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Investigation of the neuroleptic drug haloperidol and its metabolites using tandem mass spectrometry
International Journal of Mass Spectrometry and Zon Processes, 122 (1992) 121-131 Elsevier Science Publishers B.V., Amsterdam
121
Investigation of the neuroleptic drug haloperidol and its metabolites using tandem mass spectrometry* Jian
Fang”,
John
W. Gorroda, Mahmud Kajbafb, John H. Lambb and
Stephen Naylor’ “Chelsea Departmentof Pharmacy, King’s College, University of London, London S W3 6LX (UK) bMRC Toxicology Unit, Carshalton. Surrey SMS 4EF (UK) ‘Mass Speclrometry Facility, Departments of Biochemistry and Molecular Biology, and Pharmacology, Mayo Clinic, Rochester, MN 55905 (USA)
(First received 3 June 1992; in final form 30 June 1992)
ABSTRACT The in vitro metabolism of haloperidol, a clinically utilized neuroleptic drug, was investigated using guinea pig derived hepatic microsomal incubates. By employing a combination of reversed phase HPLC and tandem mass spectrometry, it was revealed that haloperidol was metabolized to at least eight different compounds, including the proposed dopaminergic toxin 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4oxobutyll-pyridinium species and an intermediate metabolite 4-(4-chlorophenyl)-l-[4-(4-fluorophenyl)-4oxobutyll-1,2,3,6_tetrahydropyridine. Keywor&
tandem mass spectrometry; haloperidol.
INTRODUCTION
Haloperidol (4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-4-piperidinol, HAL) see Fig. 1) is a neuroleptic agent that has proven clinically useful and belongs to the important butyrophenone class of such drugs [l]. It is widely used in current clinical practice but has a number of neurological side-effects associated with its use. In particular, it causes tardive dyskinesias and produces Parkinsonian symptoms in both humans and animals [2,3]. The metabolic fate of HAL has not until recently been extensively studied, even though the parent compound is a clinically useful drug. Previous reports have suggested that oxidative N-dealkylation and ketone reduction are the major metabolic pathways. Furthermore, recent evidence linking the nigrostriatal neurotoxin N-methyl-4-phenyl-1,2,3,6_tetrahydropyridine (MPTP) (see Fig. 2) with the induction of Parkinsonian symptoms [7] has led to the Correspondence to: S. Naylor, Mass Spectrometry Facility, Departments of Biochemistry and Molecular Biology, and Pharmacology, Mayo Clinic, Rochester, MN 55905, USA. * Dedicated to the memory of Professor Michael Barber, a friend and colleague. 0168-l 176/92/%05.00
0
1992 Elsevier Science Publishers
B.V. All rights reserved.
122
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
123
MPDP’
MPP’
Fig. 2. Chemical structures of the neurotoxin MPP+.
MPTP and its two metabolites MPDP+, and
that HAL, which is structurally similar, or more likely one of its analogous metabolites, also operates via a similar mechanism [8,9]. It has been demonstrated the MPTP is first metabolized to iV-methyl-4phenyl-2,3_dihydropyridinium (MPDP+ ) and subsequently to the N-methyl4-phenylpyridinium (MPP+ ) species [ 10,111, as shown in Fig. 2. MPP+ is believed to exert its neurotoxic effect by inhibiting mitochondrial respiration. Recently, Castagnoli and co-workers have reported the detection of the putative neurotoxic pyridinium metabolite (C(Qchlorophenyl)- 1-[4-(4fluorophenyl)-4-oxobutyll-pyridinium, HP+), comparable to MPP+, derived from HAL in both the urine and brain tissue of haloperidol-treated rats [ 12,131and the urine of haloperidol-treated humans [ 141.Two of us [8,9] have also reported the presence of HP+ in an in vitro mouse microsomal incubation mixture as well as the HAL-1,2,3,6-tetrahydropyridine analogue (HTP), which is structurally very similar to MPTP. In the present study, we have utilized a combination of reversed phase HPLC and tandem mass spectrometry (MS-MS) [15] to further investigate the metabolic pathways of HAL. In the process, we have identified and characterized eight metabolites of the parent drug HAL, obtained from in vitro guinea pig liver microsomal incubations. suggestion
EXPERIMENTAL
Materials HAL was obtained from the Sigma Chemical Company, St. Louis, MO, USA; HPLC acetonitrile (far UV grade) was purchased from FSA Laboratory Supplies.
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J. Fang et aLlint. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
4-(4-Chlorophenyl)-1-[4-(4-fluorophenyl)-4-hydroxybutyl]-4-piperidinol (RHAL) was prepared according to Oida et al. [16]. HTPi, HP+, 4-(4-chlorophenyl)-l-[4-(4-fluorophenyl)-4-oxobutyl]-4-piperidinol-N-oxide (HNO), 4(4-chlorophenyl)- 1-[4-(4-fluorophenyl)-4-oxobutyll1,2,3,6-tetrahydropyridine-N-oxide (HTPNO) were synthesised in this laboratory (J.F. and J.W.G.). Details of these syntheses will be described in a separate publication. All other chemicals, enzymes, and cofactors were obtained from Sigma Chemical Co., Poole, UK. Methods Dunkin-Hartley guinea pigs were obtained from the Animal Supply Unit of King’s College, University of London. Animals were fasted overnight before sacrifice. Hepatic microsomal preparations were prepared using the centrifugation method described previously [17]. Incubation procedures were as follows: a reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating system consisting of the sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+ ) (2 pmol), glucose-6-phosphate disodium salt (10 pmol), glucose-6-phosphate dehydrogenase (1 unit), and MgCl, (2 mg) all in 2 ml phosphate buffer (0.2 M, pH 7.4) was preincubated for 5 min. Enzymatic reactions were initiated by addition of HAL (2pmol) and microsomal preparations equivalent to 0.5 g original tissue. In control incubates, heat inactivated microsomes were used instead of fresh microsomal preparations. Incubations were carried out for 30min at 37°C. Enzymic reactions were terminated by addition of ZnSO, (200mg) to the incubation mixture. The precipitated proteins were removed by centrifugation (MSE Centaur 2, 3000 rev. min-’ , 20min). The supernatant was passed through a preconditioned (methanol (4 ml) followed by distilled water (4 ml)) Sep-Pak C,, cartridge. Excess ZnSO, was removed by washing with distilled water (4 ml). The retained compounds were eluted by methanol (4 ml), which was subsequently evaporated to dryness at 45°C under nitrogen [18]. The residues were reconstituted in methanol (50~1) and subjected to HPLC separation (10 ~1 injections). The HPLC chromatographic system comprised a Waters M-45 pump and a Rapiscan SA6508 detector set to record at 220,450 and 360nm. A Tandon TM7002 computer was used to record chromatograms. The HPLC system used a 5 pm Hypersil CPS-5 column (4.6 mm x 25 cm). The mobile phase was a combination of acetonitrile (67%) and ammonium acetate buffer (10 mM) adjusted to pH 5.4 with acetic acid. The solvent was delivered at 1 ml min-‘. The incubation extract was injected into the HPLC system, and peaks of interest were collected. Control incubation extracts were also subjected to
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
125
HPLC, and fractions were collected at the same time intervals as that of the sample. The collected elutes were evaporated to dryness using a vacuum freeze dryer. Mass spectrometry
All mass spectra were obtained on a VG 70-SEQ instrument of EBQ,Q2 configuration, where E is an electrostatic analyser, B is the magnet, Q, is an r.f.-only quadrupole collision cell, and Q2 is a mass-filter quadrupole. EB and Q2 correspond to mass spectrometer one (MS,) and two (MS,), respectively. All synthetic standards and microsomal incubate mixtures as well as control samples were ionised by either (i) positive ion fast atom bombardment mass spectrometry (FAB-MS), using xenon atoms from a model Bl 1N saddle-field fast-atom gun (Ion Tech, Teddington, UK) or (ii) direct insertion probe (DIP) electron ionization-MS (EI-MS) where the probe was heated to 260°C after 30 s in the source. In both cases, the ions produced were accelerated out of the source region to an energy of 8 keV and analyzed in MS, using a scan speed of 10s per decade over the mass range 50-800 at a resolution of x 2000. Product ion spectra-Protonated molecular ions (MH+) in the case of FAB or molecular ions (M’+) in EI (in both cases also referred to as precursor ions) of synthetic standards, microsomal incubate and control samples were selected with a resolution of z 1000 using EB and subjected to collision activated dissociation using argon as the collision gas in Q,. Collision energy was optimized to give maximum fragmentation of the precursor ion at = 220 eV with a gas pressure in the Q, housing of M 10e5 mbar. The resulting fragment or product ions were mass analyzed in Q2 and a product ion spectrum acquired by scanning Q2 over the range m/z40-450 with 10 scans being obtained in the multi-channel analysis (MCA) mode. Sample preparation for mass spectrometry
Usually 1~1 of the x 10~1methanol solution containing the HPLC fraction of the HAL metabolite(s) was removed by a Hamilton syringe. In FAB-MS, the 1~1 of methanol was thoroughly mixed with z 1.5 ~1 of 3nitrobenzylalcoho1 (NBA) on the stainless steel probe tip and inserted into the source. In EI-MS, the 1~1 solution was added to the glass vial on the probe tip and gently heated to remove solvent before inserting into the mass spectrometer. RESULTS AND DISCUSSION
Initial studies focused on determining optimal FAB matrix conditions for detecting HAL and three synthetic derivatives (RHAL, HTP, and 4-fluoro-
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benzoylpropionic acid (FBPA)). A variety of FAB matrices were used including glycerol, thioglycerol, glycerol-thioglycerol (1 : l), thiodiglycol, dithiothreitol-dithioerythreitol (3 : l), and 3-nitrobenzylalcohol (NBA). In each case for HAL, RHAL, and HTP, NBA proved to afford the most abundant signal-to-noise ratio for the respective protonated molecular ions (MH+). In particular, it was possible to detect x 750pmol of HAL on the probe tip in positive ion FAB-MS using NBA as the matrix. In the case of the fluorophenyl standard FBPA, FAB-MS proved to be relatively insensitive as an ionization method. It was possible to detect only x 3 nmol of FBPA on the FAB probe tip using NBA. (All other matrices afforded either similar or less sensitivity.) However, using the direct insertion probe with EI as the ionization mode, it was possible to detect x 200 pmol of the synthetic standard FBPA. Product ion spectra of synthetic standards
The FAB product ion spectral data of the synthetic standards HAL, RHAL, HTP, HTPNO, HP+, 4-chlorophenyl-4-hydroxypiperidine (CPHP), and HNO (structures shown in Fig. 1) and the product ions observed when ionized using the DIP for FBPA are shown in Table 1. In all cases, the collision energy in the gas cell (Q, ) was optimized to afford maximum fragmentation of the precursor ion at x 220eV with an argon gas pressure of x lo-‘mbar. These conditions are known to bring about multiple collisions between selected precursor ions and the collision gas [19,20] but do not cause maximum scattering of the resulting fragment product ions [21]. In the case of piperidinol containing compounds, namely HAL, RHAL, and HNO, the FAB-MS-MS product ion spectra contained abundant ions at [MH - 18]+ corresponding to loss of H,O from the piperidinol ring. Furthermore, the product ion spectra of all the synthetic standards HAL, RHAL, HTP, HTPNO, HP+, and HNO were relatively simple and contained few product ions. The predominate bond cleavage occurred between the piperidine nitrogen and the alkyl carbon of the hydrocarbon chain. The resulting fragmentation afforded two classes of product ions. Firstly, the compounds listed above all contained a product ion corresponding to the chlorophenyl-piperidine (or derivative thereof) portion of the molecule, at m/z212 (HAL, RHAL); 194 (HAL, RHAL, HTP); 210 (HTPNO); 190 (HP+); and 228 (HNO). Furthermore, there was no evidence of fragmentation occurring between the chlorophenyl and the piperidine (or derivative) ring systems. This was further confirmed by inspection of the product ion spectrum of synthetic CPHP which only contained a single product ion at m/z 194, denoting loss of H,O, and the ring systems remained intact. Secondly, all the fluorobenzoyl containing compounds (HAL, RHAL, HTP, HTPNO,
J. Fang et al./Int. J. Mass Spectrom. ion Processes 122 (1992) 121-131
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TABLE 1A Product ion data obtained from FAB-MS-MS Ion abundance
(%)
for synthetic standards Ion assignment
HAL 376 358 212 194 165
100 80 5 25 90
zH*O)+ [HNCS H90-CsH4Cl + H]’ [HNCS H, -CBH&I + H] +
RHAL 378 360 212 194 165 149
100 65 5 10 5 5
[HNC, H90-C,H&l + H]+ [HNCSH,-CsH4Cl + HI+
HTP 358 194 165
100 100 60
ECS~,-C6~El
HTPNO 374 210 165
100 45 85
E;o)c~H,-C,H,C~
HP+ 354 190 165
PX-Wl+
F-Go~,oOl+ WY-M+
kk,Hi -C6~4Cl]+
;8 100 80
HNO 392 374 228 165
100 20 15 80
[F-W-WI+ E:H~o]+
[HNCsH902-C6H4C1 + HI+
PGJ-&oOl+
TABLE 1B Production
ion data for synthetic standards obtained from El-MS-MS Ion abundance
FBPA 196 178 152 123
100 10 25 60
+ HI+
W&J-Wl+
100
CPHP 212 194
+ H]+
PX-4@1+
(%)
Ion assignment
M’+ [M-18]‘+ [M-COJ+ [F-C6H,-CO]‘+
128
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
100
ui 9
0 I
I
0
”
I
I
10
5
1 15
Min.
Fig. 3. HPLC chromatogram of haloperidol metabolite mixture obtained following guinea pig hepatic microsomal incubation. UV absorption recorded at 220 nm. IS is an internal standard.
HP+, and HNO) displayed a product ion at m/z 165, corresponding to a charge-initiated fragmentation of the alkyl carbon-nitrogen bond with expulsion of the nitrogen-containing moiety as a neutral [14]. Although the product ion spectra are simple, the two types of ions produced give information on whether modilication has occurred at either end of the metabolite. The product ions from the EI-MS-MS for FBPA are recorded in Table 1B and display fragmentation loss from the oxidized aliphatic chain. Analyses of microsomal incubations by HPLC,
UV, and mass spectrometry
Guinea pig liver microsomal incubates were subjected to reverse phase HPLC, after precipitation and removal of microsomal proteins [18], and the HPLC chromatogram is shown in Fig. 3. Individual fractions were collected, and after lyophilization and redissolving in a minimum of methanol (lO,~l), were analyzed directly by either FAB-MS or EI-MS. Specific HPLC fractions when subjected to positive ion FAB-MS displayed ions containing a distinctive chlorine isotope pattern at m/2212(214) and 392(394) (6-7.5min); 194(196), 374(376), and 378(380) (7.5-8.2 min); 376(378) (8.2-10.7 min); and 354(356) and 358(360) (11.4-12.8min). No ions with the exception of m/z
J. Fang et al/Id.
J. Mass Spectrom. Ion Processes 122 (1992) 121-131
129
376(378) (8.2-10.7 min), corresponding to unmetabolized drug substrate HAL, were detected in HPLC fractions collected from control microsomal incubate injections. When the HPLC fraction collected between 3-5min was subjected to EI-MS, ions at m/z 180 and 196 were observed, which were not present in control samples subjected to the same ionization method. The FAB product ion spectra of ions at m/z 212,374,378,376,354, and 358 were almost identical to the product ion spectra of synthetic standards CPHP, HTPNO, RHAL, HAL, HP+, and HTP, respectively. These observations in conjunction with the fact that the HPLC retention times and UV spectra of synthetic standards closely matched the microsomal incubate fractions confirmed that the parent drug HAL undergoes the following metabolic processes; oxidative N-dealkylation (to give CPHP), ketone reduction (RHAL), dehydration (HTP), followed by either N-oxidation (HTPNO) or ring reduction, to afford the pyridinium species HP+. Previously, we [8] and Castagnoli and co-workers [12,13] have noted that the pyridinium analogue, HP+, of HAL is a major metabolite from microsomal preparations for a variety of animal sources, including rats and mice. Furthermore, we have shown using FAB-MS-MS that the dehydration product HTP is also formed and is the intermediate in the pathway to HP+. This confirms our previous tentative conclusions using thermospray-MS [9]. However, both in this study and our previous studies [8,9], it has not been possible to isolate and detect the 2,3-dihydropyridinium analogue HDP+ (shown in Fig. 1). However, further studies using on-line HPLC continuous flow liquid secondary ionization MS to try to detect this reactive intermediate are in progress. The FAB product ion spectrum obtained from the ion at m/z392 [67.5min] shown in Fig. 4 was almost identical to that obtained from the synthetic standard HNO (detailed in Table 1A). Also, the UV spectra of synthetic HAL and HNO were identical (&,,, = 246 nm in phosphate buffer) to the sample containing the ion at m/2392. However, the retention time of synthetic HNO is NN1 min longer than the HPLC fraction containing the ion at m/z 392. Based on the product ion spectrum of m/z 392, either N-oxidation to give the N-oxide, or C-oxidation to give the chloro-phenol has occurred. Moreover, since a product ion at m/z 165 is observed, no oxidation of the fluorophenyl aromatic ring nor the aliphatic chain could have taken place. Furthermore, since the UV spectrum of the fraction containing the ion at m/z 392 is almost identical to HNO, this suggests that no oxidation of the chlorophenyl ring system has occurred, since such an oxidation would result in a +5-8nm shift in the UV [22]. Therefore, it appears likely that Noxidation has occurred to afford the N-oxide. However, HNO may exist as two stereoisomers, i.e. cis and trans [23], and it is possible that the metabolite
130
J. Fang et aLlInt. J. Mass Spectrom.
Ion Processes 122 (1992) 121-131
374 228
Fig. 4. The FAB product ion spectrum of m/z 392 obtained in the 6-7.5 min HPLC fraction of a guinea pig hepatic microsomal incubation. The spectrum was obtained using argon as the collision gas ( 10m5mbar) at a collision energy of x 220eV and ten scans were collected in the MCA mode.
isolated is a stereoisomer of synthetic HNO. Further work is in progress to determine the reason for the difference in retention times observed with the synthetic and biosynthetic N-oxides. The HPLC fraction 7.5-8.2 min contained an ion in positive FAB-MS at m/z 194 with an isotope contribution at m/z 196 indicative of chlorine being present in the ion. A FAB product ion spectrum of m/z 194 did not produce any detectable product ions at a variety of collision gas energies (lo-300 eV). Based on the previous discussion that the product ion spectrum of CPHP (Table 1A) only gave a product ion corresponding to loss of H,O, and that all other synthetic standards containing the chlorophenyl piperidine (or derivative) system did not show any fragmentation related to bond cleavage between the two rings, the structure CPTP shown in Fig. 1 is consistent with these observations. This has been tentatively suggested as a metabolite from HTP in our previous work [9]. The EI product ion spectrum of the ion at m/z 196 observed in the 3-5 min HPLC fraction closely resembled that observed for synthetic standard FBPA. Furthermore, the HPLC retention time and UV spectrum of the sample containing the ion at m/z 196 were identical to synthetic FBPA. Also present in the 3-5 min HPLC fraction was an ion at m/z 180, which could correspond to the intermediate aldehyde 4-fluorobenzoylpropional ( FBALD) (see Fig. l),
J. Fang et aLlInt. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
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but the EI product ion spectrum was inconclusive, and further studies are ongoing to fully establish the structure of this metabolite. CONCLUSION
Using a combination of reversed-phase HPLC and MS-MS, it was possible to isolate and identify eight metabolites of the neuroleptic agent HAL from a guinea pig hepatic microsomal incubate. Based on these results, it has been possible to develop a clearer understanding of the major metabolic pathways by which HAL undergoes biotransformation. REFERENCES 1 A.G. Goodman, L.S. Goodman and A. Gilman, in A.G. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 6th Edn., MacMillan, New York, 1980, pp. 391-418. 2 J.L. Waddington, Psychopharmacology, 101 (1990) 431. 3 R.E. See and G. Ellison, Psychopharmacology, 100 (1990) 404. 4 W. Soudijn, I. Van Wijngaarden and F. Allewijn, Eur. J. Pharmacol., 1 (1967) 47. 5 A. Forsman, G. Folsch, M. Larrson and R. Ohman, Curr. Ther. Res., 21 (1977) 606. 6 E.R. Korpi, D.T. Costakos and R.J. Wyatt, Biochem. Pharmacol., 34 (1985) 2923. 7 I.J. Kopin and S.P. Markey, Ann. Rev. Neurosci., 11 (1989) 81. 8 J. Fang and J.W. Gorrod, Toxicol. Lett., 59 (1991) 117. 9 J. Fang and J.W. Gorrod, Med. Sci. Res., 20 (1992) 175. 10 E. Wu, T. Shinka, P. Caldera-Munoz, H. Yoshizumi, A. Trevor and N. Castagnoli Jr., Chem. Res. Toxicol., 1 (1988) 186. 11 H. Rollema, B. Subramanyam, M. Skolnik, J. d’Engelbronner and N. Castagnoli Jr., in H. Rollema, B.H.C. Westerink and W.J. Drijfout (Eds.), Monitoring Molecules in Neurosciences, Proc. 5th International Conference on in vivo Methods, Noordwijkerhout, The Netherlands, 1991, pp. 367-369. 12 B. Subramanyam, T. Woolf and N. Castagnoli Jr., Chem. Res. Toxicol., 4 (1991) 123. 13 B. Subramanyam, H. Rollema, T. Woolf and N. Castagnoli Jr., Biochem. Biophys. Res. Commun., 166 (1990) 238. 14 B. Subramanyam, S.M. Pond, D.W. Eyles, H.A. Whiteford, H.G. Fouda and N. Castagnoli Jr., Biochem. Biophys. Res. Commun., 181 (1991) 573. 15 K.L. Busch, G.L. Glish and S.A. McLuckey, Mass Spectrometry/Mass Spectrometry: Principles and Applications of Tandem Mass Spectrometry, VCH, New York, 1988. 16 T. Oida, Y. Terauchi, K. Yoshida, A. Kagemoto and Y. Sekine, Xenobiotica, 19 (1989) 781. 17 J.W. Gorrod, D.J. Temple and A.H. Beckett, Xenobiotica, 5 (1975) 453. 18 M. Kajbaf, M. Jahanshahi, K. Pattichis, J.W. Gorrod and S. Naylor, J. Chromatogr. Biomed. Appl., 575 (1992) 75. 19 P.J. Todd and F.W. McLafferty, Int. J. Mass Spectrom. Ion Phys., 38 (1981) 371. 20 K.L. Schey, H.I. Kenttamaa, V.H. Wysocki and R.G. Cooks, Int. J. Mass Spectrom. Ion Processes, 90 (1989) 71. 21 A.J. Alexander, E.W. Dyer and P.K. Boyd, Rapid Commun. Mass Spectrom., 3 (1989) 364. 22 R.M. Silverstein, G.C. Bassler and T.C. Morrill, Spectrometric Identification of Organic Compounds, 4th Edn., 1981, pp. 323-325. 23 J. Fang, Ph.D. Thesis, University of London, (1992).
121
Investigation of the neuroleptic drug haloperidol and its metabolites using tandem mass spectrometry* Jian
Fang”,
John
W. Gorroda, Mahmud Kajbafb, John H. Lambb and
Stephen Naylor’ “Chelsea Departmentof Pharmacy, King’s College, University of London, London S W3 6LX (UK) bMRC Toxicology Unit, Carshalton. Surrey SMS 4EF (UK) ‘Mass Speclrometry Facility, Departments of Biochemistry and Molecular Biology, and Pharmacology, Mayo Clinic, Rochester, MN 55905 (USA)
(First received 3 June 1992; in final form 30 June 1992)
ABSTRACT The in vitro metabolism of haloperidol, a clinically utilized neuroleptic drug, was investigated using guinea pig derived hepatic microsomal incubates. By employing a combination of reversed phase HPLC and tandem mass spectrometry, it was revealed that haloperidol was metabolized to at least eight different compounds, including the proposed dopaminergic toxin 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4oxobutyll-pyridinium species and an intermediate metabolite 4-(4-chlorophenyl)-l-[4-(4-fluorophenyl)-4oxobutyll-1,2,3,6_tetrahydropyridine. Keywor&
tandem mass spectrometry; haloperidol.
INTRODUCTION
Haloperidol (4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-4-piperidinol, HAL) see Fig. 1) is a neuroleptic agent that has proven clinically useful and belongs to the important butyrophenone class of such drugs [l]. It is widely used in current clinical practice but has a number of neurological side-effects associated with its use. In particular, it causes tardive dyskinesias and produces Parkinsonian symptoms in both humans and animals [2,3]. The metabolic fate of HAL has not until recently been extensively studied, even though the parent compound is a clinically useful drug. Previous reports have suggested that oxidative N-dealkylation and ketone reduction are the major metabolic pathways. Furthermore, recent evidence linking the nigrostriatal neurotoxin N-methyl-4-phenyl-1,2,3,6_tetrahydropyridine (MPTP) (see Fig. 2) with the induction of Parkinsonian symptoms [7] has led to the Correspondence to: S. Naylor, Mass Spectrometry Facility, Departments of Biochemistry and Molecular Biology, and Pharmacology, Mayo Clinic, Rochester, MN 55905, USA. * Dedicated to the memory of Professor Michael Barber, a friend and colleague. 0168-l 176/92/%05.00
0
1992 Elsevier Science Publishers
B.V. All rights reserved.
122
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
123
MPDP’
MPP’
Fig. 2. Chemical structures of the neurotoxin MPP+.
MPTP and its two metabolites MPDP+, and
that HAL, which is structurally similar, or more likely one of its analogous metabolites, also operates via a similar mechanism [8,9]. It has been demonstrated the MPTP is first metabolized to iV-methyl-4phenyl-2,3_dihydropyridinium (MPDP+ ) and subsequently to the N-methyl4-phenylpyridinium (MPP+ ) species [ 10,111, as shown in Fig. 2. MPP+ is believed to exert its neurotoxic effect by inhibiting mitochondrial respiration. Recently, Castagnoli and co-workers have reported the detection of the putative neurotoxic pyridinium metabolite (C(Qchlorophenyl)- 1-[4-(4fluorophenyl)-4-oxobutyll-pyridinium, HP+), comparable to MPP+, derived from HAL in both the urine and brain tissue of haloperidol-treated rats [ 12,131and the urine of haloperidol-treated humans [ 141.Two of us [8,9] have also reported the presence of HP+ in an in vitro mouse microsomal incubation mixture as well as the HAL-1,2,3,6-tetrahydropyridine analogue (HTP), which is structurally very similar to MPTP. In the present study, we have utilized a combination of reversed phase HPLC and tandem mass spectrometry (MS-MS) [15] to further investigate the metabolic pathways of HAL. In the process, we have identified and characterized eight metabolites of the parent drug HAL, obtained from in vitro guinea pig liver microsomal incubations. suggestion
EXPERIMENTAL
Materials HAL was obtained from the Sigma Chemical Company, St. Louis, MO, USA; HPLC acetonitrile (far UV grade) was purchased from FSA Laboratory Supplies.
124
J. Fang et aLlint. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
4-(4-Chlorophenyl)-1-[4-(4-fluorophenyl)-4-hydroxybutyl]-4-piperidinol (RHAL) was prepared according to Oida et al. [16]. HTPi, HP+, 4-(4-chlorophenyl)-l-[4-(4-fluorophenyl)-4-oxobutyl]-4-piperidinol-N-oxide (HNO), 4(4-chlorophenyl)- 1-[4-(4-fluorophenyl)-4-oxobutyll1,2,3,6-tetrahydropyridine-N-oxide (HTPNO) were synthesised in this laboratory (J.F. and J.W.G.). Details of these syntheses will be described in a separate publication. All other chemicals, enzymes, and cofactors were obtained from Sigma Chemical Co., Poole, UK. Methods Dunkin-Hartley guinea pigs were obtained from the Animal Supply Unit of King’s College, University of London. Animals were fasted overnight before sacrifice. Hepatic microsomal preparations were prepared using the centrifugation method described previously [17]. Incubation procedures were as follows: a reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating system consisting of the sodium salt of nicotinamide adenine dinucleotide phosphate (NADP+ ) (2 pmol), glucose-6-phosphate disodium salt (10 pmol), glucose-6-phosphate dehydrogenase (1 unit), and MgCl, (2 mg) all in 2 ml phosphate buffer (0.2 M, pH 7.4) was preincubated for 5 min. Enzymatic reactions were initiated by addition of HAL (2pmol) and microsomal preparations equivalent to 0.5 g original tissue. In control incubates, heat inactivated microsomes were used instead of fresh microsomal preparations. Incubations were carried out for 30min at 37°C. Enzymic reactions were terminated by addition of ZnSO, (200mg) to the incubation mixture. The precipitated proteins were removed by centrifugation (MSE Centaur 2, 3000 rev. min-’ , 20min). The supernatant was passed through a preconditioned (methanol (4 ml) followed by distilled water (4 ml)) Sep-Pak C,, cartridge. Excess ZnSO, was removed by washing with distilled water (4 ml). The retained compounds were eluted by methanol (4 ml), which was subsequently evaporated to dryness at 45°C under nitrogen [18]. The residues were reconstituted in methanol (50~1) and subjected to HPLC separation (10 ~1 injections). The HPLC chromatographic system comprised a Waters M-45 pump and a Rapiscan SA6508 detector set to record at 220,450 and 360nm. A Tandon TM7002 computer was used to record chromatograms. The HPLC system used a 5 pm Hypersil CPS-5 column (4.6 mm x 25 cm). The mobile phase was a combination of acetonitrile (67%) and ammonium acetate buffer (10 mM) adjusted to pH 5.4 with acetic acid. The solvent was delivered at 1 ml min-‘. The incubation extract was injected into the HPLC system, and peaks of interest were collected. Control incubation extracts were also subjected to
J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
125
HPLC, and fractions were collected at the same time intervals as that of the sample. The collected elutes were evaporated to dryness using a vacuum freeze dryer. Mass spectrometry
All mass spectra were obtained on a VG 70-SEQ instrument of EBQ,Q2 configuration, where E is an electrostatic analyser, B is the magnet, Q, is an r.f.-only quadrupole collision cell, and Q2 is a mass-filter quadrupole. EB and Q2 correspond to mass spectrometer one (MS,) and two (MS,), respectively. All synthetic standards and microsomal incubate mixtures as well as control samples were ionised by either (i) positive ion fast atom bombardment mass spectrometry (FAB-MS), using xenon atoms from a model Bl 1N saddle-field fast-atom gun (Ion Tech, Teddington, UK) or (ii) direct insertion probe (DIP) electron ionization-MS (EI-MS) where the probe was heated to 260°C after 30 s in the source. In both cases, the ions produced were accelerated out of the source region to an energy of 8 keV and analyzed in MS, using a scan speed of 10s per decade over the mass range 50-800 at a resolution of x 2000. Product ion spectra-Protonated molecular ions (MH+) in the case of FAB or molecular ions (M’+) in EI (in both cases also referred to as precursor ions) of synthetic standards, microsomal incubate and control samples were selected with a resolution of z 1000 using EB and subjected to collision activated dissociation using argon as the collision gas in Q,. Collision energy was optimized to give maximum fragmentation of the precursor ion at = 220 eV with a gas pressure in the Q, housing of M 10e5 mbar. The resulting fragment or product ions were mass analyzed in Q2 and a product ion spectrum acquired by scanning Q2 over the range m/z40-450 with 10 scans being obtained in the multi-channel analysis (MCA) mode. Sample preparation for mass spectrometry
Usually 1~1 of the x 10~1methanol solution containing the HPLC fraction of the HAL metabolite(s) was removed by a Hamilton syringe. In FAB-MS, the 1~1 of methanol was thoroughly mixed with z 1.5 ~1 of 3nitrobenzylalcoho1 (NBA) on the stainless steel probe tip and inserted into the source. In EI-MS, the 1~1 solution was added to the glass vial on the probe tip and gently heated to remove solvent before inserting into the mass spectrometer. RESULTS AND DISCUSSION
Initial studies focused on determining optimal FAB matrix conditions for detecting HAL and three synthetic derivatives (RHAL, HTP, and 4-fluoro-
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benzoylpropionic acid (FBPA)). A variety of FAB matrices were used including glycerol, thioglycerol, glycerol-thioglycerol (1 : l), thiodiglycol, dithiothreitol-dithioerythreitol (3 : l), and 3-nitrobenzylalcohol (NBA). In each case for HAL, RHAL, and HTP, NBA proved to afford the most abundant signal-to-noise ratio for the respective protonated molecular ions (MH+). In particular, it was possible to detect x 750pmol of HAL on the probe tip in positive ion FAB-MS using NBA as the matrix. In the case of the fluorophenyl standard FBPA, FAB-MS proved to be relatively insensitive as an ionization method. It was possible to detect only x 3 nmol of FBPA on the FAB probe tip using NBA. (All other matrices afforded either similar or less sensitivity.) However, using the direct insertion probe with EI as the ionization mode, it was possible to detect x 200 pmol of the synthetic standard FBPA. Product ion spectra of synthetic standards
The FAB product ion spectral data of the synthetic standards HAL, RHAL, HTP, HTPNO, HP+, 4-chlorophenyl-4-hydroxypiperidine (CPHP), and HNO (structures shown in Fig. 1) and the product ions observed when ionized using the DIP for FBPA are shown in Table 1. In all cases, the collision energy in the gas cell (Q, ) was optimized to afford maximum fragmentation of the precursor ion at x 220eV with an argon gas pressure of x lo-‘mbar. These conditions are known to bring about multiple collisions between selected precursor ions and the collision gas [19,20] but do not cause maximum scattering of the resulting fragment product ions [21]. In the case of piperidinol containing compounds, namely HAL, RHAL, and HNO, the FAB-MS-MS product ion spectra contained abundant ions at [MH - 18]+ corresponding to loss of H,O from the piperidinol ring. Furthermore, the product ion spectra of all the synthetic standards HAL, RHAL, HTP, HTPNO, HP+, and HNO were relatively simple and contained few product ions. The predominate bond cleavage occurred between the piperidine nitrogen and the alkyl carbon of the hydrocarbon chain. The resulting fragmentation afforded two classes of product ions. Firstly, the compounds listed above all contained a product ion corresponding to the chlorophenyl-piperidine (or derivative thereof) portion of the molecule, at m/z212 (HAL, RHAL); 194 (HAL, RHAL, HTP); 210 (HTPNO); 190 (HP+); and 228 (HNO). Furthermore, there was no evidence of fragmentation occurring between the chlorophenyl and the piperidine (or derivative) ring systems. This was further confirmed by inspection of the product ion spectrum of synthetic CPHP which only contained a single product ion at m/z 194, denoting loss of H,O, and the ring systems remained intact. Secondly, all the fluorobenzoyl containing compounds (HAL, RHAL, HTP, HTPNO,
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TABLE 1A Product ion data obtained from FAB-MS-MS Ion abundance
(%)
for synthetic standards Ion assignment
HAL 376 358 212 194 165
100 80 5 25 90
zH*O)+ [HNCS H90-CsH4Cl + H]’ [HNCS H, -CBH&I + H] +
RHAL 378 360 212 194 165 149
100 65 5 10 5 5
[HNC, H90-C,H&l + H]+ [HNCSH,-CsH4Cl + HI+
HTP 358 194 165
100 100 60
ECS~,-C6~El
HTPNO 374 210 165
100 45 85
E;o)c~H,-C,H,C~
HP+ 354 190 165
PX-Wl+
F-Go~,oOl+ WY-M+
kk,Hi -C6~4Cl]+
;8 100 80
HNO 392 374 228 165
100 20 15 80
[F-W-WI+ E:H~o]+
[HNCsH902-C6H4C1 + HI+
PGJ-&oOl+
TABLE 1B Production
ion data for synthetic standards obtained from El-MS-MS Ion abundance
FBPA 196 178 152 123
100 10 25 60
+ HI+
W&J-Wl+
100
CPHP 212 194
+ H]+
PX-4@1+
(%)
Ion assignment
M’+ [M-18]‘+ [M-COJ+ [F-C6H,-CO]‘+
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J. Fang et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 121-131
100
ui 9
0 I
I
0
”
I
I
10
5
1 15
Min.
Fig. 3. HPLC chromatogram of haloperidol metabolite mixture obtained following guinea pig hepatic microsomal incubation. UV absorption recorded at 220 nm. IS is an internal standard.
HP+, and HNO) displayed a product ion at m/z 165, corresponding to a charge-initiated fragmentation of the alkyl carbon-nitrogen bond with expulsion of the nitrogen-containing moiety as a neutral [14]. Although the product ion spectra are simple, the two types of ions produced give information on whether modilication has occurred at either end of the metabolite. The product ions from the EI-MS-MS for FBPA are recorded in Table 1B and display fragmentation loss from the oxidized aliphatic chain. Analyses of microsomal incubations by HPLC,
UV, and mass spectrometry
Guinea pig liver microsomal incubates were subjected to reverse phase HPLC, after precipitation and removal of microsomal proteins [18], and the HPLC chromatogram is shown in Fig. 3. Individual fractions were collected, and after lyophilization and redissolving in a minimum of methanol (lO,~l), were analyzed directly by either FAB-MS or EI-MS. Specific HPLC fractions when subjected to positive ion FAB-MS displayed ions containing a distinctive chlorine isotope pattern at m/2212(214) and 392(394) (6-7.5min); 194(196), 374(376), and 378(380) (7.5-8.2 min); 376(378) (8.2-10.7 min); and 354(356) and 358(360) (11.4-12.8min). No ions with the exception of m/z
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376(378) (8.2-10.7 min), corresponding to unmetabolized drug substrate HAL, were detected in HPLC fractions collected from control microsomal incubate injections. When the HPLC fraction collected between 3-5min was subjected to EI-MS, ions at m/z 180 and 196 were observed, which were not present in control samples subjected to the same ionization method. The FAB product ion spectra of ions at m/z 212,374,378,376,354, and 358 were almost identical to the product ion spectra of synthetic standards CPHP, HTPNO, RHAL, HAL, HP+, and HTP, respectively. These observations in conjunction with the fact that the HPLC retention times and UV spectra of synthetic standards closely matched the microsomal incubate fractions confirmed that the parent drug HAL undergoes the following metabolic processes; oxidative N-dealkylation (to give CPHP), ketone reduction (RHAL), dehydration (HTP), followed by either N-oxidation (HTPNO) or ring reduction, to afford the pyridinium species HP+. Previously, we [8] and Castagnoli and co-workers [12,13] have noted that the pyridinium analogue, HP+, of HAL is a major metabolite from microsomal preparations for a variety of animal sources, including rats and mice. Furthermore, we have shown using FAB-MS-MS that the dehydration product HTP is also formed and is the intermediate in the pathway to HP+. This confirms our previous tentative conclusions using thermospray-MS [9]. However, both in this study and our previous studies [8,9], it has not been possible to isolate and detect the 2,3-dihydropyridinium analogue HDP+ (shown in Fig. 1). However, further studies using on-line HPLC continuous flow liquid secondary ionization MS to try to detect this reactive intermediate are in progress. The FAB product ion spectrum obtained from the ion at m/z392 [67.5min] shown in Fig. 4 was almost identical to that obtained from the synthetic standard HNO (detailed in Table 1A). Also, the UV spectra of synthetic HAL and HNO were identical (&,,, = 246 nm in phosphate buffer) to the sample containing the ion at m/2392. However, the retention time of synthetic HNO is NN1 min longer than the HPLC fraction containing the ion at m/z 392. Based on the product ion spectrum of m/z 392, either N-oxidation to give the N-oxide, or C-oxidation to give the chloro-phenol has occurred. Moreover, since a product ion at m/z 165 is observed, no oxidation of the fluorophenyl aromatic ring nor the aliphatic chain could have taken place. Furthermore, since the UV spectrum of the fraction containing the ion at m/z 392 is almost identical to HNO, this suggests that no oxidation of the chlorophenyl ring system has occurred, since such an oxidation would result in a +5-8nm shift in the UV [22]. Therefore, it appears likely that Noxidation has occurred to afford the N-oxide. However, HNO may exist as two stereoisomers, i.e. cis and trans [23], and it is possible that the metabolite
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Ion Processes 122 (1992) 121-131
374 228
Fig. 4. The FAB product ion spectrum of m/z 392 obtained in the 6-7.5 min HPLC fraction of a guinea pig hepatic microsomal incubation. The spectrum was obtained using argon as the collision gas ( 10m5mbar) at a collision energy of x 220eV and ten scans were collected in the MCA mode.
isolated is a stereoisomer of synthetic HNO. Further work is in progress to determine the reason for the difference in retention times observed with the synthetic and biosynthetic N-oxides. The HPLC fraction 7.5-8.2 min contained an ion in positive FAB-MS at m/z 194 with an isotope contribution at m/z 196 indicative of chlorine being present in the ion. A FAB product ion spectrum of m/z 194 did not produce any detectable product ions at a variety of collision gas energies (lo-300 eV). Based on the previous discussion that the product ion spectrum of CPHP (Table 1A) only gave a product ion corresponding to loss of H,O, and that all other synthetic standards containing the chlorophenyl piperidine (or derivative) system did not show any fragmentation related to bond cleavage between the two rings, the structure CPTP shown in Fig. 1 is consistent with these observations. This has been tentatively suggested as a metabolite from HTP in our previous work [9]. The EI product ion spectrum of the ion at m/z 196 observed in the 3-5 min HPLC fraction closely resembled that observed for synthetic standard FBPA. Furthermore, the HPLC retention time and UV spectrum of the sample containing the ion at m/z 196 were identical to synthetic FBPA. Also present in the 3-5 min HPLC fraction was an ion at m/z 180, which could correspond to the intermediate aldehyde 4-fluorobenzoylpropional ( FBALD) (see Fig. l),
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but the EI product ion spectrum was inconclusive, and further studies are ongoing to fully establish the structure of this metabolite. CONCLUSION
Using a combination of reversed-phase HPLC and MS-MS, it was possible to isolate and identify eight metabolites of the neuroleptic agent HAL from a guinea pig hepatic microsomal incubate. Based on these results, it has been possible to develop a clearer understanding of the major metabolic pathways by which HAL undergoes biotransformation. REFERENCES 1 A.G. Goodman, L.S. Goodman and A. Gilman, in A.G. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 6th Edn., MacMillan, New York, 1980, pp. 391-418. 2 J.L. Waddington, Psychopharmacology, 101 (1990) 431. 3 R.E. See and G. Ellison, Psychopharmacology, 100 (1990) 404. 4 W. Soudijn, I. Van Wijngaarden and F. Allewijn, Eur. J. Pharmacol., 1 (1967) 47. 5 A. Forsman, G. Folsch, M. Larrson and R. Ohman, Curr. Ther. Res., 21 (1977) 606. 6 E.R. Korpi, D.T. Costakos and R.J. Wyatt, Biochem. Pharmacol., 34 (1985) 2923. 7 I.J. Kopin and S.P. Markey, Ann. Rev. Neurosci., 11 (1989) 81. 8 J. Fang and J.W. Gorrod, Toxicol. Lett., 59 (1991) 117. 9 J. Fang and J.W. Gorrod, Med. Sci. Res., 20 (1992) 175. 10 E. Wu, T. Shinka, P. Caldera-Munoz, H. Yoshizumi, A. Trevor and N. Castagnoli Jr., Chem. Res. Toxicol., 1 (1988) 186. 11 H. Rollema, B. Subramanyam, M. Skolnik, J. d’Engelbronner and N. Castagnoli Jr., in H. Rollema, B.H.C. Westerink and W.J. Drijfout (Eds.), Monitoring Molecules in Neurosciences, Proc. 5th International Conference on in vivo Methods, Noordwijkerhout, The Netherlands, 1991, pp. 367-369. 12 B. Subramanyam, T. Woolf and N. Castagnoli Jr., Chem. Res. Toxicol., 4 (1991) 123. 13 B. Subramanyam, H. Rollema, T. Woolf and N. Castagnoli Jr., Biochem. Biophys. Res. Commun., 166 (1990) 238. 14 B. Subramanyam, S.M. Pond, D.W. Eyles, H.A. Whiteford, H.G. Fouda and N. Castagnoli Jr., Biochem. Biophys. Res. Commun., 181 (1991) 573. 15 K.L. Busch, G.L. Glish and S.A. McLuckey, Mass Spectrometry/Mass Spectrometry: Principles and Applications of Tandem Mass Spectrometry, VCH, New York, 1988. 16 T. Oida, Y. Terauchi, K. Yoshida, A. Kagemoto and Y. Sekine, Xenobiotica, 19 (1989) 781. 17 J.W. Gorrod, D.J. Temple and A.H. Beckett, Xenobiotica, 5 (1975) 453. 18 M. Kajbaf, M. Jahanshahi, K. Pattichis, J.W. Gorrod and S. Naylor, J. Chromatogr. Biomed. Appl., 575 (1992) 75. 19 P.J. Todd and F.W. McLafferty, Int. J. Mass Spectrom. Ion Phys., 38 (1981) 371. 20 K.L. Schey, H.I. Kenttamaa, V.H. Wysocki and R.G. Cooks, Int. J. Mass Spectrom. Ion Processes, 90 (1989) 71. 21 A.J. Alexander, E.W. Dyer and P.K. Boyd, Rapid Commun. Mass Spectrom., 3 (1989) 364. 22 R.M. Silverstein, G.C. Bassler and T.C. Morrill, Spectrometric Identification of Organic Compounds, 4th Edn., 1981, pp. 323-325. 23 J. Fang, Ph.D. Thesis, University of London, (1992).