ANALYTICAL
25, 532448 (1968)
BIOCHEMISTRY
Mass
Fragmentography
Identification of Chlorpromazine and Its Metabolites in Human Blood by a New Method
CARL-GUSTAF
HAMMAR
AND BO HOLMSTEDT
Department of Toxicology, Swedish Medical Research Council, Karolinska Znstitutet, Stockholm 60, Sweden AND
RAGNAR Department
of Mass Spectrometry,
RYHAGE
Karolinska
Znstitutet,
Stockholm
60, Sweden
Received January 22, 1968
Chlorpromazine, one of the most important drugs in the treatment of mental illness, has been used empirically since 1952. Evidence for its metabolism in man rests mainly on the determination of degradation products and conjugates in urine. These analyses have been performed using both traditional and more unconventional methods (l-11). With the present emphasis on the correlation of blood levels of drugs to therapeutic effects and side effects, it is desirable to have an analytical technique with a sufliciently good separation power and sensitivity to permit the determination in blood of chlorpromazine and its metabolites (12-13). Recent reports describe for this purpose the use of gas chromatography with the electron capture detector (1415). In this paper we present results obtained with a combination of gas chromatography and mass spectrometry (GLC-MS) (16). We have also introduced a new technique, which we call mass fragmentography (17). The major metabolic pathways of chlorpromazine are summarized in Figure 1. In human urine the parent compound and its metabolites have been found and sometimes quantitated by use of a variety of methods including spectrophotometry, paper chromatography, thin-layer chromatography, electrophoresis, and gas chromatography with flame ionization or microcoulometric detectors (l-11). In analyses of blood as compared to urine there is a need for higher sensitivity due to the small volumes available and the low concentrations of the metabolites. According to Forrest the number of theoretically possible metabolites is 168 of which more than 27 have been postulated in the literature (13). Johnson et oz. 542
MASS
0
REACT ION
+2
533
FRAGMEXTOGRAPHT
PRODUCT
OXIDATION
SULFOXIDE NITROGENOXIDE
zs-bo 9N+O
HYDROXYLATION
HYDROXIDE
-OH
I
0
C
N
N”
;i
J
G A T E D
EOR
&I*
DESMETHYLATION
AMINE
A T E D
Cl”2 I OXIDATIVE DEAMINATION
ACID
- COOH
0-R
9 CONJUGAflON FIG,
1. Metabolic
0”
pathways
C
I:
of chlorpromazine.
were able to find 10 nonconjugated metabolites in human urine by using gas chromatography (microcoulometric detector) (7). In recent years GLC-MS has been successfully used for the identification of, e.g., steroids, hormones, lipids, urinary acids, and terpenes (1924). To our knowledge the method has not previously been used for the characterization of drugs and their metabolites. In spite of not containing a high resolution mass spectrometer the combined instrument has proved to be extremely useful. Solving the structure of an unknown may present difficulties. With natural products these may be overcome by the accumulation of a fund of knowledge about the mass spectra and fragmentation pattern of related compounds (25). This is even more the case with modern drugs where the chemical structure is always known and synthetic analogs as a rule are readily available. This knowledge forms the basis for the interpretation of the structure of the drug metabolites. METHODS
: DOSAGE
AND
EXTRACTION
PROCEDURES
The individual dosage of chlorpromazine varies considerably. Usually 50 mg of chlorpromazine is given three times a day, but 300 mg and even more may sometimes be required. In order to be able to trap chlorpromazine and its metabolites we used 10-20 ml of blood, taken at various time intervals after each administration of the drug. After centrifugation both plasma and erythrocytes were used for analysis. Only blood from people on chronic treatment were used in the investigation. Samples of 5-10 ml of plasma were i&a&d in two ways. (I) The pH of the plasma was adjusted to 12 and it was extracted twice with dichloromethane. For chlorpromazine itself we have also used extraction with heptane containing 1.5% isoamyl alcohol (26). This is followed by washing with acetate buffer at pH 5.6 and extraction into
534
HAMMAR,
HOLMSTEDT,
AND
RYHAGE
Fra. 2A. Gas chromatogram of extracts of “normal” plasma and patient plasma and synthetic reference. Patient receiving 200 mg chlorpromazine daily. Extracts and reference treated with diazomethane. GLC conditions: Aerograph 204 equipped with ECD. 3% Versamid 900 on silanized Gas Chrom P (109-12OM). Column length 1.2 m and 1.8 mm i.d. Carrier gas nitrogen. Flow 20 ml/min. Injector temperature 23O”C, column temperature 210”, and detector temperature 225”.
MASS
535
FRAGMENTOGRAPHY
3
100, 90 se, 2 .5 c
70
.5 2
60
0 50 .-P ‘;; 40 5 = 30 20 IO 50
150
200
250
300
m/e
L350
cp- prop.-Ma-aster cxq Ch
+
IOO1 so$eo.+r .+r ?O?O22 w w E E ..50. 50. z z -= -= 4040-0 -0 w aL 30Kw 3020-
+t c‘OCH, 0”
100
319
232
I
I
246Lnt
SO-
!!
IO50
100
100
r 150
200
250
300
350
m/e Fro. 2B. Mass spectra of a compound in plasma extract and synthetic reference. Patient receiving 200 mg chlorpromazine daily. Extract and reference treated with diazomethane. Conditions: Instrument LKB-9000. GLC conditions as in Figure 2A. Carrier gas helium. Flow 20 ml/mm. Separator temperature 23O”C, ion source temperature 290”, ionization potential 70 eV, trap current 120 .uA, multiplier voltage 3.1 kV. Upper panel: Mass spectrum of compound from plasma extract. Lower panel: Mass spectrum of synthetic methyl-2-chlorophenothiazinyl propionate.
536
HAMMAR,
HOLMSTEDT,
AND
RYHAGE
0.1 N HCl.
The acid water phase is made alkaline and extracted with toluene containing 15% isoamyl alcohol. (II) The pH was adjusted to 3.8 and the plasma sample incubated with p-glucuronidase overnight at 37°C. The pH was then adjusted first to 12 and then to 1.5 and the plasma extracted at each pH with dichloromethane. The extract from pH 12 was treated with trifluoroacetic anhydride (TFAA) and that from pH 1.5 with diaxomethane. After these treatments both dichloromethane extracts are suitable for GLC. These extracts are supposed to contain the metabolites freed from possible conjugation. Erythrocytes were washed twice with saline and hemolyzed with water to the same volume as the plasma, incubated with p-glucuronidase as in the case of plasma, and extracted at pH 12. EXPERIMENTAL
Identification
AND
RESULTS
of b-Chlorophenothiazinylpropionic
Acid
The acid extract from patients on chlorpromazine gave a peak after esterification in the gas chromatogram that was never observed in normal plasma (Fig. 2A). In order to identify the metabolite a mass spectrum was recorded (Fig. 2B, upper panel). Search among reference spectra revealed a close similarity to methyl-2-chlorophenothiazinylpropionate (Cp-prop.-Me-ester) (Fig. 2B, lower panel). 2-Chlorophenothiazinylpropionic acid has recently been found to be a metabolite in urine (27). The analysis was repeated with the reference compound. The peak in plasma had exactly the same retention time as that of the synthetic substance. (The retention time of the ester relative to chlorpromazine is 1.6.) Mass spectra were also recorded before and after the plasma peak. No traces of Cp-prop.-Me-ester were detected in these mass spectra. The resemblance of the spectrum of the reference substance to that of the compound in plasma is such that they must be identical. The relative abundance of the two spectra is similar and no mass numbers are missing in the peak from plasma except that representing the small fragment m/e = 216. This probably results from background interference. A second plasma extract (from another patient) was treated in the same way and gave exactly the same result. However it should be remarked that 2-chlorophenothiazinylpropionic acid was completely absent from the plasma of still other patients. No effort has yet been made to estimate quantitatively the amount of the acid present in t,he plasma. Fragmentation Pattern of d-Chlorophenothiuzinyl
Derivatives
For reference purposes the mass spectra of the following compounds were recorded: chlorpromazine (as base), des- and dides-methylchlor-
MASS
537
FRAGMENTOGRAPHY
promazine (DMCP and DDMCP, respectively), 2-chlorophenothiazine (as bases and trifluoroacetates) , 2-chlorophenothiazinyl-propionic and -acetic acids (as methyl esters), 7-hydroxychlorpromazine and its desmethylated analogs (as bases or treated with diazomethane in order to convert the hydroxy group into a methoxy group. The two desmethylated compounds were subsequently reacted with trifluoroacetic anhydride). The mass spectra of these compounds invariably show, for each molecular ion and some fragments, a second mass number two mass units up scale, with an intensity of about 40% of the molecular ion or the parent fragment ion in question (Fig. 3). This is partly due to the fact that 100 90 ,<
80
f‘ z c 2 .E e, >
70
f
z CK
r I i I
60 50
[ CH II 2 Y\ 8 ,c H
40-
CH 12 CHZ +2
/ 0% A JC H E’3
30‘CF3 20-
69
IO-
1+
cF3
126
‘54
/ 50
100
150
200
250
300
350
400
m/e FIG. 3. Mass spectrum and fragmentation methylchlorpromazine (DDMCP-TFA).
pattern
of trifluoroacetate
of dide5
s6C1 has a naturally occurring isotope with a mass of 37. About 25% of the chlorine atoms have this higher mass. The content of sulfur, however, also contributes to the intensity of the isotope mass number (95% of the sulfur atoms have a mass of 32 and 4.2% a mass of 34; in addition a mass of 33 is represented but only to 0.75%). The contribution to intensity of the mass number one unit up scale stems from 13C. About 1.1% of t.he carbon atoms have this mass. This means that an isotope peak always appears one mass unit up scale relative to the mass number of a parent fragment or the molecular ion. The intensities of these isotope peaks increase with increasing number of carbon atoms. The combined isotope ions of the ring system including chlorine, sulfur, and carbon results in a group of four easily observed mass numbers. The fourth peak, with the highest mass number, is mainly due to the combined content of
538
HAMMAB,
HOLMSTEDT,
AND
RYHAGE
a%1 and 13C. This characteristic pattern and particularly the presence of chlorine isotopes is helpful in the identification of the chlorpromazine metabolites. The fragmentation patterns of the compounds have certain features in common. A fragment representing the 2-chloropl~enotl~iazinyl skeleton is invariably present with the parent mass number 232. An additional substituent in the ring skeleton such as hydroxy or methoxy as in 7-hydroxychlorpromazine does not affect the position of this bond cleavage but will naturally increase the mass of the fragment corresponding to that of the substituent. We also always found the ring skeleton with one methylene group attached to the ring nitrogen representing a parent mass number of 246 or 246 plus the contribution due to a hydroxyl or mcthoxyl group. These fragments and the molecular ion have the characteristic group of isotope mass numbers. Chlorpromazine and the trifluoroacetatcs of desmethyland didesmethyl-chlorpromazine give fragments representing the whole side chain (m/e = 86, 168, and 154, respectively) as well as the side chain nitrogen with its substituents and one methylene group attachetl to it (nz/e = 58, 140, and 126). Substitution in the ring skeleton does not affect these side chain cleavages. There are also unknown fragments in these spectra and fragments with low intensities which we have not investigated. Mass Pragmentography A knowledge of the fragmentation pattern and the technical possibilities of focusing on three mass numbers allow a rcfincmcnt of the dctermination. The mass spectra of the tnetabolic products in estracts at basic pH were impossible to interpret because of the small amounts available and the relatively high background. This led us to mass fragmentography, which is essentially a technique in which the mass spectrometer is used as a chromatographic detector and advantage is taken of the physicochemical characteristics of the compounds in order to achieve separation and specificity. The availability of the Accelerating Voltage Alternator unit (LKBl AVA) makes it possible to record the ion intensity of three mass numbers within a short time interval (Fig. 4). Depending upon the duration of the peak emerging from the gas chromatograph, higher or IoTver frequencies of alternation of the accelerating voltage can be chosen. A low switching frequency allows a filter with long-time constancy to increase the signal/noise ratio, which means that with maintenance of an acceptable noise level sensitivity can be further increased. ’ LKB-Produkter
AB, Fack, 16125 Brommal, Sweden.
MASS
539
FRAGMENTOGRAPHY
Knowing the fragmentation pattern of the chlorophenothiazines as described above, it is easy to see tbnt the mass numbers 232,234, and 240 are suitable for focusing upon (II4‘g 3). The accelerating voltage altcrnator can be used to record any mass number within a mass range of 10% and the above-mentioned numbers fall within this range. The fragments selected have different intensities and are easily recognizable.
FIG. 4. Schematic voltage alternator.
diagram
of mass spectrometer
equipped
with
accelerating
From the practical point of view the following procedure is used: A reference substance is introduced through the direct inlet probe of the mass spectrometer and a whole spectrum is recorded. In order to get a starting point in the spectrum an easily recognized peak, with a mass number as close to the desired fragment as possible, is probed by manua1 scanning. From there on, the fragment with the lowest mass number of the two or three desired ions is brought into focus by manual changing of the magnetic field under maintenance of full accelerating voltage (3500 V). By keeping the magnetic field constant and decreasing the accelerating voltage with first one then another potentiometer the desired ions representing higher mass numbers arc then selected. The mass spectrometer is thus set to monitor continuously the three mass numbers of the compounds emerging from the gas chromatograph.
540
HAMMAB,
HOLMSTEDT,
AND
RYHAGE
DDMCP-TFA
6
4
1
IO
MINUTES Fro. 5. Mass fragmentogram of extract of red blood cells treated with p-glucuronidase. Patient receiving 75 mg chlorpromazine daily. Conditions: Instrument LKB9000. GLC on 0.75% Versamid 900 on silanized Chromosorb G (100-120 M). Column length 12m and 18 mm i.d. Carrier gas helium. Flow 20 ml/mm. Injector temperature 24O”C, column temperature 236”, separator temperature 250”, ion source temperature 290”. Ionization potential 50 eV, trap current 240 PA, multiplier voltage 2.5 kV. Mass fragmentogram recorded by means of accelerating voltage alternator (AVA). Focus on fragments corresponding to m/e = 232, 234, and 246 (see Fig. 3). Upper panel: The original mass fragmentogram. Lower panel: The three curves, each representing a mass number, are drawn from the original fragmentogram. (DMCP-TFA and DDMCP-TFA = trifluoroacetates of des- and dides-methylchlorpromazines.)
The recording is made on W-sensitive paper run at slow speed (2.5 cm/min). The resulting curve has certain similarities to an ordinary gas chromatogram (Fig. 5, upper panel). However three deflection lines become visible as a result of the alternating accelerating voltage. To
MASS
FEAGMENTOGRAPHY
541
make the original recording more readable a transparent paper is superimposed and the curves are drawn (Fig. 5, lower panel). The lowest mass number is recorded with a frequency double that of the other ones (Emax E, E,,, E, E,,,, where E,, represents full, E, and E, reduced, accelerating voltage). The middle mass number can be distinguished from the highest one by omitting every fourth galvanometer deflection (Fig. 5, upper panel). Two sets of galvanometers with different sensitivities are used (ratio 1:lO). The high voltage is inversely proportional to the mass number and must be reduced by 10% in order to obtain maximum mass difference between the masses studied. In this case the ion intensity of the highest mass will be reduced about 10% since the intensity is roughly proportional to the accelerating voltage. A typical example of a mass fragmentogram obtained from a plasma of a patient treated with chlorpromazine is presented in Figure 6A, lower panel. Two metabolites of chlorpromazine, monodesmethyland didesmethyl-chlorpromazine, are easily recognized by their retention times and by the relative intensities of the three mass numbers. Earlier in the fragmentogram an increased intensity of mass number 246 can be observed. Since the other two mass numbers do not increase correspondingly, this response cannot be due to any derivatives of the 2-chlorophenothiazinyl compounds already discussed. The beginning of the mass fragmentogram shows compounds that are incompletely resolved. Temperature programming resolves the accumulation of intensities in the beginning, but does not reveal any compounds with the characteristic relative intensities of the three selected mass numbers. None of the peaks resolved shows a simultaneous rise of all three mass numbers. The upper panel in Figure 6A shows a mass fragmentogram of the first extract at PH 12. The mass number 234 has a higher intensity than the others. This can very likely be explained by supposing that some other product in the plasma extract is retained by the column and is slowly bleeding from it. This gives a background activity with the mass number 234. The interfering substance is not present when the plasma is reextracted at pH 12 after treatment with ,Q-glucuronidase. By these facts, combined with refocusing on the trifluoroacetylated side chains of desmethylchlorpromazine and didesmethylchlorpromazine, the specificity can be further increased (Fig. 6B). The side chains differ in one methylene group, e.g., 14 mass units. A slight increase of the intensity of the mass numbers that do not represent the compound in question can also be observed when the references are run in an identical way. The reason for this is unknown at the moment. In Figure 6C the molecular ion and its isotope have been focused upon (desmethylchlorpromazine, left fragmentogram, and didesmethylchlorPromazine, right fragmentogram) . By adding the information provided
542
HAMMAR,
HOLMSTEDT,
AND
RYHAGE
by the different mass fragmentograms one obtains, e.g., for the compound with the retention time of desmethylchlorpromazine, the following mass numbers: 168, 232, 234, 246, 400, and 402. The relative intensities are the same as for those of the reference compound. This means that, apart from the retention time, a complete agreement exists between the reference compound and the metabolite with regard to the fragments representing the side chain, the ring skeleton and its chlorine isotope, the ring skeleton plus one methylene group, and the molecular ion with its isotope. When these requirements are fulfilled it can be firmly stated that there is a complete identity between a reference and a metabolite, especially since none of the mass numbers used gives rise to peaks when plasma from people not given chlorpromazine is treated in the same way. The other metabolite didesmethylchlorpromazine also gives a complete agreement with the reference with regard to retention time and the fragments present including the molecular ion. In the same way chlorpromazine
FIQ. 6A. Mass fragmentograms of plasma extracts. Patient receiving 200 mg chlorpromazine daily. Conditions: As in Figure 5 except for injector temperature 23O”C, column temperature 220”, separator temperature 240”, ion source temperature 290”, ionization potential 70 eV, trap current 120 PA, multiplier voltage 2.9 kV. Upper panel: 10 ml of plasma extracted at pH = 12 with dichloromethane and treated with TFAA. Whole extract of nonconjugated metabolites injected. Lower panel: The same plasma, but after treatment with p-glucuronidase. Extraction and treatment as in upper panel: one-fourth of the extract injected.
MASS
0
543
FRAGMENTOGRAPHY
5
15
IO MINUTES
FIG. 6B. Mass fragmentogram of plasma extract. Patient receiving 400 mg chlorpromazine daily. Conditions: Instrument LKB-9000. GLC conditions as in Figure 6A. Column temperature 232”C, injector temperature 240”, separator temperature 250”, ion source temperature 290”. Ionization potential 50 eV, trap current 240 pA, multiplier voltage 3.3 kV. Focus upon the m/e values corresponding to side chains of the two trifluoroacetylated desmethylated chlorpromazine metabolites. C DDMCP-TFA DMCP-TFA
0
5
0
5
IO
MINLITES
FIG. 6C. Mass fragmentogram of same basic extract as in Figure 6B. GLC and MF conditions: As in Figure 6B except for column temperature, which was kept at 238°C. Focus upon m/e values corresponding to molecular ions and isotopic mass numbers of trifluoroacetylated desmethylchlorpromazine (left) and didesmethylchlorpromazine (right).
itself (Fig. 7) and 2-chlorophenothiazinylpropionic acid have been identified in the plasma extracts. Thus chlorpromazine has been identified in plasma before treatment with &glucuronidase and desmethylchlorpromazine, and didesmethylchlorpromazine both before and after treatment. The two desmethylated metabolites have also been identified in red blood cells after treatment with P-glucuronidase. 2-Chlorophenothiazinylpropionic acid has been identified by both mass spectrometry and mass fragmentography.
544
HAMMAR,
Plasma
4ng
HOLMSTJCDT,
AND
EYHAGl
extract.
CP
r 0
5 MINUTES
Fm. 7. Mass fragmentogram of plasma extract (extraction procedure I under “Methods”). Patient receiving 100 mg chlorpromazine daily. Blood drawn 1 hr after administration. Conditions: Instrument LKB-9000. GLC on 3% Versamid 900 on silaniaed Gas Chrom P (IOO-120M). Column length 1.2 m and 18 mm id. Carrier gas helium. Flow 20 ml/min. Injector temperature 23O”C, column temperature 210”, separator temperature 250”, ion source temperature 290”. Ionization potential 50 eV, trap current 129 @A, multiplier voltage 2.7 kV. Focus upon m/e = 318 and 320 corresponding to the molecular ion and the isotopic mass number of chlorpromazine. Upper panel: Untreated plasma extract. Middle panel: Extract treated with trifluoroacetic anhydride (TFAA). Lower panel: 4 nanogram (4 X10-O gm) chlorpromazine (CP).
Whether the 2-chlorophenothiazinylpropionic acid exists free, proteinbound, conjugated with glucuronic acid, or in two or all three of these forms we do not know at the moment. The extraction after treatment with P-glucuronidase has been performed at pH 1.5 only. The desmethylated products shown to occur after this treatment could be N-glucuronides or merely bound to protein (15). DISCUSSION
Alternation of the accelerating voltage in the mass spectrometer was introduced by Sweeley et al. (28), who used this technique in order to determine the composition of unresolved or partially resolved components in the mixture from the gas chromatographic effluent. Two mass numbers were used and the isotope abundance in the mixture of penta-o-trimeth-
MASS
FBAGMENTOGRAPHY
545
ylsilyl derivatives of glucose and glucose-d, was determined. It was possible to achieve partial separation and quantitation of these compounds by a very rapid alternation of the accelerating voltage between the two isotopes. Also mixtures of the trimethylsilyl derivatives of epiandrosterone and dehydroepiandrosterone were investigated by recording two different ions. Amounts down to 20 nanograms of the compounds were detected. As shown by the present work the alternation of the accelerating voltage representing two or three mass numbers can also be taken advantage of in other ways, namely, to characterize molecules with regard to both their representative fragments and molecular ions. When representative fragments of chlorpromazine are recorded in this way both the parent compound and desmethylated and oxidatively deaminated metabolites couId be identified in the plasma of patients given chlorpromazine. By refocusing, “partial mass spectra” characteristic of the compounds could be obtained. This may seem to be an indirect way of obtaining identification but on the other hand a gain in sensitivity is obtained. When this technique is used and two or three mass numbers are recorded, the following characterize a compound: the presence of all the investigated mass numbers, a characteristic ratio between their intensities, and the retention time of the compound. The sensitivity is of course dependent upon the intensity of the fragment (s) in focus. Generally mass fragmentography seems to be a more sensitive method than gas chromatography with most ordinary detectors. This is due to the high selectivity of the method and the fact that the signals are amplified by an electron multiplier, while signals from other detectors including the so-called “total ion current” are amplified in the usual manner. By use of a lower switching frequency of the voltage it is possible to utilize a filter with long-time constancy to increase the signal/noise ratio and also to increase the sensitivity. Thus by this technique we have been able to detect lo-‘* gm (1 pg = 3 X lo-l5 mole) of chlorpromazine by focusing upon the molecular ion and its isotope peak (m/e = 318 and 320). The relative intensity of m/e = 318 is about 20%. With the use of the electron capture detector lob9 gm of chlorpromazine can be detected (15). When the so-called “total ion current” in the mass spectrometer (16, 29) is used for the recording of a gas chromatogram, 10-S gm of chlorpromazine can be detected. Smaller amounts of the compounds are thus required for mass frngmentography than when a whole mass spectrum is scanned and recorded. When the high sensitivity of the mass fragmentographic technique is utilized the result is naturally contingent upon adsorption and &sorp-
546
HAMMAR,
HOLMSTEDT,
AND
RYHAGE
tion effects in the gas chromatographic system. These conditions also limit the sensitivity of mass fragmentography when subnanogram quantities of drugs and metabolites are analyzed. Improvements in supports, liquid phases, packings, and columns are thus likely to become even more important in the future. The possibility of utilizing certain fragments or ions for detection allows a unique means of selectivity, which can easily be changed in such a way that either a single compound or a family of related compounds is recorded on the UV-sensitive paper. The high selectivity naturally increases the exactness of the identification of the compounds. Simultaneously the need to obtain “pure extract” is diminished which allows less time-consuming means of extraction and group separation. When mass numbers are used for continuous monitoring, the peak areas obtained for a specific compound should be proportional to the abundance of the fragments in question, and should lend themselves to quantitation. When organic compounds are qualitatively analyzed by MS in the LKB-9000 the bleeding from the column gives a certain background which has to be subtracted from the spectrum either manually or automatically by a computer. Mass fragmentography gives a very steady baseline but naturally interference by a compound containing a fragment with any of the mass numbers monitored makes it essential to determine the exact relative intensities of these numbers. Deviations from this clearly indicate interference by an unknown. Such peaks cannot be used off-hand for identification, but require confirmation by refocusing. Temperature programming may be useful in resolving “clusters of mass numbers.” Naturally occurring isotopes further help in the identification provided that they do not occur in a too low concentration; indeed with the technique that we have described labeled compounds with stable nonradioactive isotopes may become important in future studies of drug metabolism. The use of retention times, mass spectra, relative intensities between selected mass numbers, and isotope relationships has allowed us to identify in plasma and erythrocytes from patients given chlorpromazine both the parent drug and a series of its metabolites. No doubt further metabolites may be found and it is not unlikely that in the future the pattern of circulating metabolites of any given drug may be as important as the plasma levels of a single compound when therapeutic effects and side effects are estimated. The technique described in this paper offers unique possibilities of obtaining knowledge of the major metabolic pathways of drugs in man at an early stage. Based upon this information pharmacological experiments with the right animal species can be designed. Drug metabolism represents, however, only one field in which the sensitivity and the
MASS
547
FRAGMENSTOGRAPHY
selectivity of mass fragmentography can be taken advantage of. TTery likely it will become important also in other fields of chemistry and biochemistry. SUMMARY
Chlorpromazine and some of its metabolites have been identified in human blood with the combination of gas chromatography and mass epectrometry. A new method, massfragmentography has been elaborated. It is based upon a continuous recording of up to three mass numbers characteristic of a single subst’ance or a group of compounds. With this technique both a high sensitivity and a high selectivity which can be changed according to wish are achieved. Compounds are identified by their retention times and the fact that all mass numbers are represented in characteristic relative intensities. Refocusing on other characteristic fragments and/or the molecular ion confirms the identity. Through repeated refocusing “a partial mass spectrum” of a compound can be established even when the amounts present are too small for the scanning of a complete spectrum. With this method chlorpromazine, and its desmethylated and didesmethylated metabolites have been identified in plasma. The latter two metabolites were obt.ained also after treatment of the plasma with /3-glucuronidase, as was 2-chlorophenothiazinylpropionic acid, which was found in quantities that allowed the scanning of a complete mass spectrum. In reld blood cells the two desmethylated metabolites could be identified with mass fragmentography only after treatment with flglucuronidase. The use of the method is discussed, part,icularly with regard to blood levels of drugs as related to therapeutic effects and side effects. ACKNOWLEDGMENTS This work has been supported by a grant from the Sational Institute of General Medical Scirnces (GM 13978) to Professor B. Holmstedt and Associate Professor F. SjGqvist. We thank Assistant Professor S. M&tens for making available blood samples from chlorpromazine-treated patients and Miss V. Lepviikman and Engineer A. Akerlind for skillful assistance. The reference substances wepe kindly put at our disposal by Dr. D. H. Efron, Pharmacology Program, NIMH. REFERENCES 1. FORREST, 2. BECKETT,
779-94
3.
POSNER,
377-91
I. S., AND FORREST, F. M., Cl&. Chem. 6, 11-15 (1960). A. H., BEATEN, M. A., AND ROBINSON, A. E., Biochem.
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12,
Therap.
141,
(1963).
H. S.. (1963).
CTJLPAN,
R.,
AND
LEVINE,
J., J. Pharmacol.
Esptl.
548
HAMMAR,
4. DRNCOLL, 2, 109-114
5. ROSE, R. (1964).
J.
L., MARTIN,
HOLMSTED$ H.
F.,
.~ND
AND GTJDZINOWICZ,
BYHAGE B.
J.,
J.
Gas
Chromatog.
(1964).
M., DIMASCIO,
A.,
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