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Anal. Chem. 1982, 5 4 , 1466-1471
(19) Beckey, H. D. "Prlnclples of Field Ionization and Field Desorption Mass Spectrometry"; Pergamon: Oxford, 1977. (20) Brent, D. A.; Rouse, D. J.; Sammons, M. C.; Bursey, M. M. Tetrahedron Lett. 1973, 4127. (21) Schulten, H.-R.; Rollgen, F. W. Org. Mass Spectrom. 1975, 70, 649. (22) Schulten, H A . ; Rollgen, F. W. Angew. Chem., Int. M .Engl.. 1975, 74, 561. (23) Veith, H. J. Org. Mass Spectrom. 1978, 11, 629. (24) Sanders, R. A.; De Stefano, A. J.; Keough. T. Org. Mass Spectrom. 1980, 15, 348. (25) Glerllch, H. H.; Rollgen, F. W.; Borchers, F.; Levsen, K. Org. Mass Spectrom. 1977, 12, 387. (26) Velth. H. J. Org. Mass Spectrom. 1978, 73,280. (27) VeRh, H. J. Adv. Mass Spectrom. 1980, 8, 766.
(28) Flscher, M.; Velth, H. J. Helv. Chim. Acta 1978, 67, 3038. (29) Large, R.; Knof, H. J . Chem. Soc., Chem. Commun. 1974, 935. (30) Schulten, H A . ; Kiimmler, D. Z . Anal. Chem. 1978, 278, 13.
RECEIVED for review December 29,1981. Accepted March 3, 1982. This investigation was supported by the "Wissenschaftsministerium Dusseldorf", the "Fonds der Chemischen Industrie, Frankfurt", the Foundation for Fundamental Research on Matter (FOM), and the Netherlands Organization for the Advancement of Pure Research (ZWO).
Structural Elucidation of Drug Metabolites by Triple-Quadrupole Mass Spectrometry Robert J. Perchalskl Research Service, Veterans Admlnistratlon Medical Center, and College of Pharmacy, University of Florida, Galnesville, Florida 32602
Richard A. Yost* Department of Chemistty, University of Florida, Gainesville, Florida 326 1 1
B. J. Wilder Neurology Service, Veterans Administration Medical Center, and College of Medicine, University of Florida, Galnesville, Florida 32602
A tandem quadrupole mass spectrometer wlth a center quadrupole colllslon chamber Is used to determine structures of drug metabolltes by analysts of one plasma or urlne extract. After a chemical Ionization spectrum of the pure drug Is obtalned, a daughter Ion experlment Is run, passing the molecular Ion and characterlstlc fragment Ions through quad 1. Since metabolltes generally contaln a large portlon of the parent drug In their structure, one or more of the daughter ions of the pure drug should be present In each metabollte spectrum. An extract of plasma or hydrolyzed urlne Is then run elther In the parent Ion mode or In the neutral loss mode to obtain, respectively, a spectrum of all parent Ions that fragment to produce the daughter Ions characteristic of the pure drug or a spectruni of parent Ions that fragment by loss of the selected mass difference. Finally, the extract Is run In the daughter Ion mode, to obtain complete daughter spectra of each parent. By appllcatlon of a knowledge of typlcal metabolic pathways, most, If not all, of the metabolltes of a drug can be found In minutes or hours. The valldlty and utlllty of thls method are shown for prlmldone, chromide, and phenytoln.
The marriage of chromatographic methods, particularly gas-liquid chromatography, with mass spectrometry has long been used to successfully determine structures of drug metabolites. Numerous examples of the techniques employed can be found in the continuing series of Gudzinowicz and Gudzinowicz ( I ) and the volume edited by Frigerio and Ghisalberti (2). Methods generally involve some pretreatment of the sample to free conjugated or protein-bound species, followed by liquid-liquid or liquid-solid extraction to separate basic, acidic, and neutral components and endogenous in0003-2700/82/0354-1466$01.25/0
terferences. Since many metabolites are polar molecules, derivatization may be employed to increase volatility, thermal stability, and chromatographic compatibility. This final item is important because compounds that have dissimilar functional groups may not all be detectable under a single set of chromatographic conditions. Therefore, in a metabolite search in which the compounds sought are unknown, use of only one set of conditions may preclude the discovery of one or more species, and developing multiple chromatographic systems takes considerable time. The advent of mixture analysis by tandem mass spectrometry (MS/MS) pioneered in the laboratories of Cooks ( 3 , 4 ) and of McLafferty (5) with the reversed-geometry, doublefocusing mass spectrometer has obviated the need for introduction of a pure sample into the mass spectrometer (chromatographically or on solids probe) and has made possible the analysis of mass-separated ions by a second, on-line technique. In the instrument developed by Cooks and coworkers, the second stage is a kinetic energy analyzer (electric sector) giving rise to the pseudonym MIKES or mass-analyzed ion kinetic energy spectrometry. MS/MS has now been made relatively easy and practica1 by the triple-quadrupole mass spectrometer, introduced by Yost and Enke (6). This instrument consists of an ion source, three quadrupole mass filters, and an ion detector, all in series. The first and third quadrupoles can be set to scan a range of masses or to select one or more molecular or fragment ions. The second quadrupole is enclosed in a cylinder and allows all masses to pass. A collision gas can be introduced into quad 2 to cause fragmentation of ions passed through quad 1 by collisionally activated decomposition (CAD). The daughter ions are then mass analyzed in quad 3. The primary modes of operation are listed in Table I. The quadrupoles can be scanned rapidly, and the various operational modes can be selected by computer at will. These 0 1982 American Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
Table I. Common Operational Modes of the Triple-Stage Quadrupole Mass Spectrometer mode quad 1 quad 2 quad 3 (1)single stage mass pass all m / z no collision gas; scan m / z spectrometera pass all m / z (2) parent ion experiment
scan m/z
collision gas;& pass all m / z
select m / z (up to 25 ions)c
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spectrum normal mass spectrum (CI, EI, positive and/or negative ion) spectrum of all ions that fragment to give selected m / z spectrum of all ions that arise from fragmentation of selected m/z
(a) scan m / z or (b) select up to 4c daughter ions for each ion passed through quad Id spectrum of all daughter (4)neutral loss experiment scan m / z collision gas ;b scan m / z at same ions that result from rate as quad 1but (linked scan) pass all m / z with a constant loss of Mc mass difference, M aAlternatively, quad 1 may be scanned while quad 3 passes all m / z . bIf no collision gas is present, the unimolecular dissociation products (mietastable ions) will be observed. Characteristic of Finnigan triple quadrupole MS/MS data system software. dSelected reaction monitoring. (3) daughter ion
experiment
select m / z (up to 25 ions)c
collision gas ;b pass all m/z
capabilities make the instrument ideally suited to determining the structures of drug metabolites. Since most metabolites retain a relatively large part of the parent drug structure, it is reasonable to assume that their daughter iepectra will contain one or more of the fragments that appear in the daughter spectrum of the pure drug. Therefore, if an extract of urine or plasma from a person taking a particulrv drug is analyzed by scanning quad 1 and selecting these corresponding fragmenta in quad 3, the resulting parent spectrum should contain all molecular ions that produce the common fragments. Then, by reversing the instrument and scanning quad 3 while selecting these molecular ions in quad 1, complete daughter spectra are obtained that can be used to identify each selected ion. In some instances, drugs and metabolites may contain identical groups or atomm that are easily lost. In a neutral loss experiment, the first and third quadrupoles are scanned with a particular mass difference. The resulting spectrum gives all parent ions (drug and metabolite molecular ions) that fragment by loss of the selected mass. The daughter spectrum of each parent ion is then obtained for identification. Because all components of a sample are ionized under a single set of conditions, there is no need to spend time developing chromatographic methods or waiting for components of interest to elute. In this report, we describe this method that allows determination of metabolite structure from one extract of urine (2 mL) or plasma (1 mL)i in as little as 30 min, and we give examples that show the power of the special operational modes of the tandem mass spectrometer.
EXPERIMENTAL SECTION Apparatus. A Finnigan triple-stage quadrupole mass spectrometer/data system was1 used for all studies. Samples were introduced on a solids probe that was heated ballistically from 25 to 350 OC in 2 min. Spectra were recorded over the temperature range of interest for each sample. Instrumental parameters were optimized for peak shape and sensitivity by using perfluorotributylamine. Electron eneirgies of 70 and 100 eV were used for electron impact (EI) and clhemical ionization (CI), respectively. The CI reagent gas was methane at an ionizer pressure of 40 Pa. Nitrogen collision gas was introduced into quad 2 at a pressure of 0.27 Pa. Collision energy was set to 18-20 eV. The continuous dynode electron multiplier was typically run a t -1500 V with the positive and negative conversion dynodes at h3000 V. Reagents. All chemicals were analytical reagent grade. Primidone, phenytoin, and phenobarbital were recrystallized from methanol, ethanol, and an ethanol-water mixture, respectively. Phenylethylmalonamide and 5-(p-hydroxyphenyl)-5-phenylhydantoin were used as received from Aldrich Chemical Co. Chromide (3-bromo-N-ethyllamamide),3-bromocinnamamide, and 3-bromocinnamic acid were gifts from the Burroughs-
Wellcome Co. Samples of plasma and urine were obtained from Neurological Enterprises, Inc., Gainesville, FL, or the Veterans Administration Medical Center, Neurology Clinic. Procedure. The plasma samples were extracted by a previously published method (7). The urine sample was hydrolyzed at 100 "C for 30 min with an equal volume of concentrated HC1, adjusted to a pH of about 2 and extracted with 10 mL of ethyl acetate. Residues from the extracts were dissolved in chloroform (200 p L ) and a 2-pL aliquot was evaporated in a probe vial for insertion into the mass spectrometer. All MS operations were under computer control. During operations that involve selection of a number of specific ions to pass through quads 1 or 3, the instrument selects ions in succession and scans as required, starting over after the last ion is passed. During data analysis, the spectra can be summed over all scans to give an output that shows results for all ions or can be split to give the sum of selected scans for each specific ion. The full procedure for metabolite identificationconsists of the following steps: (1) Run a normal CI spectrum for the pure parent drug. Chemical ionization is used in preference to electron impact because of increased sensitivity and spectral simplicity. (2) Obtain daughter spectra for the molecular ion and most representative fragment ions of the pure parent drug. Include the primary CI addition product (e.g., [M + C2H5]+for methane reagent gas). (3) Evaluate the summed (all parents) and split (single parent) daughter spectra and choose the most abundant daughter ions for the parent ion experiment. (4) Run the parent ion experiment on the plasma or urine extract, selecting the daughter ions chosen in step 3. (5) Evaluate the split parent spectra and choose, for the daughter ion experiment, ions that have masses greater than the mass of the selected daughter but are not contained in the normal mass spectrum of the pure drug. (6) Run the daughter ion experiment on the plasma or urine extract, selecting the parent ions chosen in step 5. (7) Determine the structure of each parent from the split daughter spectrum either by interpretation or by comparison with the daughter spectrum of the pure reference standard. In certain instances in which the parent drug contains an atom or group of atoms that is easily lost in the mass spectrometer but not by metabolism (e.g., halogen), steps 3-5 can be replaced by the following: (3) Run one or more neutral loss experiments on the plasma or urine extract, setting the mass difference between quads 1 and 3 to that of the lost fragment (e.g., 36Cl,s7Cl). In some instances, daughter ions may be found in the final daughter ion experiment that cannot be assigned to any specific parent. In this case, steps 4 through 7 may be repeated. RESULTS AND DISCUSSION To show the utility and applicability of this method and to illustrate the procedure, we have chosen three examples
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
oo H
'sH5 'ZH5
"
0
PRIMIDONE, M.W. 218
'gH5
N-H
'ZH5
c 6 H z c 1 1 C2H5
0
PHENOBARBITAL
PHENYLETHYLMALONAMIDE M.W. 2 0 6
M.W. 232
Flgure 1. Prlmldone and ,metabolites.
E
11
I
QUAD ONE PASSING
19
I
207
151 d
M/Z
M/Z
100
150
Id,
.I
200
Flgure 3. Daughter spectra of pure primidone (A) and primidone from plasma extract (B).
'
,
,g[J/
I
!,,,.,,,
134 145 100
/?g,
150
250
200
1
IO0
233
200
150
,
247
I
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, / ,
174
250
D
1
I233 QUAD THREE PASSING 162 219
I I . M/Z
I
IO0
I50
I
, I d 200
250
Flgure 2. C I (A), daughter (B) and parent spectra (C,D) of prlmldone.
of drugs metabolized by different but common metabolic pathways. In two cases the metabolites are present in plasma,
and in the third case the metabolite is largely present in urine as a glucuronic acid conjugate. Example 1. Primidone. Primidone is a barbiturate anticonvulsant that is metabolized to phenobarbital and phenylethylmalonamide (Figure 1). All three are physiologically active and present in the plasma of patients taking primidone. Figure 2A,B shows the results of steps 1 and 2. Spectrum A is the CI spectrum of pure primidone and spectrum B is the summation of all scans accumulated during the vaporization of primidone while quad 1 was passing the indicated ions (daughter spectrum). Figure 2C,D shows the results of steps 3-5 (parent ion experiment, plasma extract). Spectrum C is the summation of scans accumulated while passing masses 91, 106, 119, and 162 through quad 3. Of the ions to be selected for the parent experiment, those that are contained in the daughter spectrum of the molecular ion of the pure drug are weighted more heavily than those of fragment ions. Daughter ions contained in the daughter spectra of CI fragments are included in the parent experiment only if they are relatively abundant and therefore represent a particularly stable structure. Spectrum D is the parent spectrum of the most abundant fragment, 162. Mass 190 is in the normal CI spectrum of the pure drug, so it is probably not a metabolite. This was confirmed by matching the daughter spectrum of 190 from step 2 with that obtained in step 6. The major parents are as follows: 207, (M + H)+ for phenylethylmalonamide; 219, (M + H)+ for primidone; and 233, (M + H)' for phenobarbital. Additional proof that these are true molecular ions is given by the obvious (M C2H5)+addition product for each. The daughter spectrum of each of these shows the appropriate molecular ion plus corresponding fragmentation. Figures 3-5 show the daughter spectra of the pure drug and metabolites along with those obtained by selecting the molecular ions of each in the plasma extractdaughter ion experiment (step 6). An obvious application of parent spectra that is evident from Figure 2D is quantitation of drugs and metabolites from the parent spectrum of the most abundant common daughter. The simultaneous generation of all signals for quantitation from a single fragment should maximize precision. An appropriate internal standard, possibly a stable-isotope-enriched analogue of the parent drug, labeled at some position within the lost fragment, would also give the same daughter ion.
+
ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
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Table 11. CI and Daughter Spectra of Chromide (mol w t 253, 255) daughter ions (abundance, %) (M t ]-I)+ (M + 29)' (M t 41)' spectrum 257 (17), 255 (17), 211 (4), 209 ( 4 ) 254 (1lOO) 282 (12) 294 (2) CI 256 (9'7) 284 (12) 296 (2) 183 (7), 181 (6), 175 ( l l ) , 174 (ll),131 (6), 103 ( l l ) , 72 (17) 282 (11) 257 (15), 255 (15), 212 (5), 211 (44) daughteP 254 (1100) 256 (9cB) 284 (11) 210 (5), 209 (46), 183 (4), 181 (3), 175 (7), 174 (8), 132 (7), 131 (4), 130 (2), 103 (7) aSummed spectrum. Quad 1 passing parrent ions 254, 256, 256, 257, 282, and 284. Table 111. Daughter Spectra of 3-Bromocinnamamide (mol wt 225, 227)a sample parent ion, daughter ions (abundance, %) pure 226 (100) 209 (73), 184 (3), 183 (7), 181 (ll),147 (6), 146 (14), 130 (2), 104 (l),103 (2), 102 (1) extract 226 (100) 225 (20), 209 (56), 184 (2), 183 (7), 181 (6), 147 (16), 146 (6), 131 ( l ) 130 , ( l ) , 104 (3), 103 ( l ) 1012 , (1) 211 (73), 186 (3), 185 (7), 183 (ll),147 (6), 146 (14), 130 (2), 104 (6), 103 (2), 102 (1) Pure 228 (100) extract 228 (100) 211 (30), 210 (44), 186 (5), 185 (lo), 183 (3), 182 (7), 149 ( I ) , 147 (lo), 146 (6), 131 ( l ) , 130 (3), 129 (3), 104 (3), 103 (3), 102 (1) a Quad 1 passing (M t If)+.
OUAD ONE PASSING 233
2 33
177
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I
119
134 145 M/Z
100
174 190
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2)j
105 'I9
205
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91
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OUAD ONE PASSING 233
,Il, ++-%A IO6
;;5
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~
,
,
100
M/Z
150
,
1
I,,,I,,, I; 1
105
,
: ;I,
M/Z
200
134
150
100
Flgure 4. Daughter spectra (of pure phenylethylrnalonamide (A) and phenylethylmalonamide from plasma extract (B).
Figure 5. Daughter spectra of pure phenobarbital (A) and phenobarbltal from plasma extract (B).
Example 2. Cinromidt: Until recently, cinromide was an experimental, broad-specitrum anticonvulsant being used in clinical trials in the United States and Canada. Plasma concentrations of cinromide and ita major metabolites (Figure 6), 3-bromocinnamamide and 3-bromocinnamic acid, are usually less than 1 pg/mLl, 5 bg/mL, and 50 pg/mL, respectively, in patients taking therapeutic doses. All three are physiologically active; however, they are ncrt equally potent. The loss of bromine, evident in the CI and daughter spectra of pure cinromide (Table 11) as m/z 175 (loss of 7DBrand slBr from mlz 254 and 256, respectively), makes this drug a prime candidate for the neutral loss experiment. Figure 7A shows, for the plasma extract, the neutral loss spectirum derived from a linked scan of quads 1 arnd 3 with a mass1 offset of 79. In addition to the ions that are characteristic of the parent drug, there is a new group at m/z 226,227,228. Another neutral-loss experiment, run with a mass offset of 81, gave a similar spectrum that showed ionm a t m / z 228, 229, and 230. The parent spectrum (Figure 7H) of the plasma extract run with the instrument in the parent ion mode, selecting the daughter ions characteristic of pure chromide in quad 3, shows the same group of ions at m/z 226-4330.
ClNROMlDE
,,/ ' L . W .
N - deal kyl at i on
d
2 5 3 , 255
q C H = C H C ' N H \2
L hydrolysis
e'r M.W.
\hydrolysis
o
225, 227
~
H O'>CH=CH,Q
Eir M.W. 2 2 6 , 2 2 8
Figure 8. Chromide and metabolltes.
Tables I11 and IV, respectively, compare the CAD fragmentation patterns of pure 3-bromocinnamamide and 3bromocinnamic acid with those obtained from the plasma extracts. These show that the parent ions a t m/z 226, 228 arise from the amide, and those at m/z 227,229 arise from the acid. The parent ion at mlz 230 is an isotope peak at (M + 2)' of 3-bromocinnami~-~~Br acid that is primarily due to the contribution of 13C. Figure 8 shows the daughter spectra of m/z 230 (A), 226 (B), and 228 (C) and verifies that the
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
i
I
MAS S OFFSET
227
OUAD ONE PASSING 254
79
I
226
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209
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1
209
I I03
174
132
/ .
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150
200
226
I .
250
Flgure 7. Neutral loss (mass offset: 79) (A) and parent spectra (B) of cinromlde plasma extract.
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QUAD ONE PASSING 230
PHENYTOIN, M.W. 2 5 2
Y
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-Yd
0
p -rlYDROXY
M.W
1
2 61
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1 QUAD ONE PASSING
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'r I 100
150
268
Flgure 10. Phenytoin and metabolite.
+
M/Z
PHENY TOIN
200
Flgure 8. Daughter spectra of m l z 230 (A), m l z 226 (B), and m l z 228 (C) from plasma extract. parent ion at m / z 228 is actually a composite of the characteristics of m/z 226 and m / z 230. Fragmentation indicates both loss of HzO (characteristic of the acid) and loss of NH3 (characteristic of the amide). The high concentration of the acid metabolite (70 wg/mL of plasma in this sample) gives an
+
isotope peak (M 2)+of greater intensity than the (M H)+ ion of the amide. Figure 9 shows the daughter spectra of the 79Brmolecular ions (mlz 254) obtained from a sample of pure compound (A) and the corresponding molecular ion from the plasma extract (B) for cinromide. The spectra show identical fragmentation patterns, except that for the ion at m / z 226 from the plasma extract. This fragment indicates that part of the molecular ion intensity of the plasma extract is due to the (M + C2Hs)+ addition product of the 3-bromocinnamamide (mol wt 225). The spectra of the 81Brmolecular ions (m/z 256) of cinromide showed a similar fragment at mlz 228. The difference in the relative intensity of the parent and fragment ions in the two spectra can be attributed to concentration or matrix effects. Example 3. Phenytoin. Phenytoin is a hydantoin anticonvulsant that is metabolized primarily by parahydroxylation (Figure 10). The metabolite is conjugated with glucuronic acid and excreted in the urine. Other metabolites are known (catechol, hydantoic acid); however, acid hydrolysis converts these to 5-(p-hydroxyphenyl)-5-phenylhydantoin. This example is straightforward and is included to show use of this procedure with a urine hydrolysate, normally considered to be a fairly complex sample. Figure 11 shows the summed daughter spectrum of pure phenytoin and the parent spectrum of the m / z 175 daughter from the urine extract. The molecular ions, as well as CzH5+ and C3H5+addition products at m / z 253, 281, and 293, respectively, for phenytoin and m / z 269, 297, and 309 for the
ANALYTICAL CHEMISTRY, VOL. 54, NO. 9, AUGUST 1982
1471
Table IV, Daughter Spectra of 3-Bromocinnamic Acid (mol wt 226,228)" sample parent ion daughter ions (abundance, %) pure 227 (44) 209 (loo), 185 (24),181 (ll), 157 (3),148 (27),147 (12),130 (2),104 ( l ) ,102 (1) extract 227 (4'1) 209 (loo), 199 (l),191 (3),185 (28),181 (lo), 157 (3),148 (31),147 ( 8 ) , 130 (2), 104 (l),102 (1) 211 (loci), 187 (23),183 (lo), 159 (3),148 (27),147 (12),130 (2),104 (l), 102 (1) pure 229 (44) extract 229 (58) 211 (lOOl), 201 (2),187 (27),183 (13),159 (4),148 (35),147 (4),130 (2),104 ( l ) , 103 (4),102 (1) aQuad 1passing (M t 13)'. Table V. Daughter Spectra of Phenytoin (mol wt 252)and 5-(p-Hydroxyphenyl)-5-phenylhydantoin(mol wt 268)a sample parent ion daughter ions (abundance, %) phenytoin pure 253 (31) 225 (14),210 (13),182 (loo), 175 (32),132 (3),104 (3) extract 253 (59) 235 (7),225 (15),210 (13),182 (loo), 175 (35),132 (4),104 (4), plus other fragments < 5% p-hydroxy phenytoin pure
251 (9),241 (16),226 (3),208 ( 8 ) , 198 (loo), 191 (3),175 (28), 148 (l), 132 (l), 104 (1) 251 (8),241 (18),226 (3),208 (7),198 (loo), 191 (3),175 (24), 148 (4),132 (l), 104 (2)
269 (25)
extract
269 (27)
=Quad 1 passing (M
+ €I)+. mild deproteinization of plasma, and hydrolysis and extraction of urine are necessary to obtain free, volatile species in a volatile solvent. This simple work-up takes less time than the evaporation of 3 KL of plasma or urine in a solids probe vial at room temperature and gives a sample that is much easier to handle. The tandem mass spectrometer is a unique and versatile instrument that can be used to great advantage in some areas where gas-liquid and high-pressure liquid chromatography are now applied. In addition to metabolite studies, we are currently expanding the field of application of patent and neutral loss spectra to include kinetic and mechanistic studies of fast chemical reactions, such as on-column derivatization reactions for gas chromatography, and simultaneous quantitation of multiple parent ions from single daughter ions.
182 QUAD ONE PASSING 175. 182. 210, 225. 253, 281. 293
1
1, M/Z
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,
,I14 ,172, , IO0
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, I ] , 1, Jzr,
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200
'
300
I 259 OUAD THREE
PASSING
1
ACKNOWLEDGMENT The authors thank Dora M. Mitchell for technical assistance, Dean D. Fetterolf and Harry 0. Brotherton for helpful discussions, and Alice Cullu for editorial assistance.
LITERATURE CITED Figure 11. Daughter spectrum of pure phenytoin spectrum of m l z 175 from urine extract (B).
(A)
and parent
hydroxylated metabolite are obvious. Table V gives the CAD fragmentation patterns obtained for the molecular ions of phenytoin and its metabolite from pure standards and the plasma extract. This method can be app tied generally to in vivo and in vitro chemical systems that involve transformation of one compound into one or more structurally related compounds. In metabolite studies, a knowledge of common metabolic pathways is helpful (8). It should be possible in some cases to perform this analysis directly on plasma or urine without sample pretreatment. However, under normal circumstances in which drugs and metabolites are bound to plasma protein, or conjugated with glucuronic acid or sulfate, extraction or
(1) Gudzlnowicz, B. J., Gudzinowicz, M. J., Eds. "Analysis of Drugs and Metabolites by Gas Chromatograph-Mass Spectrometry"; Marcel Dekker: New York, 1977, Vols 1-3; 1978, Vols 4 and 5; 1979, Vol 6; 1960, Vol 7. (2) Frigerlo, A., Ghlsalberti, E. L., Eds. "Mass Spectrometry in Drug Metabolism"; Plenum Press: New York, 1977. (3) Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1978, 50, 81A-92A. (4) Glish, G. L.; Shaddock, V. M.; Harmon, K.; Cooks, R. G. Anal. Chem. 1980, 52,165-167. (5) McLafferty, F. W.; Bockhoff, F . M. Anal. Chern. 1978, 5 0 , 69-76. (6) Yost, R. A.; Enke, C. G. Anal. Chem. 1979, 51, 1251A-1264A. (7) Perchalskl, R. J.; Bruni, J.; Wilder, B. J.; Willmore, L. J. J . Chromatogr. 1979, 163, 187-193. (8) Fingl, E.; Woodbury, D. M. "The Pharmacological Basis of Therapeutics", 5th ed.; Goodman, L. S., Goodman, A,, Eds.; MacMilIan: New York, 1975; Chapter 1, pp 11-18.
RECEIVED for review February
22, 1982. Accepted April 27, 1982. This work was supported by the Medical Research Service of the Veterans Administration, the Epilepsy Research Foundation of Florida, Inc., and the National Science Foundation, Grant No. CHE-8106533.
