which on rearrangement may be written as
tions x10 and X I . On putting x10 and X I , in turn, equal to zero, Equation 22 reduces to the expressions
or (19b) Substituting Equation 19a in the expression for xl/x10 given in Equation 15, we get
(20) NOW1 - (1 - Rz)xz0 = Rz[l + i(1 - R z ) / R 2 l (1 which on combining with Equations 19b and 20 gives 1 - (1 - R,)x,O = ( R , / R , ) [ l - (1 - R , ) x , l
~ Z O ) ] ,
(21)
Combining Equations 17d and 21, we finally obtain LTxi'bxi
=
R1U;
11 - (1 - R l ) x l ] [ l- (1 - R 1 ) ~ l O (22) ]
This constitutes a band occurring in two different concentrations of the same component, where U X I ~ * Xrepre~ sents the velocity of the boundary between the concentra-
which are the velocities of component 1 a t concentrations and x l O , respectively. Thus, from theoretical considerations, a single band a t different concentrations can exist, although with time and the downstream passage, the concentrations produce a single homogeneous band of longer or shorter length depending on whether XI > or < x1O. On applying the condition RI = Rz[l - (1 - R I ) X I ~to ] the expression for x~ given in Equation 16, we arrive a t x z = 0. Thus, a single band in two concentrations may be produced in two ways, either by placing two plugs of different concentrations in contact or by letting a two-component band travel down the column and then a t an appropriate time withdrawing instantaneously from the mixed region all the molecules of one of the species which may be regarded to act as a kinematic catalyst. x1
Received for review April 6, 1972. Accepted February 22, 1973. One of us (M. J.) wishes to express thanks to the University of Islamabad for the award of a research fellowship.
Gas Chromatographic Degradation of Several Drugs and Their Metabolites Albert0 Frigerio, Kenneth
M. Baker,
and Giorgio Belvedere
lstituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea, 62 20157 Milano, ltaly
The gas chromatographic behavior of some drugs and their metabolites has been examined, particularly with regard to decompositions and, hence, misidentification. The structures of decomposition products were elucidated using gas chromatography-mass spectrometry, or in some cases collection and identification. Drugs which have been studied are oxazepam, N-methyloxazepam, nitrooxazepam. o-chlorooxazepam. carbamazepine, carbamazepine-10.11-epoxide, 1 0 , l l -dihydroxycarbamazepine, and floropipamide. Each of the drugs oxazepam, ochlorooxazepam, and nitrooxazepam underwent a decomposition to give 4-phenylquinazoline-2-carboxaldehyde derivatives while N-methyloxazepam is stable. This reaction also occurred on heating the compounds to 200 "C. Carbamazepine partly decomposes when injected as a methanol solution to give iminostilbene and 9-methylacridine. Carbamazepine-10.11 -epoxide decomposes completely to 9-acridinecarboxaldehyde. while the same degradiation product is obtained from 10.11-dihydroxycarbamazepine. Floropipamide undergoes a thermal dehydration of the amide position of the molecule to give a nitrile. Mechansims for most of the reactions are discussed.
Gas chromatography (GLC) is widely used for separating complex mixtures arising from biological sources ( I , 2 ) ; however, the technique suffers from the fact that it 1846
permits determination of only one property of a compound uiz. its retention time. Since many compounds may have the same retention time for a given set of conditions, the exact identity of a material cannot be established. Hence GLC is more valuable when it does not give a peak, rather than when it does; however, when this concept is used, it is possible to have a misidentification of drugs ( 3 ) . The rearrangement or degradation on the gas chromatographic column of the material under investigation can also give false information concerning the presence of a molecular species. The combination of GLC with mass spectrometry overcomes the problem of positively identifying drugs and their metabolites present in a biological system ( I , 4-9). Hammar, E. Holmstedt, J.-E. Lindgren, and R . Tham. Advan. Pharmacoi. Chemother.. 7. 53 (1969). (2) S. P. Cram and R. S. Juvet. Jr., Anal. Chem.. 44. 213R (1972). (3) L. R. Goldbaum, E. H. Johnston, and J. M . Blumberg, J. Forensic Sci.. 8 . 286 (1963). (4) C. Merritt, Jr.. "Applied Spectroscopy Reviews," Vol. 3, Marcel Dekker, New York, N.Y.. 1970, p 263. (5) E. C. Horning and M . G. Horning, J. Chromatogr. Sci.. 9. 129 (1971). (6) C. J. W. erooks. "Mass Spectrometry," Vol. 1. 0 . H. Williams Ed.. The Chemical Society, London, 1971, p 288. (7) G. A . Junk, Int. J , Mass Spectrom. / o n Phys., 8 . 1 (1972). (8) A. L. Burlingame and G. A. Johanson, Anal. Chem.. 44. 337R (1972). (9) C. J. W. Brooks, A. R. Thawley, P. Rocher, E. S. Middleditch., G. M. Anthony. and W. G. Stilwell. Advan. Chromatogr. Proc. Int. Symp., 6th. 262 (1970). (1)
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
k:$)b"
100-
r,
.-e E e
80.
c'
0
60-
-H
H
-co
OXAZEPAM
DIS MW
286
C
.-eE
40.
I
-a Lo
a
20-
286 I* 1
60
80
I1
I,
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
360
380
400
mie 268
100.
80-
.-m 5
205 -CI
e
-
239
G LC 60-
E
.-e
40-
Lo
Q
a
20.
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
mie
Figure 1. M a s s s p e c t r u m of oxazepam Upper by direct injection system; lower by GLC
This combined technique can also show if, and to what extent, a compound is degraded or chemically changed during GLC analysis. Many examples of such degradations are known, but few have been reported (IO). The documentation of these degradations is particularly important with regard to analyzing and identifying drugs and their metabolites and this paper will present some exOXAZEPAM IV R , ICI , n z = H I R, = C I , R p = H amples related to 3-hydroxy-1,4-benzodiazepin-2-ones, II R , = C I , R Z I C I 0-CW LOROOXAZEPAY V R, = C I , Rz:CI carbamazepine (5H-dibenz[b,flazepine-5-carboxamide) M l T I O O X A Z E P A ~ V I R, :NO2 , Rz I H Ill R, :NOz , R z = H and its metabolites, and floropipamide. These compounds Figure 2. M e c h a n i s m for the t h e r m a l degradation of 3-hydroxyhave various medical uses. The benzodiazepines are wide1,4-benzodiazepin-2-ones ly used as psychotropic drugs ( I I ) , carbamazepine is used as an antiepileptic agent (12, 13) or in the treatment of trigeminal neuralgia ( I 4 ) , while floropipamide is one of the butyrophenone class of neuroleptic drugs used in the methyloxazepam 110 "C, nitrooxazepam (111) 160 "C, o-chlorooxatreatment of mental diseases (15, 16). zepam (11) 110 "C, carbamazepine 100 "C, carbamazepine-10,ll-
(y
EXPERIMENTAL Apparatus. An LKB 9000 gas chromatograph-mass spectrometer was used under the following conditions: molecular separator 290 "C, ion source 290 "C, electron energy 70 eV, trap current 60 &A, multiplier voltage 3.5 kV. Introduction of samples by the direct injection technique was carried out under the same conditions using the following temperatures: oxazepam (I) 140 "C, N (10) 6 . J. Millard, Advan. Drug Res.. 6. 180 (1971). (11) G . Zbinden and L. 0. Randal1,Advan. Pharmacoi.. 5, 213 (1967). (12) S. Livingstone. "Drug Therapy for Epilepsy," Charles C Thomas, Springfield, Ill., 1966, p 125. (13) H. Fichsel and R. Heyer. Deuf. Med. Wochenschr.. 95. 2367 (1970). (14) D. J . Dalessio and K . H. Abbott. Headache. 5 . 103 (1966). (15) G . E. Crane. Inf. J . Neuropsychiat. Suppi.. 3. 110 (1967). (16) T . Ban, "Psychopharmacology," Williams and Wilkins, Baltimore, M d . , 1969, p 238.
b"'
epoxide 100 "C, l0,ll-dihydroxycarbamazepine120 "C, and floropipamide 120°C. In all cases a GLC column of dimensions 2 m X 2 mm packed with 3% OV-17 on Gas Chrom Q (100-120 mesh), with a helium carrier gas flow rate of 30 ml/min, was used. Operating temperatures were as follows: benzodiazepines, column temperature 280 "C, injection port 300 "C; carbamazepine and derivatives, column temperature 200-260 "C, injection port 250-280 "C; floropipamide, column temperature 270 "C, injection port 300 "C. Materials. Oxazepam (7-chloro-1,3-dihydro-3-hydroxy-5-phenyl-2H- 1,4-benzodiazepin-2-one)(I), N-methyloxazepam (7chloro-l-methyl-1,3-dihydro-3-hydroxy-5-phenyl2H-1,4-benzodiazepin-2-one) (VII), nitrooxazepam (7-nitro-1,3-dihydro-3-hydroxy-5-phenyl-2H-l,4-benzodiazepin-2-one) (III), and o-chlorooxazepam (7-chloro-1,3-dihydro-3-hydroxy-5-(2~chlorophenyl)2H- 1,4-benzodiazepin-2-one)(11) were supplied by Ravizza S.p.A., Muggib, Milan; carbamazepine (5H-dibenzo[b,f]azepine5-carboxamide) (VIII) was supplied by Ciba-Geigy, Basel, Switzer-
ANALYTICAL CHEMISTRY, VOL. 45. NO. 11, SEPTEMBER 1 9 7 3
1847
METHYL OXAZEPAM
I
60
80
100
120
140
1 -H
160
180
200
220
240
260
280
DI S
300
320
340
380
380
400
380
400
m/e 271
1
lo01
G LC
60
80
100
120
140
160
180
200
220
240
260
260
300
320
340
360
mie
Figure 3. Mass
spectrum of N-methyloxazepam
Upper by direct injection system; lower by GLC
land; carbamazepine-10,ll-epoxide (5H-dibenzo[b,f]azepine-5- the loss of the formyl moiety from the structure IV, to carboxamide-l0,ll-epoxide)(1x1, l0,ll-dihydroxycarbamazepine give the ion a t m l e 239. As a result of the high tempera(10,ll-dihydro-1 0 , l l-dihydxoxy-5H-dibenzo[b,flazepine-5-carbox- ture conditions of the column, the seven-membered ring amide) (X). and 9-ariidinecarboxaldehyde (XI) were supplied by G . Pifferi, Itakeber, Milan; and floropipamide (4-(4-carbamido-4- of oxazepam had rearranged to a six-membered ring with the loss of a water molecule to form a very stable compiperidino piperidin0)-p-fluorobutyrophenone](XIV) was supplied pletely aromatic system (Figure 2). by P. A. J. Jannsen, Jannsen Laboratoria, Beerse, Belgium. By using compounds labeled with deuterium ( I @ , it has RESULTS AND DISCUSSION been shown by several workers in this laboratory that the 3-Hydroxy-1 ,&benzodiazepin-2-ones. The quantitative rearrangement involves the hydrogen bound to N1 and the microdetermination of this class of drugs in biolpgical mahydrogen in the hydroxyl group a t C3 in the loss of the water molecule. Investigation of the mass spectra of the terials is widely carried out using GLC (17). GLC of oxGLC peaks produced by other drugs of this class showed azepam (I) gave a peak with a much shorter retention time than other less polar benzodiazepines indicating, that this that the rearrangement appeared to be a general one, e.g., o-chlorooxazepam (11) and nitrooxazepam (111) were shown peak may have been due to a degradation product of oxato rearrange under the GLC conditions, although N zepam (18. 19). By comparison of the mass spectra obtained by direct injection and by gas chromatographymethyloxazepam (VII) was quite stable. The mass spectra mass spectrometry (Figure l ) , this peak was shown to corof o-chlorooxazepam (11) and nitrooxazepam (111) obtained by direct injection mass spectrometry had molecular ions respond to oxazepam less a water molecule. Analysis of the fragmentation patLern in the mass spectrum showed a t m l e 320 and 297 while those resulting from a GLCthat the compound was 6-chloro-4-phenylquinazolin-2-car- mass spectrum had molecular ions a t m l e 302 and 279 boxaldehyde (IV). corresponding to the loss of water in each case to give 4The mass spectrum showed a molecular ion a t m l e 268 phenylquinazolin-2-carboxaldehydederivatives ( V and VI, which is 18 mass units lower than that of the parent oxrespectively) (Figure 2). Heating each of the benzodiazepines I, 11, and I11 in a azepam (I) and showed a loss of 29 mass units, this being melting point apparatus to 200 "C produced the same (17) F. Marcucci. R . Fanelii, and E. Mussini, J . Chromatogr.. 37. 318 (1968). (18) A. Forgione. P. Martelli. F. Marcucci. R . Fanelli, E. M u s s i n i , and G . C. Jornmi, J . Chromatogr.. 59. 163 (1971 ) . (19) W. Sadee and E. V a n der Kleijn. J . Fharm. Sci.. 60. 135 (1971).
1848
(20) A. Forgione, A. Frigerio. and P. Martelli Proc. Int. Symp. Gas Chromatog.-Mass Spectrom., Isle of Elba, Italy, May 77-19, 1972, p 213 (1972).
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
180 100-
*=
80.
.-m LI
-
k0
60-
I
C
-
.-E
CARBAMAZEPINE 40.
10,ll-EPOXIDE
-
223
(P
M.W. 2 5 2
IX
252
0
=
- NH2
20.
r L
L
L
r
,I,,
1 1 *
I
.
GLC
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
mm Figure 4. Mass spectrum of carbamazepine-l0,ll-epoxide Upper by direct injection system; lower by GLC
rearrangement with loss of water, and each of the 4phenylquinazolin-2-carboxaldehydes thus prepared had the same retention time on GLC and the same mass spectra by direct injection as the parent benzodiazepines analyzed by GLC-mass spectrometry ( 2 0 ) . In the case of N-methyloxazepam (VII), the molecule is quite stable to gas chromatographic analysis (Figure 3). Carbamazepine. Carbamazepine (VIII), and its metabolites carbamazepine-10,ll-epoxide (IX) and 10,11dihydroxycarbamazepine ( X ) all undergo rearrangements and or degradations during GLC. Carbamazepine-10,llepoxide (IX) was identified by direct injection mass spectrometry as a metabolite in the urine of patients who had been administered carbamazepine orally (21, 22). We have shown by mass spectrometry that attempted separation of this metabolite by GLC gives a relatively nonpolar material which elutes from the column not as the epoxide, but as a rearranged, degraded product ( 2 3 ) . The mass spectrum (Figure 4) of the eluted compound established the molecular weight of this material as 207 with fragment ions corresponding to the loss of 28 amu to give a n intense peak a t m / e 179, which underwent very little further fragmentation suggesting the formation of the very stable acridine radical ion. The structure 9-acridinecarboxaldehyde (XI) was proposed for this material.
21) A. Frigerio, R . Fanelli. P. Biandrate. G. Passerini. P. L. Morselli. and S. Garattini. J Pharm. S c i . . 61. 1144 (1972).
EO", IX
XI
Figure 5. Mechanism for the degradation of carbamazepine1 0 , l l -epoxide during GLC analysis
m a OH
\
N'
I
CONH, VI11
OH
h.
I CONHI
x
Separation of the product by preparative CLC using a 2-m OV-17 column a t 220 "C and comparison of its physical properties with an authentic sample established 9acridinecarboxaidehyde as the correct assignment. A mechanism rationalizing the rearrangement is shown in Figure 5. The process is probably c a d y z e d by the (22) P. L. Morselii, P. Biandrate, A. Frigerio, M . Gerna. and G. Tognoni. "Proceedings Workshop on the Determination of Anti-Epileptic Drugs in Body Fluiils.' Noordwijkerhout. The Netherlands. April 1972, in press. (23) K . M. Baker, A. Frigerio, P. L. Morselli, and G . Pifferi. J . Pharm. Sci.. in press.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMEER 1973
1849
M-44
FLOROPIPAMIDE
I
XIV
331
I
M.W. 3 7 5 d 165 .
D.I.S.
i
..
G + H
\H,
138
I
I
I
I
b 123
201
(M+
375
A
'"1
GLC.
Figure 6.
Mass spectrum of floropipamide
Upper by direct injection system; lower by GLC
slightly acidic characteristics of the column a t the high temperatures employed and then the intermediate amide decomposes to give the much more stable, completely aromatic system, 9-acridinecarboxaldehyde. 10,ll-Dihydroxycarbamazepine(X), another metabolite of carbamazepine recently found in our laboratory, also undergoes a degradation on an OV-17 column to the same 9-acridinecarboxaldehyde (XI). This rearrangement to give the aldehyde is an example of the pinacol type and is again catalyzed by the acidic conditions of the column, although the reaction is not so facile, as the rearrangement will not take place on an SE-30 column which is less acidic. Carbamazepine, the parent drug, also undergoes a degradation to some extent when injected as a methanol solution onto an OV-17 column. Besides a large peak due to carbamazepine, there are also minor components of lower polarity identified as iminostilbene (XII) and 9-methylacridine (XIII).
These were identified by mass spectrometry and comparison of their mass spectra with those of authentic samples. This degradation takes place only when carbamazepine is injected onto the column as a methanol solution. Under the high-temperature conditions (220-260 "C), the alcohol acts as an acid ( 2 4 ) . Iminostilbene (XII) is produced by methanolysis of the amide linkage, while the 9-methylacridine (XIII) requires a rearrangement of the carbamazepine skeleton under the acidic conditions. Floropipamide. Comparison of the mass spectrum of floropipamide (XIV) obtained by direct injection and by gas chromatography showed they were different (Figure 6). During GLC floropipamide undergoes the well known thermal dehydration to give a nitrile ( 2 5 ) . The mass spectra for floropipamide obtained by both methods of introduction show molecular ions a t mle 375 and 357, i.e., a difference of 18 amu (HzO) between the two spectra. In addition the mass spectrum of floropipamide obtained by direct injection contains the ion a a t mle 224 (Figure 6). resulting from a /3-bond fission with respect to the piperidine ring, a fragmentation common to this class of drugs (26). This ion ( m / e 224) is not present in the spectrum ol Chem. Soc.. 9 2 . 598E (1970). (25) T. Mukaiyama, M. Tokizawa, and H. Takei, J Org. Chem.. 27. 80: (1962). (26) A. Frigerio, A. Forgione. P. Martelli, and R. Faneill. "Atti I t o Con. vegno di Spettrornetria di Massa." Ispra. Italy. Sept 1-3, 1971, Eu. ratom, 1971, p 401 (24) J . I. Braurnan and L. K. Blair, J. Amer
H
1850
XI1
XI11
iminostilbene
9-methylacridine
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
the nitrile obtained as a result of dehydration during gas chromatography. In conclusion, the application of GLC to the investigation of small quantities of material available in drug metabolism studies should be approached with great care. The correlation with standards and identification of peaks by GLC should be carried out bearing in mind that degradations can occur on the column. Where possible the identity of the peaks should be checked by other physical methods.
ACKNOWLEDGMENT The authors wish to thank S. Garattini, G. C. Jommi, and E. Mussini, A. Forgione,p. Martelli, and p, L. Morselli for helpful discussions and suggestions. Received for review October 31, 1972. Accepted February 28, 1973. This work was supported by N. I. H. Grant No. 1 PO1 GM18376-02 P T R and a Welcome Trust Fellowship to one of us (K. M. B,).
Role of the Liquid Phase in Gas-Liquid-Solid Chromatography and Its Influence on Column Performance-An Experimental Approach Fabrizio Bruner, Paolo Ciccioli, Giancarlo Crescentini, and Maria Teresa Pistolesi Laboratorio sull'lnquinamento Afmosferico del C. N. R . , c / o lstitufo di Chimica Analitica, Citta Universitaria, 00185 Roma, ltaly
The changes occurring in heats of adsorption, separation factors, HETP YS. u curves and retention characteristics when different amounts of liquid phase are added to graphltized carbon black Sterling FT have been studied. Squalane and glycerol have been used as extreme cases of liquid phases, and particular separation problems have been taken as examples. Changes in chromatographic features are related to the changes in surface characteristics. Examples of analytical applications are given.
Although several analytical applications of gas-liquidsolid chromatography (GLSC) have been published recently (1-3) and some estimation of the influence of the amount of liquid phase on retention parameters has been attempted, a detailed study of the role played by the liquid phase in modifying the Chromatographic properties of adsorbents has not yet been performed. The efficiency of etched glass capillaries coated with different amounts of squalane was investigated some years ago (4), while the use of liquid phases as "tail reducers" in gas adsorption chromatography was introduced by Eggertsen et al. (5, 6) opening the way to a wider use of adsorbents in gas chromatography. The effect of water in the carrier gas as the modifier of chromatographic properties and of nitrogen adsorption isotherms was also studied (7). All these works contributed to the solution of particular analytical problems by linearizing the adsorption isotherms of hydrocarbons and polar compounds, showing A. Di Corcia, D. Fritz, and F. Bruner, Anal. Chem., 42, 1500 (1970). F. Bruner. P. Ciccioli, and A. Di Corcia, Anal. Chem., 44, 894 (1972). F. Bruner, A. Liberti, M. Possanzini. and 1 . Allegrini, Anal. Chem., 44,2070 (1972), F. A. Bruner and G. P. Cartoni, Anal. Chem., 36, 1522 (1964). F. T. Eggertsen, H. S. Knight, and S. Groennings. Ana/. Chem., 28, 303 (1956). F. T. Eggertsen and H. S. Knight, Anal. Chem., 30, 15 (1958). G. Aiberini, F. Bruner, and G. Devitofrancesco, Anal. Chem., 41, 1940 (1969).
that GLSC can be a powerful tool to fully exploit the advantages of gas adsorption chromatography. Physical homogeneity, nonspecificity, and the possibility of obtaining uniform coatings with any type of liquid phase are the basic chromatographic features of graphitized carbon blacks. The practical absence of chemically active groups on their surface and the selectivity, based on geometrical structure and molecular polarizability, are the reasons for the wide application they had in our and other laboratories (8, 9). By changing the type and the amount of liquid phase, a n extremely wide number of stationary phases can be obtained with the same carbon black as matrix. Furthermore, the advantages of both GLC and GSC can be attained by GLSC with graphitized carbon blacks, ie., high selectivity, low HETP, thermal stability, and applicability to every kind of compound. In this paper, we report the results of a detailed study on the changes in the chromatographic parameters such as separation factors, HETP curves, and thermodynamic functions, induced by modifying the adsorbtive surface of Sterling FT with various amounts of different liquid phases. The choice of Sterling FT among the large variety of graphitized carbon blacks has been made by the considerations: (1) surface area (15 m2/g) is good for reliable coatings a t 0 < 1, where 0 is the ratio between covered and uncovered surface, but still allows the elution of rather high-boiling compounds; (2) good mechanical resistance to ensure reproducibility of columns made with the same mesh range. The choice of squalane, phenanthrene, and glycerol as modifiers was made because of the well defined chemical structure of these compounds with respect to more common liquid phases for GLC and because the decisive difference between squalane (nonpolar) and glycerol (very (8) G. M. Petov and K. D. Scherbakova, "Gas Chromatography 1966," A. B. Littlewood, Ed., institute of Petroleum, London, 1967, p 74. (9) C. Vidal-Madjar, J. Ganansia. and G. Guiochon in "Gas Chromatography 1970," R . Stock, Ed., Institute of Petroleum, London, 1971, p 20.
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1973
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