r i s swept over the sausage and c a r r i e d i n t o the source o f the mass spectrometer. Ions are formed from the v o l a t i l e constituents, and the f i r s t analyzer o f the instrument scans (5 s) t o provide a mass spectrum o f the mixture. A p a r t i c u l a r i o n i s selected from a l l o f those formed, excited by c o l l i s i o n , and i t s fragment ions analyzed by a second mass analyzer (5 s ) . The MS/MS spectrum thus obtained i s compared t o the spectrum o f the authentic compound (contained i n the laboratory l i b r a r y ) and the i d e n t i t y o f the compound established (10 s ) . The t o t a l time f o r a n a l y s i s by MS/MS i s under a minute, including the time required t o load the sausage i n t o the sample 0097-6156/85/ 0289-0121 $06.00/ 0 © 1985 American Chemical Society

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boat. A p a r t i c u l a r i o n i c component present i n the mass spectrum can be i d e n t i f i e d i n only a few seconds. Since a l l ions are a v a i l a b l e continuously, the a c q u i s i t i o n o f data can be t a i l o r e d t o the i n t e n s i t y o f the s i g n a l . For strong s i g n a l s , a few seconds s u f f i c e s ; f o r weak s i g n a l s , the i n t e g r a t i o n time can be lengthened appropriately. Note t h a t the i o n chosen f o r the MS/MS experiment at m/z 163 i s only a minor component i n the mass spectrum. The analyst has the freedom t o examine any i o n formed i n the source i n any order, u n l i k e GC/MS, which allows examination o f the sample only i n a short "time window" established by the chromatography. To re-examine a compound i n GC/MS, the e n t i r e sample must be reintroduced t o the gas chromatograph. In MS/MS, the o r i g i n a l i o n i s simply again selected by the f i r s t mass analyzer. F i n a l l y , t h e time evolution o f a number o f compounds can be followed d i r e c t l y w i t h MS/MS, f o r example, as the sample i s heated. In GC/MS t h i s simple experiment generates a number o f samples, each o f which must be d i s c r e t e l y analyzed. Background. As an a n a l y t i c a l technique, tandem mass spectrometry i s j u s t entering i t s second decade o f development. The v a r i e t y o f reported a p p l i c a t i o n s b e l i e s i t s r e l a t i v e youth. Tandem mass spectrometry (MS/MS) grew out o f e a r l y work which used met e s t a b l e ion t r a n s i t i o n s i n order t o e s t a b l i s h i o n structures and i n t e r r e l a t i o n s h i p s . A f t e r extensive applications t o i o n s t r u c t u r a l studies, i t s usefulness i n d i r e c t complex mixture analysis became apparent w i t h the e a r l y work o f Cooks (1-3). I t s successes i n problem s o l v i n g are summarized i n a recent book edited by McLafferty (4). Now, w i t h several commercial instruments a v a i l a b l e , MS/MS i s being evaluated f o r a p p l i c a t i o n i n several new areas, i n c l u d i n g biochemical a n a l y s i s , forensic chemistry, and food and f l a v o r analyses. The p r i n c i p l e s o f MS/MS w i l l be summarized i n the f i r s t p a r t o f t h i s chapter. The second part o f the chapter w i l l deal w i t h the reported a p p l i c a t i o n s o f MS/MS t o f l a v o r analysis. Principles Ion Processing. As mentioned, MS/MS began w i t h the study o f meta stable ions (_5 ). Metastable t r a n s i t i o n s are observed from ions which undergo a d i s s o c i a t i o n while i n t r a n s i t through the instrument. The t r a n s i t i o n i s a chemical reaction c h a r a c t e r i s t i c of the nature o f the i o n . In MS/MS, the instrument i s modified so that the reactions occur more frequently and the masses o f the reacting i o n and the product i o n can be established. The approach t o MS/MS i s thus quite d i f f e r e n t from that f o r high r e s o l u t i o n mass spectrometry. There the exact mass measurement which provides the empirical formula o f the i o n i s a p h y s i c a l measurement. In metastable i o n studies, the focus i s on the nature o f the i n d i v i d u a l chemical reactions. Each metastable ion represents an i n d i v i d u a l l y defined t r a n s i t i o n f o r which masses and abundances o f both products and reactants can be s p e c i f i e d . The a n a l y t i c a l advantage t h a t accrues i s based on the greater information inherent i n a chemical rather than a p h y s i c a l

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MS-MS Applied to Flavor Compound Characterization

BUSCH AND KROHA

approach. An i o n once formed i s not simply measured t o e s t a b l i s h i t s mass and i t s r e l a t i v e abundance, but rather i s processed i n experiments designed t o define i t s chemical r e a c t i v i t y . Independent sequential analyses. In order t o explain the b a s i s o f an MS/MS experiment consider the b a s i c r e a c t i o n sequence shown i n r e a c t i o n 1. M]+

+

>

N

m 2

+

+

1TV3

+ Ν

(1)

The goal i s t o e s t a b l i s h the masses o f both the products and reactants o f t h i s chemical r e a c t i o n . We thus require an analyzer t o e s t a b l i s h Μ^ , and a second analyzer t o e s t a b l i s h rrç*. The mass of the neutral i s defined by difference. The r e a c t i o n takes place between the two analyzers, aided by energy added t o the reactant i o n i n t h i s region (vide i n f r a ) . With the a d d i t i o n o f a source (S) and the detector (D), and denoting the r e a c t i o n as *, the block diagram of a simple MS/MS instrument i s established (Figure 2). This diagram a l s o explains the various experiments a v a i l a b l e i n MS/MS. The s a l i e n t points are: 1) there are two mass analyzers t o characterize the r e a c t i o n ; 2) the t r a n s i t i o n from reactant t o product occurs between the analyzers; 3) the analyzers operate independently. There must be a source o f energy i n order t o i n i t i a t e the r e a c t i o n between the analyzers beyond the inherent metastable i o n abundances. T y p i c a l l y the interanalyzer region i s f i t t e d w i t h a c o l l i s i o n c e l l which contains about a m i l l i t o r r of target gas Ν (often nitrogen or helium). The incoming i o n c o l l i d e s w i t h the target gas, transforming some f r a c t i o n o f the k i n e t i c energy of the i o n i n t o i n t e r n a l energy which then causes fragmentation. +

Resolution. In MS/MS, each o f the two independent mass analyzers can be operated a t a low r e s o l u t i o n while r e t a i n i n g a high o v e r a l l s e l e c t i v i t y . Since e x t r a c t i o n of the highest r e s o l u t i o n from a given analyzer requires a disproportionate e f f o r t , the s o l u t i o n o f demanding a n a l y t i c a l problems i s s i m p l i f i e d . By analogy, a chromatographer reduces the performance requirements o f a s i n g l e stage separation of a complex mixture by adding a simple sample p r e f r a c t i o n a t i o n . The same general p r i n c i p l e i s apparent i n MS/MS. Signal-to noise. Mass spectrometers are e x t r a o r d i n a r i l y s e n s i t i v e devices, having the a b i l i t y t o analyze nanogram amounts o f sample. MS/MS, as discussed above, deals w i t h the chemistry o f i o n i c reactions, and thus i t i s o f t e n chemical rather than e l e c t r o n i c noise t h a t establishes the l i m i t o f detection (6). Chemical noise i s produced by matrix constituents other than the sample, the reactions o f which may be i n s u f f i c i e n t l y resolved from the sample reaction o f i n t e r e s t . The r o l e o f the a n a l y t i c a l chemist i s t o design the MS/MS experiment t o provide the best p o s s i b l e d i s c r i m i n a t i o n against the chemical noise i n the system. The problem i s a s i g n i f i c a n t one; i n complex mixtures, the matrix constituents are present i n great excess, and t h e i r reactions are unknown. However, the use o f several stages o f independent mass a n a l y s i s can provide a very high signal-to-noise r a t i o . Table I summarizes the operation o f a generic species o f analyzers given an i n i t i a l mixture w i t h equal parts o f s i g n a l and noise. For t h i s example, each analyzer passes 50% o f the s i g n a l but only 10% o f the

123

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SAUSAGE χ—

^STREAM

CONCENTRATE NJECT

QC/M8 26i

fU

Mi

1M Itl

Jl

•ft

•a!

It*

M E C T ANALY88 MS/MS

10·

ΊΊ

20·

ΜΙΑ·

Figure 1. Comparison o f the time scales o f the procedures o f GC/MS and MS/MS a n a l y s i s o f v o l a t i l e f l a v o r compounds emitted from sausage samples (19).

Figure 2. Simple diagram o f an MS/MS instrument and three scanning modes based on changes i n mass between the parent and the daughter i o n . See t e x t f o r d e t a i l s .

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noise. Even w i t h t h i s crude d i f f e r e n t i a t i o n , a f t e r two stages o f a n a l y s i s , the s i g n a l t o noise r a t i o has increased from u n i t y t o 25:1. With further a n a l y s i s , i t r i s e s r a p i d l y even as the t o t a l s i g n a l l e v e l decreases. I n MS/MS, the choice o f i o n i z a t i o n method, ion p o l a r i t y , and the mass analysis i t s e l f a l l contribute t o the d i f f e r e n t i a t i o n o f s i g n a l from noise, and the stepwise enhancement i s o f t e n several hundred t o one rather than 5:1 as shown i n t h i s example. Table I .

Signal t o Noise Enhancements w i t h M u l t i p l e Analyses

Number of stages 0 1 2 3

Intensities Signal Noise 1000 500 250 125

1000 100 10 1

Signal-to-noise ratio 1 5 25 125

Types o f Experiments The a n a l y s i s i n a mass spectrometer i s not based on the mass o f an ion, but rather on i t s mass t o charge r a t i o , m/z. Thus i f e i t h e r the mass o r the charge o f the i o n i s a l t e r e d i n an MS/MS experiment, the change can be followed by the second mass analysis. The experiments o f MS/MS can be subdivided i n t o those which involve changes i n mass o r changes i n charge. There e x i s t s a t h i r d category o f experiments which involve changes i n r e a c t i v i t y independent o f mass and charge, but these experiments w i l l not be discussed i n t h i s chapter. Changes i n mass. The most ccnrnon experiments i n MS/MS are based on changes i n mass. These are summarized i n Figure 2. Assume a complex mixture has been introduced i n t o the source, and that ions are formed corresponding t o each constituent o f the mixture. The f i r s t mass analyzer s e l e c t s ions o f a s p e c i f i e d mass which are passed i n t o the c o l l i s i o n region between the analyzers. Here the a d d i t i o n a l energy imparted by c o l l i s i o n causes the breakup o f t h i s parent i o n i n t o smaller fragment ions. The masses o f the fragment ions, termed daughter ions, i s established by scanning the second mass analyzer. The r e s u l t i n g spectrum i s c a l l e d a daughter i o n MS/MS spectrum, and consists o f a l l o f the fragment ions from a selected parent i o n (Figure 2a). Since the mass analyzers operate independently, i t a l s o i s possible t o set the second mass analyzer t o pass only daughter ions of a selected mass t o the detector. The f i r s t mass analyzer i s then scanned across the mass range. A s i g n a l a t the detector i s noted when the f i r s t mass analyzer passes a parent i o n that fragments t o the s p e c i f i e d daughter i o n . The spectrum that i s obtained i s c a l l e d a parent i o n MS/MS spectrum, and consists o f a l l the precursor ions o f a s p e c i f i e d daughter i o n (Figure 2b). I f both mass analyzers are scanned a t the same rate w i t h a

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COMPOUNDS

constant mass o f f s e t χ between them, then signals w i l l be observed at the detector whenever a parent i o n passing through the f i r s t mass analyzer produces a daughter i o n w i t h a mass χ daltons l e s s than the parent i o n . The spectrum obtained i s c a l l e d the constant neutral loss MS/MS spectrum, and consists o f a l l the parent ions i n the parent ion/daughter i o n p a i r s r e l a t e d by the l o s s o f a neutral of s p e c i f i e d mass (Figure 2c). The three MS/MS experiments described above provide d i f f e r e n t information i n complex mixture a n a l y s i s . The daughter i o n MS/MS spectrum i s often obtained when targeted compound analysis i s performed. The parent i o n selected corresponds t o the targeted component, and the daughter i o n spectrum obtained from the mixture i s compared t o that obtained f o r the authentic targeted compound introduced t o the source under the same conditions. In t h i s way, the presence o f the target can be established and o f t e n quantitated. Parent and constant neutral l o s s MS/MS spectra are more o f t e n used f o r i d e n t i f i c a t i o n o f functional groups, and can be used f o r both targeted compound a n a l y s i s , o r f o r a completely unknown mixture. Experience often shows that there are c h a r a c t e r i s t i c daughter ions o r neutral losses that occur f o r s p e c i f i c functional groups. For instance, 149 as a daughter i o n i s c h a r a c t e r i s t i c i n the MS/MS spectra o f phthalates. A scan f o r parent ions o f the s p e c i f i e d daughter i o n 149 would thus pinpoint a l l o f the various phthalates present i n a mixture, regardless o f whether each was known t o be present o r not. S i m i l a r l y , the protonated molecular ions o f carboxylic acids t y p i c a l l y form daughter ions by l o s s o f carbon dioxide. A constant n e u t r a l l o s s MS/MS spectrum w i t h the o f f s e t s p e c i f i e d as 44 daltons (the weight o f the neutral fragment OO2) w i l l pinpoint parent ion/daughter i o n p a i r s that undergo a chemical reaction t y p i c a l o f carboxylic acids, again without p r i o r knowledge o f t h e i r presence. +

+

Changes i n charge. The c o l l i s i o n s used t o add energy t o ions and cause fragmentation a l s o may cause changes i n charge o f the i o n . Singly-charged ions can be oxidized t o doubly-charged ions i f the energy imparted by the c o l l i s i o n i s higher than the second i o n i z a t i o n p o t e n t i a l o f the molecule (charge s t r i p p i n g , Equation 2 ). Doubly-charged ions passed i n t o the c o l l i s i o n c e l l can be reduced t o the singly-charged i o n (Equation 3) i n a process known as charge exchange; the n e u t r a l involved i n the c o l l i s i o n acquires the balancing charge. F i n a l l y , negative ions can be converted i n t o the corresponding p o s i t i v e ions i n the oxidation process known as charge inversion (Equation 4 ) . Μ]+ M!

+ Ν 2+

+ Ν

M" + Ν

2

> M ] * + e" + Ν > M]* + N+ > M+ + 2e~ + Ν

(2) (3)

(4)

The energetic requirements f o r these reactions are d i f f e r e n t from those o f the reactions which involve changes i n mass. For the most part, these reactions are observed i n high energy c o l l i s i o n s . They have predominately been used f o r studies o f i o n structure, but have r e c e n t l y been used f o r complex mixture a n a l y s i s . This expansion i s

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based on the r e l a t i o n s h i p between i o n s t r u c t u r e and the f a c i l i t y o f charge changing reactions. Nitrogen-containing compounds f o r instance, form doubly-charged ions more r e a d i l y than other classes o f compounds. An experiment based on the r e a c t i o n of doubly charged ions can thus be s p e c i f i c f o r nitrogen-containing ions. #

A n a l y t i c a l C h a r a c t e r i s t i c s o f MS/MS MS/MS was f i r s t considered as a replacement f o r GC/MS. I t s true character as a complement t o t h a t method i s now r e a l i z e d , and the most demanding of a n a l y t i c a l problems often require the f u l l d i f f e r e n t i a t i n g power o f a GC/MS/MS combination. The choice between GC/MS o r MS/MS f o r a p a r t i c u l a r a p p l i c a t i o n must r e s t on r e l a t i v e merits o f s e n s i t i v i t y , s e l e c t i v i t y , and speed, each o f which w i l l now be b r i e f l y discussed. S e n s i t i v i t y . I t i s misleading t o assign a s i n g l e value o f s e n s i t i v i t y f o r e i t h e r GC/MS or MS/MS without s p e c i f y i n g the problem, the instrument, the experiment, the spectrum, and the operator. In general, f o r modern instruments, analyses o f compounds a t the nanogram l e v e l can be considered routine f o r e i t h e r technique. Lower l i m i t s o f detection are a v a i l a b l e w i t h s p e c i a l a t t e n t i o n t o the experiment. For example, i n GC/MS, selected i o n monitoring i s used t o increase s e n s i t i v i t y . In t h i s experiment, the mass analyzer no longer scans across the f u l l mass range, but rather integrates signal* i n a few mass windows corresponding t o ions o f i n t e r e s t . The e f f e c t i v e r e s o l u t i o n o f the chromatographic separation i s u s u a l l y increased. The selected i o n monitoring technique i s u s e f u l when compounds o f p a r t i c u l a r i n t e r e s t are known t o produce c h a r a c t e r i s t i c ions i n the source. Thus Harvey {!) demonstrated that the t r i m e t h y l s i l y l (IMS) d e r i v a t i v e s o f diphenylpropanoids form c h a r a c t e r i s t i c ions a t m/z 266 (Scheme 1). S e t t i n g the mass analyzer o f the spectrometer t o pass only m/z 266 pinpoints the e l u t i o n o f such compounds from the gas chromatographic column. S e n s i t i v i t y f o r selected i o n monitoring experiments t y p i c a l l y i s reported i n the low picogram l e v e l , although i n favorable cases, low femtogram s e n s i t i v i t y can be achieved. In MS/MS, the selected i o n monitoring experiment i s transformed i n t o selected r e a c t i o n monitoring. Both mass analyzers are set t o pass s p e c i f i e d parent ion/daughter i o n p a i r s . As i n selected i o n monitoring, there i s a time advantage as the mass analyzers are not scanned but rather integrate s i g n a l . There i s an added s p e c i f i c i t y over selected i o n monitoring i n t h a t both the reactant and the product are s p e c i f i e d . I f sample i n t r o d u c t i o n i s v i a the d i r e c t probe, the v a p o r i z a t i o n p r o f i l e provides a t h i r d parameter v i a which the compound can be i d e n t i f i e d . MS/MS has been used f o r the i d e n t i f i c a t i o n o f targeted compounds i n complex mixtures a t the nanogram l e v e l (8). A t lower l e v e l s , matrix constituents a f f e c t the p r e c i s i o n of the response, n e c e s s i t a t i n g e i t h e r higher r e s o l u t i o n measurements or sample cleanup. The l a t t e r route has been s u c c e s s f u l l y pursued under the guise o f GC/MS/MS down t o l e v e l s o f 10-100 pg i n pharmaceutical a p p l i c a t i o n s (£).

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S p e c i f i c i t y . Yost (10) has compared the r e l a t i v e informing power of GC/MS and MS/MS. The informing power o f an a n a l y t i c a l procedure i s expressed mathematically i n terms o f the number o f " b i t s . " A c a p i l l a r y gas chromatography column o f 1 0 p l a t e s combined w i t h a quadrupole mass spectrometer w i t h u n i t mass r e s o l u t i o n up t o 1000 daltons provides an informing power o f 6.6 χ 1 0 b i t s . An MS/MS instrument comprised o f two sequential quadrupole mass analyzers o f the same performance provides an informing power o f 1.2 χ b i t s . Within the l i m i t o f the assumptions made, the informing powers are i d e n t i c a l . There are, however, various experimental parameters i n MS/MS which can be used as a d d i t i o n a l r e s o l u t i o n elements. These include energy resolved, pressure resolved, and angle resolved MS/MS experiments ( 4 ) . These parameters are balanced by v a r i a t i o n o f gas chromatographic conditions, such as the stationary phase chosen, the temperature program followed, and a d d i t i o n a l steps o f sample p u r i f i c a t i o n and pretreatment. I n summary, both GC/MS and MS/MS are powerful enough t o solve most a n a l y t i c a l problems, both generating the extensive data sets c h a r a c t e r i s t i c o f such combined methods ( 1L1 ). 5

6

Speed o f Analysis. The introductory example focussed on the speed of MS/MS a n a l y s i s o f v o l a t i l e compounds. However, there are several aspects o f a n a l y t i c a l speed o f i n t e r e s t i n MS/MS. The f i r s t , and that which has received the most a t t e n t i o n , i s the time required f o r a n a l y s i s . The analogy between GC/MS and MS/MS involves the comparison o f r e t e n t i o n times through the gas chromatograph w i t h i o n f l i g h t times through the f i r s t mass analyzer. The former occupies between 10* and 10^ s, and the l a t t e r on the order o f microseconds. I n the s p e c i f i c example o f targeted compound a n a l y s i s i n a complex mixture, MS/MS can o f f e r a s i g n i f i c a n t time advantage i n the examination o f a large number o f samples. G l i s h has shown t h a t the a n a l y s i s o f mixtures using a preset protocol o f selected reaction monitoring can occur a t near the r a t e o f sample introduction i n t o the source o f the instrument (12). The time advantage o f MS/MS a l s o i s exemplified by the a b i l i t y to s e l e c t from a mixture o f ions i n the source any parent i o n , i n any order, and t o return as necessary t o that parent i o n f o r p r e c i s e measurements. This independence o f access p e r s i s t s f o r the duration o f the sample residence time i n the source. This i s i n marked contrast t o the s i t u a t i o n i n GC/MS, where each sample i s a v a i l a b l e f o r examination by the mass spectrometer only during the r e t e n t i o n time window. To repeat the measurement, the e n t i r e sample must be r e i n j e c t e d . The source residence time f o r most samples introduced v i a the d i r e c t i n s e r t i o n probe i s on the order of a minute o r two, depending on the temperature o f the source and the r a t e o f heating o f the probe t i p i t s e l f . The f i n a l aspect o f speed t o be considered i s the information f l u x . In MS/MS, the sample may be a v a i l a b l e f o r minutes rather than the seconds corresponding t o the width o f a gas chromatographic peak. I n the absence o f a preset protocol, experimental decisions must be made i n r e a l time. For example, what parent i o n should be selected f o r a daughter i o n MS/MS spectrum? Does the spectrum o f parent ions from the source change w i t h probe temperature? What should the c o l l i s i o n energy and

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pressure be? The newest generation o f data systems can make such decisions automatically w i t h i n c e r t a i n preset l i m i t s ( 13 ). The r a t e a t which information must be obtained and processed i n these l a t t e r MS/MS experiments i s much higher than t h a t i n a GC/MS experiment. Applications t o Flavor Compounds The use o f gas chromatography/mass spectrometry i n food and f l a v o r analyses i s now well-established, and reviews are p l e n t i f u l (14-16). By contrast, the use o f MS/MS i n t h i s area i s l e s s widespread. In part t h i s has been due t o the longer a v a i l a b i l i t y o f commercial GC/MS instruments as opposed t o MS/MS instruments, but a l s o i n no small p a r t due t o the enormous success o f the GC/MS method i t s e l f . Food and f l a v o r analyses deal perforce with the i d e n t i f i c a t i o n and q u a n t i t a t i o n o f v o l a t i l e components o f complex mixtures. Gas chromatography, e s p e c i a l l y i n c a p i l l a r y form, i s able t o separate such compounds w i t h high e f f i c i e n c y . The a d d i t i o n o f the mass spectrometer allows the i d e n t i f i c a t i o n o f the eluted compound a t the very low l e v e l s found i n many food and f l a v o r mixtures. As i n GC/MS, the analyst using MS/MS must be concerned w i t h sample handling ( c o l l e c t i o n , treatment, and œntandnation) and sample a n a l y s i s ( i o n i z a t i o n method and mass measurement). Sample c o l l e c t i o n , treatment, and contamination. In GC/MS, sample treatment i s o f t e n extensive. In preparation f o r GC/MS a n a l y s i s o f nutmeg, Harvey (7) ground 100 mg o f nutmeg t o a f i n e powder and extracted f o r an hour w i t h e t h y l acetate. The f i l t e r e d e x t r a c t was frozen t o p r e c i p i t a t e t r i g l y c e r i d e s , f i l t e r e d again, and then d e r i v a t i z e d overnight w i t h a standard t r i m e t h y l s i l y l a t i o n reaction. By contrast, i n the MS/MS a n a l y s i s o f nutmeg by Davis (17), 10-50 mg o f ground nutmeg are loaded i n t o a glass c a p i l l a r y , introduced d i r e c t l y i n t o the source o f the mass spectrometer, and vaporized by a short heating program. A constant concern i n the a n a l y s i s o f f l a v o r components i s a l t e r a t i o n and contamination o f the sample. Losses o f v o l a t i l e components are a major problem. The extensive sample preparation involved i n GC/MS o f f e r s ample opportunity f o r transformations and losses because o f sample handling and exposure t o chemical d e r i v a t i z i n g reagents. In MS/MS, sample handling i s o f t e n reduced and the chances f o r outside contantination minimized. Sample carryover, a problem during e x t r a c t i o n procedures f o r GC/MS, i s not eliminated i n MS/MS, but evolves i n t o a problem o f source œntamination. This problem was severe i n some e a r l y MS/MS work, but now seems under c o n t r o l w i t h the use o f removable i o n volumes i n the source. The concentration and homogenization o f sample that occurs i n GC/MS pretreatment i s not a v a i l a b l e i n MS/MS. Sample inhomogeneities thus become o f much greater concern. Sample t o sample v a r i a t i o n i s already f a i r l y high i n samples o f n a t u r a l o r i g i n , and the extensive a p p l i c a t i o n o f MS/MS may require more c a r e f u l sampling procedures than c u r r e n t l y employed. For t r a c e analyses, a simple form o f sample pretreatment i s often employed i n MS/MS t o concentrate the sample and t o preserve the c l e a n l i n e s s o f the i o n i z a t i o n source.

129

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Sample i o n i z a t i o n . Requirements f o r sample i o n i z a t i o n are much more severe i n MS/MS than i n GC/MS. For MS/MS, the i o n i z a t i o n method should create one i o n f o r each component, and the structure o f the i o n should be the same as that o f the neutral surrogate. Electron i o n i z a t i o n u s u a l l y does not f u l f i l l these requirements, since the ions formed often include those from rearrangement reactions, and the degree o f fragmentation i s excessive. Chemical i o n i z a t i o n provides the r e q u i s i t e s i n g l e i o n f o r each ccrnponent o f the matrix i n the form o f the quasimolecular i o n (M+H) . However, chemical i o n i z a t i o n i s s e n s i t i v e t o source parameters and matrix e f f e c t s , and these problems are exacerbated by the d i r e c t introduction o f a complex mixture i n t o the source. The e f f e c t s can be compensated t o some degree by the use o f an i s o t o p i c a l l y l a b e l l e d i n t e r n a l standard f o r q u a n t i t a t i v e work. In the analysis o f unknowns i n complex mixtures, the nature o f the source chemistry should be a constant concern. +

Sample D e r i v a t i z a t i o n . The d e r i v a t i z a t i o n o f nutmeg constituents described by Harvey (7) i s designed t o increase the v o l a t i l i t y and s t a b i l i t y o f the components so that they can be separated i n the gas chromatograph. With d i r e c t probe introduction, MS/MS i s u s u a l l y able t o deal w i t h samples o f lower v o l a t i l i t y ; hence, d e r i v a t i z a t i o n i s not required. D i r e c t probe temperatures reach as high as 400° C, vaporizing many samples d i r e c t l y i n t o the vacuum o f the mass spectrometer source. D e r i v a t i z a t i o n i s used i n MS/MS for the sanewhat d i f f e r e n t purpose o f imparting a s p e c i f i c chemical r e a c t i v i t y t o the analyte. Consider the t r i m e t h y l s i l y l d e r i v a t i v e s often used t o increase v o l a t i l i t y and s t a b i l i t y . The electron i o n i z a t i o n mass spectra o f these d e r i v a t i v e s often contain fragment ions such as the t r i m e t h y l s i l y l c a t i o n i t s e l f IMS*, o r fragment ions due t o losses o f neutral species containing the t r i m e t h y l s i l y l moiety. The same fragmentation reactions are expected i n the MS/MS spectra o f these d e r i v a t i v e s . Treatment o f a mixture w i t h a s i l y l a t i n g reagent converts free hydroxyl groups t o t h e i r -0TMS d e r i v a t i v e s , and then a second l a b e l l e d s i l y l a t i n g reagent converts amino groups t o t h e i r -NH-d9lMS d e r i v a t i v e s . A parent i o n scan o f IMS* (at m/z 73) pinpoints a l l o f the precursor ions that contained a free hydroxyl group. A parent i o n scan o f dg-TMS* pinpoints the precursor ions w i t h a r e a c t i v e amino group. Ctammon ions i n the two MS/MS spectra represent molecules t h a t contain both r e a c t i v e groups. A d e r i v a t i z a t i o n scheme i n v o l v i n g a constant neutral loss MS/MS scan has been described by Zakett (18). Phenols and amines react w i t h a c e t y l c h l o r i d e t o form acylated d e r i v a t i v e s which commonly lose the neutral fragment ketene i n the MS/MS reaction. A neutral l o s s scan f o r the l o s s o f 42 daltons w i l l thus i n d i c a t e the molecular weights o f any compound which has undergone d e r i v a t i z a t i o n . The strategy was used s u c c e s s f u l l y i n the analysis o f these f u n c t i o n a l groups i n a synthetic f u e l sample (18). Applications t o f l a v o r compounds have not y e t been reported, but w i l l undoubtedly be extensively exploited considering the d i v e r s i t y o f d e r i v a t i z a t i o n chemistry already developed.

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Applications Food aromas. Labows and Shushan (19) have reviewed the d i r e c t a n a l y s i s o f food aromas by a commercial MS/MS system using an atmospheric pressure i o n i z a t i o n source. The sample i n l e t i s a simple a l l - g l a s s device that c o l l e c t s v o l a t i l e components emitted by food materials and d i r e c t s them i n t o the mass spectrometer. Losses due t o sample preparation are minimized, as are absorption or decomposition problems associated w i t h chromatographic f r a c t i o n a t i o n . P r o f i l e s of aroma compounds obtainedfcyt h i s method are claimed t o be more accurate than those obtained using other a n a l y t i c a l methods. Because o f the high d i s c r i m i n a t i o n against chemical noise i n the MS/MS system, detection l i m i t s can be very low, reported as 0.5 ppb f o r ethyl butyrate, 0.8 ppb f o r l i n a l o o l , and 45 ppb f o r limonene. These l i m i t s were established w i t h daughter i o n MS/MS spectra. Figure 3 shows the c o r r e l a t i o n between the daughter i o n MS/MS spectrum o f authentic nootkatone (with the i o n a t m/z 219 selected as the parent ion) and the i o n a t the same mass emitted d i r e c t l y from a g r a p e f r u i t . The match between the two spectra confirm the presence of t h i s targeted compound i n the emitted v o l a t i l e s . I t a l s o has been i d e n t i f i e d i n the v o l a t i l e s frcm i n t a c t oranges. The experiment can be completed i n l e s s than a minute, without sample preparation. Note that the u n i t mass r e s o l u t i o n o f the t r i p l e quadrupole instrument allows an accurate assignment o f abundances f o r daughter ions o f adjacent mass i n t h i s MS/MS spectrum. A c l o s e examination o f the spectra show t h a t the match between the authentic and the target compound i s not p e r f e c t . E i t h e r the instrumental parameters were not constant, or there i s an a d d i t i o n a l component a t m/z 219 i n the v o l a t i l e s emitted by the g r a p e f r u i t . I t i s a t t h i s stage that a simple p r e f r a c t i o n a t i o n experiment, or an a l t e r n a t i v e i o n i z a t i o n method becomes necessary t o e s t a b l i s h the number o f components present a t t h i s mass. Other MS/MS experiments were used t o give information u s e f u l f o r functional group c h a r a c t e r i z a t i o n . Fragmentation t o m/z 18 (NH4" ") i s i n d i c a t i v e o f amines, and m/z 19 (H30 ) i s a t y p i c a l fragment i o n from alcohols. Thus a parent i o n scan f o r these daughter ions pinpoints compounds o f these groups i n the emitted v o l a t i l e s . Acetate esters produce daughter ions a t m/z 43 and m/z 61. A parent i o n scan f o r the l a t t e r produces the daughter i o n MS/MS spectrum shown i n Figure 4, which i s the sum of a l l the parent ions o f a l l of the acetate esters i n the v o l a t i l e s emitted from a banana. The base peak a t m/z 131 represents the parent i o n o f isoamylacetate, known t o have the c h a r a c t e r i s t i c banana odor. Figure 5 i s the neutral l o s s scan f o r l o s s o f 44 daltons, a f a m i l i a r loss from negative ions of c a r b o x y l i c acids. Thus the ions a t m/z 87, 89, and 121 are most l i k e l y from butanoic o r pyruvic a c i d , l a c t i c a c i d , and benzoic a c i d , respectively. The i d e n t i t y o f these ions are confirmed by examining the daughter i o n spectra of the authentic compounds and the peaks obtained i n the d i r e c t a n a l y s i s o f the sample, i n t h i s case a Teewurst sausage. D i r e c t analyses o f v o l a t i l e s has been suggested as a means o f screening food products that might otherwise pass a g r i c u l t u r a l borders. The s e n s i t i v i t y seems t o be s u f f i c i e n t l y high f o r t h i s purpose, and the MS/MS a n a l y s i s possesses the r e q u i s i t e speed and 4

+

131

132


Scheme 1.

2 1 9 * MS/MS ORANGE RA

m/z RA

m/z NOOTKATONE 219* MS/MS

Figure 3. Daughter i o n MS/MS spectra o f suspected nootkatone emitted frcm g r a p e f r u i t and the authentic compound (19). PARENTS OF 6 1 *

m/z

Figure 4. Parent i o n MS/MS spectrum f o r acetate esters emitted from a banana, representing an example o f f u n c t i o n a l group screening by MS/MS ( 1 9 ) .

m/z

Figure 5. Neutral l o s s MS/MS spectrum f o r l o s s o f 44 daltons (carbon dioxide) which pinpoints (M-H)" molecular ions f o r carboxylic acids. L a c t i c a c i d and benzoic a c i d are i d e n t i f i e d a t 89"" and 121", r e s p e c t i v e l y , although these are not the l a r g e s t peaks i n the spectrum ( 1 9 ) .

9.


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s p e c i f i c i t y f o r r e a l time a n a l y s i s . A comprehensive study o f the interferences that might be expected i n such use wDuld be needed t o evaluate the s u i t a b i l i t y o f t h i s technique. Spice a n a l y s i s . Davis has studied the composition o f nutmeg using MS/MS (17). Nutmeg has been extensively studied because o f the large number o f psychoactive species alleged t o be present. This study i s noteworthy because o f the use o f both h i g h energy and low energy MS/MS t o acquire daughter i o n spectra f o r the various compounds contained w i t h i n the nutmeg, and the use o f programmed thermal desorption from the d i r e c t i n l e t probe o f the mass spectrometer i n order t o e f f e c t a crude d i s t i l l a t i o n o f the sample. Isobutane was used as the reagent gas i n order t o ensure t h a t most o f the constituents form protonated molecular ions (M+H) i n the mass spectrum w i t h a minimum o f fragmentation. Charge exchange was used as an a l t e r n a t i v e method of i o n i z a t i o n i n order t o form parent ions i n an independent manner, and examine the daughter i o n spectra o f the complementary parent i o n Μ*"·. The isobutane chemical i o n i z a t i o n mass spectrum o f nutmeg obtained a t a probe temperature o f \SO°C d i f f e r s from that obtained a t 200°C. Higher mass v o l a t i l e s are not evaporated i n t o the source u n t i l the temperature o f the probe i s elevated t o the higher temperature. As w i t h many analyses o f t h i s type, the amount o f sample i s not a l i m i t i n g factor. The temperature can thus be held steady f o r several minutes a t a given value, allowing several independent MS/MS experiments t o be completed. At higher probe temperatures, thermal degradation o f the sample can become a problem. Comparison o f the daughter i o n MS/MS spectra o f authentic 4-allyl-2,6-dimethoxyphenol a t m/z 195 and the same mass i o n from the nutmeg sample i s presented i n Figure 6. The spectra are s u f f i c i e n t l y s i m i l a r that the presence o f t h i s compound i n nutmeg can be confirmed. Of p a r t i c u l a r value i s the sharp charge s t r i p p i n g peak a t 97.5 on the mass scale. This i s the product o f an o x i d a t i o n r e a c t i o n o f the s i n g l y charged 195 t o the doubly-charged 1 9 5 as a r e s u l t o f the h i g h energy c o l l i s i o n . This r e a c t i o n occurs frequently with nitrogen- and oxygen-containing compounds. Two points should be noted. F i r s t , the match, although close, i s not exact. This indicates that not a l l o f the i o n current a t t h i s mass i s due t o t h i s compound alone, as i n the case described above. Secondly, the width o f the peaks f o r the daughter ions are very wide, compromising both the assignment o f masses and the r e l a t i v e abundances. This i s a consequence o f the instrument used, which was a reverse-geometry sector instrument (20). Daughter i o n a n a l y s i s i s accomplished w i t h a k i n e t i c energy analyzer, which mirrors the k i n e t i c energy release observed as a consequence o f the fragmentation reaction. Although t h i s value can be used as a probe o f the mechanism o f the fragmentation i t s e l f , i t i s a disadvantage i n MS/MS work f o r these reasons. I t i s known that nutmeg contains diphenylpropanoids o f c y c l i c and a c y c l i c forms. The a c y c l i c form fragments t o c h a r a c t e r i s t i c daughter ions a t m/z 193, and the c y c l i c form t o daughter ions a t m/z 203 (Scheme 2). These daughter ions can be set as products i n a parent i o n scan t o examine the e n t i r e nutmeg mixture f o r parent ions o f these classes o f diphenylpropanoids. Figure 7 shows the +

+

+

2+

133

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CHARACTERIZATION A N D M E A S U R E M E N T O F FLAVOR C O M P O U N D S

1 7

g

RA

NUTMEG 195+

MS/MS

165 154

LA RA

ι

m/z

4-ALLYL-2.5-DIMETHOXYPHENOL 195* 165 MS/MS 154

179

JL

m/z

Figure 6. Daughter i o n MS/MS spectrum o f the protonated molecular ion from 4 - a l l y l - 2 , 6-dimethoxyphenol as an authentic compared t o the spectrum obtained from an i o n o f the same mass formed d i r e c t l y from a nutmeg sample ( 17).

OCH. 203**

CYCLIC OCH

QCH,

a

OCH, 193*

1

ACYCLIC Scheme

193

PARENT ION MS/MS PARENTS OF 193 +

ACYCLIC

RII I 203

2.

357 371 I 401

PARENT ION MS/MS PARENTS OF 203 +

CYCLIC 327 357

3

7

!

221 _i

Figure 7 . Parent i o n scans f o r two isomeric forms o f diphenylpropanoids found i n nutmeg. The common ions a t m/z 355, 357, and 371 i n d i c a t e the presence o f both forms o f the compound a t those masses (17).

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r e s u l t of t h i s experiment. Parent ions at 357 , 371 , 387 , and 4 0 1 are indicated t o be a c y c l i c diphenylpropanoids. cyclic forms are i n d i c a t e d i n the parent i o n scan at 327*, 341 , 355 , 357+, 371 , and 375 . The common parent ions 355 , 357 , and 371 are c l e a r l y i n d i c a t e d as c o n s i s t i n g of both forms of diphenylpropanoid structures. The parent i o n at 355 i s thought t o be a dehydrodiphenylpropanoid d e r i v a t i v e o f m y r i s t i c i n , i d e n t i f i e d f o r the f i r s t time i n nutmeg. +

+

+

+

+

+

+

+

+

Problems and P o t e n t i a l s o f MS/MS Problems. The simultaneous i n t r o d u c t i o n of a l l the components o f a mixture i n t o the source o f the mass spectrometer c o n s t i t u t e s one o f the strengths o f MS/MS, but i s a l s o the cause of several problems. F i r s t i s the problem o f sample carryover and source c l e a n l i n e s s . One o f the tenets o f normal operation o f a mass spectrometer i s t o introduce as l i t t l e sample as p o s s i b l e , while i n MS/MS, the sample s i z e s are often quite large — up t o several mg i n trace analyses. Newer instruments are designed f o r easier source cleaning or u t i l z e i o n volumes which are replaceable through the d i r e c t probe i n l e t . I t i s p o s s i b l e t o change the e n t i r e source volume w i t h each sample and thus minimize t h i s problem. Less e a s i l y ameliorated i s the phenomenon known as the "matrix e f f e c t " . I d e a l l y , the chemical i o n i z a t i o n source produces one i o n f o r each constituent o f the mixture, and the r e l a t i v e abundances o f the ions formed are i n proportion t o the amount o f constituent present. The "matrix e f f e c t " i s a term t h a t describes enhancement or suppression o f i o n s i g n a l f o r a s i n g l e component due t o the presence of the matrix. This i s an i n s i d i o u s problem because the matrix i s not characterized, and may change from sample t o sample. For targeted compound a n a l y s i s , the usual s o l u t i o n i s t o employ an i s o t o p i c a l l y l a b e l l e d i n t e r n a l standard that i s introduced i n t o the mixture as a whole. Quantitation o f the s i g n a l o f i n t e r e s t i s then derived from the r e l a t i v e abundances o f the ions corresponding t o the l a b e l l e d and unlabelled forms o f the analyte. Since the unlabel l e d / l a b e l l e d i o n p a i r p e r s i s t s i n many o f the daughter ions formed by c o l l i s i o n , several confirming r a t i o s can be obtained i n a s i n g l e experiment. The matrix e f f e c t a l s o may be evident i n chemical noise which p e r s i s t s i n the spectrum, and i s f a r greater i n analyses near the detection l i m i t . This was h i g h l i g h t e d i n the paper by Bursey (21) i n which a matrix e f f e c t i n the determination of polychlorinated organic compounds was found. D i r e c t probe MS/MS r e s u l t s were systematically high compared w i t h those from GC/MS o r GC/MS/MS. The r a p i d throughput p o s s i b l e i n an MS/MS screening protocol i s not obtained without concomitant r i s k . MS/MS i s an empirical method of a n a l y s i s . As i s evident from the examples presented, the i n t e r p r e t a t i o n of a daughter i o n MS/MS spectrum i s o f t e n based on the same c o r r e l a t i o n p r i n c i p l e s derived from e l e c t r o n and chemical i o n i z a t i o n mass spectrometry. More often, the comparison o f the spectrum obtained t o t h a t o f the authentic compound i s used f o r i d e n t i f i c a t i o n . This i s a fundamentally u n s a t i s f y i n g procedure. While e l e c t r o n and chemical i o n i z a t i o n spectra can be compared t o a s p e c t r a l l i b r a r y which has been compiled over the past t h i r t y years, no comparable l i b r a r y o f MS/MS spectra e x i s t s . Data systems may be used w i t h i n i n d i v i d u a l

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laboratories t o create l i b r a r i e s o f s p e c t r a l data which are then searched i n the usual manner, but n a t i o n a l and i n t e r n a t i o n a l databases are nonexistent. Several obstacles must be overcome t o create such bases. F i r s t i s the d i v i s i o n o f MS/MS spectra i n t o those r e s u l t i n g from high energy c o l l i s i o n processes (as on sector mass spectrometers) and those obtained under low energy c o l l i s i o n conditions as p r e v a i l on the m u l t i p l e quadrupole instruments. The spectra thus obtained are often s i m i l a r , but perhaps not t o the p o i n t where a cross c o r r e l a t i o n can be drawn. Charge s t r i p p i n g and charge i n v e r s i o n are processes t h a t are confined l a r g e l y t o high energy MS/MS spectra, f o r example. The l e s s than u n i t mass r e s o l u t i o n o f the reverse geometry sector instruments f o r daughter ion MS/MS spectra WDuld be a problem i n reducing spectra i n t o a standard l i b r a r y form, as both the masses and the r e l a t i v e abundances o f the daughter ions are known w i t h a l i m i t e d c e r t a i n t y . F i n a l l y , the low energy daughter i o n MS/MS spectra are affected by instrument parameters such as the c o l l i s i o n energy and the c o l l i s i o n gas pressure. To date, no general standard o f operation has been accepted. While these s p e c t r a l e f f e c t s are valuable i n e x t r a c t i n g a d d i t i o n a l information frcm the MS/MS spectrum, they do represent a s i g n i f i c a n t obstacle t o the standardization of MS/MS l i b r a r i e s . The most hopeful d i r e c t i o n might be i n advanced data systems w i t h memory s u f f i c i e n t t o accept a l l spectra obtained, and searching algorithms sophisticated enough to deal w i t h m u l t i p l e spectra o f a s i n g l e compound. As mentioned e a r l i e r , q u a n t i t a t i o n w i t h MS/MS i s often c a r r i e d out w i t h i n t e r n a l standards. Without standards, the accuracy and p r e c i s i o n o f q u a n t i t a t i o n i s reduced due t o matrix e f f e c t s . Rough estimates can be q u i c k l y obtained with MS/MS. For many problems, t h i s information i s more than s u f f i c i e n t . For instance, new drugs are o f t e n derived d i r e c t l y frcm p l a n t m a t e r i a l . One o f the f i r s t questions asked i s the r e l a t i v e concentration o f the desired material i n the various p l a n t p a r t s . MS/MS has been used t o provide the approximate amounts o f the targeted compound i n roots, stems, p e t a l s , o r flowers. The p l a n t t i s s u e w i t h the highest concentration o f compound i s then extracted. In food and f l a v o r analyses, the accuracy of the q u a n t i t a t i v e data required from MS/MS may be l i m i t e d by the v a r i a b i l i t y of the sample i t s e l f . In a n a l y s i s o f a large number o f samples, MS/MS provides a quick i n d i c a t i o n o f the amounts o f compounds o f i n t e r e s t . I f v a r i a b i l i t y f a l l s outside o f a preset tolerance, then only those samples are flagged f o r more exhaustive workup and a more rigorous q u a n t i t a t i v e a n a l y s i s . This a b i l i t y t o focus a n a l y t i c a l resources on samples o f i n t e r e s t i s a valuable property of the MS/MS experiment. P o t e n t i a l . The development o f MS/MS f o r analyses o f foods and f l a v o r s w i l l f o l l o w the same growth curve as i t has i n other a p p l i c a t i o n s . A t t h i s point, only the f i r s t part o f the growth curve i s evident. Several i n d u s t r i a l l a b o r a t o r i e s are beginning t o use MS/MS on a routine b a s i s , and commercial pressure w i l l d r i v e the exp>ansion o f the method. The speed o f the MS/MS a n a l y s i s i s a strong i n i t i a l advantage. I n the long term, i t i s l i k e l y t o be the f l e x i b i l i t y of MS/MS a n a l y s i s t h a t w i l l s u s t a i n i t s use i n these areas, and j u s t i f y the high i n i t i a l cost o f the instrument. I t i s

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only a matter o f programming the experiment that d i f f e r e n t i a t e s the a n a l y s i s o f nutmeg from the a n a l y s i s o f acetate esters emitted as v o l a t i l e s frcm f r u i t . A l l these protocols can be developed and then stored w i t h i n the data system as standard methods o f a n a l y s i s , and then c a l l e d up as needed. The a b i l i t y o f MS/MS t o search f o r classes o f compounds i n a mixture w i l l be as valuable i n food and f l a v o r analyses as i t i s i n other complex mixture analyses, such as the pharmaceutical o r environmental f i e l d s . Flavors are complex mixtures, but o f t e n c o n s i s t o f groups o f chemically s i m i l a r compounds. I t i s p r e c i s e l y the i d e n t i f i c a t i o n o f these groups f o r which parent i o n and n e u t r a l l o s s MS/MS experiments are p a r t i c u l a r l y adept. This i s a c h a r a c t e r i s t i c that i s p a t e n t l y not a v a i l a b l e w i t h GC/MS, which has been the usual method o f analyses o f these mixtures. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21.

Kruger, T. L.; Litton, J. F.; Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1976, 48, 2113. Kbndrat, R. W.; McClusky, G. A.; Cooks, R. G. Anal. Chem. 1978, 50, 1222. Kruger, T. L.; Litton, J. F.; Cooks, R. G. Anal. Lett. 1976, 9, 533-542. McLafferty, F. W., Bd., "Tandem Mass Spectrometry"; Wiley: New York, 1983. Cooks, R. G., Beynon, J. H.; Caprioli, R. M.; Lester, G. R., "Metastable Ions"; Elsevier: Amsterdam, 1973. Kbndrat, R. W.; Cooks, R. G. Anal. Chem. 1978, 50, 1251A. Harvey, D. J. J. Chrom. 1975, 110, 91-102. Plattner, R. D.; Yates, S. G.; Porter, J. K. J. Agric. Food Chem. 1983, 31, 785-789. Richter, W. J.; Blum, W.; Schlunegger, U. P.; Senn, M. In "Tandem Mass Spectrometry"; McLafferty, F. W., Ed.; Wiley: New York, 1983. Fetterolf, D. D. y Yost, R. A. Int. J. Mass Spectrom. Ion Proc. 1984, 62, 33-50. Hirschfeld, T. A. Anal. Chem. 1980, 52, 297A. Glish, G. L.; Shaddock, V. M.; Harmon, K.; Cooks, R. G. Anal. Chem. 1980, 52, 165-167. Kirby, H.; Sokolow, S.; Steiner, S. In "Tandem Mass Spectrometry"; McLafferty, F. W., Ed.; Wiley: New York, 1983. Issenberg, P.; Kobayashi, A.; Mysliwy. T. J. J. Agric. Food Chem. 1969, 17, 1377-1386. Horman, I. Gazz. Chim. Italiana 1984, 114, 297-303. Horman, I. Bicmed. Mass Spectrom. 1981, 8, 384. Davis, D. V.; Cooks, R. G. J. Agric. Food Chem. 1982, 30 495-504. Zakett, D,; Cooks, R. G. In "New Appraoches in Coal Chemistry"; Blaustein, B. D.; Bockrath, B. C.; Friedman, S., Eds.; ACS SYMPOSIUM SERIES No. 169, American Chemical Society: Washington, D. C., 1981; pp. 267-288. Labows, J. N.; Shushan, B. Amer. Lab. 1983, 15(3), 56-61. Beynon, J. H.; Cooks, R. G.; Amy, J. W.; Baitinger, W. E.; Ridley, R. Y. Anal. Chem. 1973, 45, 1023A. Voyksner, R. D.; Hass, R. D.; Sovocool, G. W.; Bursey, M. M. Anal. Chem. 1983, 55, 744-749.

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Tandem Mass Spectrometry Applied to the Characterization of

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