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Identification of metabolites derived from the H2-receptor antagonist mifentidine using tandem mass spectrometry
151
Anolytrcu Chwuca km, 247 (1991) 151-159 Elsevrer Scrence Pubhshers B.V., Amsterdam
Identification of metabolites derived from the Hz-receptor antagonist mifentidine using tandem mass spectrometry M. Kajbaf
*, J.H. Lamb and S. Naylor *
MRC Toxicology Unit, Carshalton, Surrey SM5 4EF (l_J.K)
K. Patti&is and J.W. Gorrod Chelsea Department
of Pharmacy, Kmg’s College London, Umversrty of London, London SW3 6LX (U K.)
(Received 20th August 1990)
The in vrtro metabolism of mrfentidine, a second generation lustamine Hz-receptor antagomst, IS mvestrgated after hepatic microsomal mcubatron. By employing a combinatron of both daughter and parent ran scanning tandem mass spectrometry on synthetic standards and the nucrosomal incubate, it IS revealed that mrfentrdme 1s metabohsed to at least three compounds, namely the amme, formamide and urea metabohtes. The detectron and characterisatron of the three metabohtes IS carrred out with mimmal sample punhcation. Keywords.
Mass spectrometry,
Mifentrdine
Peptic ulcers are a common malady in “Western World” populations, and one of the possible causative factors is excessive secretion of gastric acid by parietal cells in the stomach [l]. Acid secretion is stimulated by the action of histamine on the Hz-receptors of parietal cells [2,3], and hence numerous H,-antagonistic drugs such as cimetidine (trade-name Tagamet) [4] and ranitidine (trade-name Zantac) [5] have been marketed worldwide to combat the effect of histamine on such receptors. Recently, a new H,-antagonist, N’-[(Cimidazolyl)phenyl]-N2-isopropylformamidine dihydrochloride (mifentidine) (1) entered phase II clinical trials [6], and it represents the prototype of the “second generation” of histamine H,-receptor antagonists. It was originally synthesised by Donetti et al. [7], and is both a potent antisecretory agent [8] and a promising anti-ulcer drug [9,10]. Preliminary in vivo experiments by Vidi [ll] have shown that it is possibly metabolised to 0003-2670/91/$03.50
0 1991 - Elsevier Science Pubhshers B.V
the amine (2) and the formamide (3) derivative. Recently Patti&is et al. [12], using in vitro guinea pig, rat, hamster and mouse hepatic microsome preparations, have tentatively identified, by UV, liquid chromatography (LC) and thin-layer chromatography the amine (2) formamide (3) and urea (4) derivatives of mifentidine. Typically, in drug metabolism studies, metabolites that are present in low concentration have to be isolated from complex biological matrices [13,14]. Such matrices can range from urine, feces and whole blood for in vivo studies, or solution mixtures that contain high concentrations of numerous cofactors, enzymes, substrates and buffers as for in vitro microsomal incubates [15,16]. Extensive purification, followed by limited analysis [UV, gas chromatography-mass spectrometry (GC-MS) and comparison of LC retention times to synthetic standards] is then performed in order to identify the unknown metabolites [17,18]. In this work we have investigated the in vitro
M KAJBAF
152
N Formamide Scheme
ET AL
HNQN
1
microsomal metabolism of mifentidine (1) using tandem MS. In particular, by utilizing both parent and daughter ion scanning techniques [19] it is possible to determine the presence, and characterise metabolites without the need to undertake extensive purification procedures.
EXPERIMENTAL
Chemicals Mifentidine (1) and all synthetic standards (24) were synthesised and supplied by the Institute De Angeli (Milan). All other materials were obtained from British Drug Houses Ltd., FSA Laboratory Supplies and Boehringer Mannheim. All FAB matrices were purchased from Aldrich, and used without further purification. Mlcrosomal incubation Hepatic microsomes from male albino DunkinHartley guinea pigs (400-600 g) were prepared by the CaCl, precipitation method described previously (201. The final pellet was suspended in phosphate buffer (0.2 mM, pH 7.4) to a concentration of 0.5 g original liver weight per ml. The substrate, mifentidine (1) (4 pmol per flask, a total of six flasks were incubated) dissolved in 0.5 ml water; plus the cofactors NADP (disodium salt), P-nicotinamide adenine dinucleotide phosphate, glucosed-phosphate (disodium salt), glucase-6-dehydrogenase, MgCl, in 2 ml phosphate buffer (0.2 mM, pH 7.4); plus the liver microsomal preparation (equivalent to 0.5 g liver resuspended in 1 ml tris-KC1 buffer) were all in-
cubated for 20 min at 37°C in a 25-ml Erlenmeyer flask as described by Gorrod et al. [21]. After incubation, the enzymic reaction was stopped by placing the flasks on ice and rapidly adding 100 ~1 of 1% ZnSO, (w/v). The contents of the Erlenmeyer flask were transferred to a 12-ml centrifuge tube and centrifuged at 3000 rpm (2000 g) for 15 min. The six supernatants were combmed (ca. 20 ml) and immediately loaded onto a pre-washed C,, reversed-phase Sep-Pak cartridge, and then eluted with 5 ml of methanol [22]. The methanol was subsequently removed under a stream of nitrogen at 37°C. The residue was redissolved in 50 ~1 of distilled methanol and subjected to mass spectrometry. Mass spectrometry All spectra were obtained on a VG70-SEQ instrument with EBQIQz configuration, where E is the electrostatic analyser, B is the magnet, Qi is an rf-only quadrupole collision cell and Qz is a mass filter quadrupole. Samples were ionized by either: (i) fast atom bombardment (FAB) using xenon as the source of fast atoms (8.5 KeV), (ii) direct insertion (probe scan) electron ionization (EI), where the probe was heated to 260°C after 30 s in the source. In both cases the ions produced were accelerated to 8 KeV from the source region and analysed in the first mass spectrometer (EB) using a scan speed of 10 s per decade over the mass range 400-60 daltons at a resolution of ca. 1000. Parent ions were selected using EB (equivalent to MS,) and subjected to collision activated dissociation (CAD) in Qi. The collision gas used was argon and the gas pressure was typically ca. 5 X
MS-MS
OF Hz-RECEPTOR
ANTAGONIST
MIFENTIDINE
153
METABOLITES
10e6 mbar. The collision energies used were lo-30 eV for both EI and FAB. Daughter ion spectra were acquired by selecting a specific ion using EB (MS,), subjecting that ion to CAD and scarming the mass filter quadrupole Q2 over a mass range of 300-30 daltons and ten scans were obtained in the multi-channel analysis (MCA) mode. Parent ion spectra were obtained using the MCA mode by selecting a specific ion to be passed through Q2 (MS,) and scanning the magnet over the mass range 400-60 daltons. MS sample preparation
Usually 1 ~1 of the 50 ~1 combined microsomal mixture in methanol was removed by a Hamilton syringe. In FAB-MS the l-p1 methanol was thoroughly mixed with 1.5 ~1 NBA on a stainless-steel probe tip and inserted into the source. In EI-MS, the l-p1 methanol 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 investigations focused on determining optimum matrix conditions, for detecting mifentidine (1) and a series of synthetic derivatives (2-4), using positive ion FAB-MS. A variety of FAB matrices were investigated including dithiothreitol-dithioerythreitol (3 : 1) (magic bullet), glycerol-thioglycerol (1: l), thiodiglycol, 0.2 M p-toluenesulfonic acid in glycerol, glycerol, 10 mM trifluoroacetic acid in glycerol, thioglycerol and 3-nitrobenzylalcohol (NBA). In all cases for each compound (l-4), NBA afforded the most abundant signal-to-noise ratio for the molecular ion (MH)+. A positive ion FAB spectrum (NBA as matrix) of an approximately equimolar mixture of pure synthetic derivatives, including the amine (2) (MH+= 160), formamide (3) (MH+= 188), and urea (4) (MH+= 245), plus mifentidine (1) (MH+ = 229) revealed molecular ions (MH)+ for all compounds, as shown in Fig. 1. The detection limit was G 2 nmol for each compound. However, a positive ion FAB spectrum of a guinea pig
m/z Fig. 1 Positive ion FAB-MS (matnx NBA) of an eqmmolar rmxture (2 nmol of each compound on the probe tip) of mifenkdine (1) and the synthetic derivatives (2,3,4). Note that “m” corresponds to matrix ions
hepatic microsomal incubate from mifentidine revealed only a distinct molecular ion (MH)+ for the substrate (l), with no indication of other molecular ions (MH)+ corresponding to expected metabolites (2-4). The failure to detect other metabolites in the microsomal incubate could be a result of two factors: (i) sensitivity of the FAB-MS method due to the abundant “chemical noise” background [23]. (ii) suppression effects on molecular ion abundance caused by the presence of salts [23] and other hydrophobic components in the mixture [24,25]. To overcome both of these problems we reverted to tandem MS analysis and used a C,, reversed-phase Sep-Pak cartridge to remove salts and any very hydrophobic contaminants. Recently, tandem MS has found increased use in the analysis of complex biological matrices with only minimal sample purification required [26,27]. In this instance the strategy developed was firstly to acquire daughter ion spectra of mifentidine (1) and a series of synthetic derivatives of (1). Secondly, using the most abundant fragment ion m/z value (obtained from the daughter ion spectra), individual parent ion scans were carried out on the microsomal incubate to determine the presence of metabolites in this complex biological
154
.
M. KAJBAF
ET AL
Fig. 2. FAB daughter 1011spectra (matrix NBA, colhsion energy 20 eV) of synthetic standards: (a) mifentldine (l), MH+= 229, (b) amine (2), MH+= 160, (c) form-de (3), MH+= 188, (d) urea (4), MH+= 245.
MS-MS
OF Hz-RECEPTOR
TABLE
ANTAGONIST
MIFENTIDINE
METABOLITES
I
Fragment ions observed synthettc standards (l-4) Fragment ton (m/z) Mifentidme 187
m the daughter
Fragment lost
of the
Possible structure of ton detected ’
(m/r) (1) 42, ]CH(CH,), i
ion spectra
-HI
170
59, [(NH-CH(CH,),)
160
69, [(CH=N-CH(CH,),)
[HIPh-N=CH -NH*]+ [HIPh-N=C]+
+Hl [HIPh-NH,]+
-HI Amme (2) 143 133
17, [NH,] 27, [HCN]
Formarmde (3) 170 18, [Hz01 160 28, WI 143 45, [CO + NH,] or [HCN + H,O] 133 55, [CO + HCN] Urea (4) 186
59, [(NH-CH(CH,),)
[HIPh-N=C]+ [HIPh-NHJ+
[HIPh-N=C=O]+
+Hl 160
85, [(C(O)-NH
[HIPh-NH,]
+
-CW’=,),)-HI ’ I = Irmdazolyl;
Ph = phenyl
mixture. Lastly, to unequivocally confirm the presence and structurally characterise ions corresponding to mifentidine metabolites in the incubate, daughter ion spectra of such ions were acquired and compared to the daughter ion spectra of synthetic standards. Initiahy FAB daughter ion spectra were obtained on mifentidine (l), plus a series of synthetic derivatives including the amine (2), formamide (3) and urea (4), which have been reported to be tentatively present in a microsomal incubate from mifentidine [12]. As shown in Fig. 2a, the daughter ion spectrum of mifentidine (1) (MH+= 229) afforded abundant fragment ions at m/z 187, 170 and 160 corresponding to loss of [CH(CH,), - H], and [(CH=N-CH [(NH-CH(CH,),) + HI (CH,),) - H] respectively (as outlined in Table 1). A daughter ion spectrum of the synthetic standard amine (2) exhibited fragment ions at m/z 143 (loss of NH,) and 133 (loss of HCkN) (see Fig. 2b
155
and Table 1); whereas the formamide (3) derivative contained daughter ions at m/z 170 [loss of H,O], 160 (loss of CO), 143 (see Table 1) and 133 (see Table 1) as shown in Fig. 2c. The synthetic urea (4) daughter ion spectrum showed fragment ions at m/z 186 {loss of (NH-CH(CH,),) + H} and at m/z 160 {loss of (-C(O)-NH-CH(CH,), - H)} (as detailed in Fig. 2d and Table 1). One of the principal factors in determining the sensitivity of parent ion scanning is the abundance of the selected daughter ion detected at the second collector [19]. Therefore, as can be seen from inspection of Fig. 2a-d, the most abundant daughter ion for mifentidine (1) at m/z 187, amine (2) at m/z 133, formamide (3) at m/z 160, and urea (4) at m/z 186 was selected to scan for parent ions using the microsomal incubate from mifentidine (1). Individual FAB parent ion scans of m/z 187, 133, 160 and 186 revealed ions at m/z 229, 160, 188 and 245 respectively, as shown in Fig. 3a-d. The parent ion at m/z 229 of m/z 187 (Fig. 3a) confirms the presence of the unmetabolised substrate, mifentidine (1). However, parent ions at m/z 160 (Fig. 3b), 188 (Fig. 3c) and 245 (Fig. 3d) correspond to the presence of the amine (2), formamide (3) and urea (4) respectively, in the microsomal mixture. It should be noted that when a control mixture (identical to the rnicrosomal mixture, but containing dead microsomes) was subjected to parent scanning of the same m/z values at m/z 133, 160 and 186, no parent ions at m/z 160, 188 and 245, respectively, were detected (results not shown). The parent ion spectrum of m/z 160, derived from the microsomal incubate (Fig. 3c) not only exhibits an ion at m/z 188 (corresponding to MH+ of the formamide (3) metabolite) but also contains abundant ions at m/z 229 {corresponding to mifentidine (l)}, and m/z 187 a corresponding to [(l) - CH(CH,), + 2H]+). In this instance a daughter ion (m/z 160) common to mifentidine (l), a mifentidine fragment ion (m/z 187) and the formamide (3) derivative in FAB-MS-MS tends a This ion is also observed m the positive ton FAB-MS
of nnfentidme (1) and ts formed durmg the fast atom bombardment process, due to fragmentation of (1).
156
M KAJBAF
ET AL.
hg. 3. FAB parent ion spectra (matnx NBA, collision energy 25 ev), of ions derived from a rmcrosomal Incubate. (a) parents of m/r 187; Ion at m/z 229 corresponds to (MH)+ of rmfentidine (I), (b) parents of m/z 133; ion at m/z 160 corresponds to (MH)+ of amme (2), (c) parents of m/z 160; ion at m/z 188 corresponds to (MH)+ of formamide (3), (d) parents of m/z 186, Ion at m/z 245 corresponds to (MH)+ of urea (4).
MS-MS
OF Hz-RECEPTOR
ANTAGONIST
MIFENTIDINE
157
METABOLITES
159
‘a (4
184
I*.
196
171
48
n.
213
1.28.
ICI 61
. ..III...I,I, *
l..llt
I II, I..,.,...I....~I,.., Is( El
I,,ll,,,,., 141
,,,,I 161
,
,
,.,. w
9
Fig. 4. EI daughter ton spectra of synthetic standards:
(a) mifentldine
to obscure the presence of (3) (MH+= 188) in the parent ion spectrum. Inspection of a daughter ion spectrum of mifentidine (l), (M’= 228), using EI as the primary ionization technique, revealed fragment ions at m/z 213, 186, 184, 171 and 159, whereas for the synthetic formamide (3) derivative (Mt= 187) only ions at m/z 160 and 159 were observed,
(l), Mt=
228; (b) formarmde (3), Mt=
as shown in Fig. 4. Subsequently an EI parent ion scan of m/z 160 (which is only present in the formamide (3) spectrum), derived from the microsomal mixture, revealed only a parent ion at m/z 187, assignable to the (Mf) of the formamide (3) metabolite (Fig. 5). To conclusively confirm the presence of unmetabolised substrate (1) and the three metabo-
10
I
1
I
6l
u
a
1
Fig. 5. EI parent Ion spectrum of ion m/z
160 dewed
187.
from a rmcrosomal
Incubate.
M
158
lites (2-4) in the microsomal mixture, individual FAB daughter ion spectra were obtained on the ions m/z 229, 160,188 and 245, derived from the
KAJBAF
ET AL
microsomal incubate and these spectra are shown in Fig. 6. With the exception of the daughter ion spectrum of mifentidine (1) (Fig. 6a), all the other 181
Im(a) 81.
170
‘7 (cl
-i&’ (d)
7
Fig. 6 Daughter nufentldine
eon spectra obtained
(1): (a) m/z
229, (b) m/z
from Ions correspondmg 160; (c) m/z
188; (d) m/z
to metabolites 245.
present
following
the rmcrosomal
metabohsm
of
MS-MS
OF H,-RECEF-TOR
ANTAGONIST
MIFENTIDINE
METABOLITES
daughter ion spectra derived from analysis of the rmcrosomal incubate, contained additional fragment ions (Fig. 6b-d) as compared to the daughter ion spectra of pure synthetic standards (2-4) shown in Fig. 2b-d. However, by comparing the daughter ion spectra obtained from ions derived from the microsomal incubate (Fig. 6) with synthetic standard spectra (Fig. 2), it is clear that all four compounds (l-4) are present in the microsomal incubate. Conclu.sion Using a combination of daughter and parent ion scanning of both synthetic standards and a microsomal incubate from the H,-antagonist drug mifentidine, three metabolites have been conclusively demonstrated to be present in the mixture. They are the amine (2) formamide (3) and urea (4) In the case of the urea (4) metabolite, it is interesting to note that this represents a rare example of the metabolism of an amidine to a urea. The method developed allows rapid determination of the presence of metabolites in a complex biological matrix such as a microsomal incubate with minimal sample purification.
REFERENCES J.J. Mtstewrcz, m A. Garner and B.J R. Whittle (Ed%), Advances in Drug Therapy of Gastrointestmal Ulceratton, Wiley, New York, 1989, pp. l-32. J W. Black, W.A.M. Duncan, C.J Durant, C.R. Ganellm and E M. Parsons, Nature, 236 (1972) 385 M I. Grossman and S.J. Konturek, Gastroenterology, 66 (1974) 517. G J. Durant, J C. Emmet, C.R. Ganelhn, E.M Parsons, H.D. Pram and G R. White, J. Med Chem., 20 (1977) 901
159 5 M.J. Daly, J M. Humphray and R. Stables, Br. J. Pharmacol., 72 (1981) 49. 6 G. Bran&r Porro, M. Lazzarom, B.P. Imbimbo, 0. Sangalet& C. Ghrrardosr and S. Damottt, Eur. J. Chn. Pharmacol., 32 (1987) 555. 7 A. Donettt, E. Cereda, E. Bellora, A. Gallaw, C. Bazzano, P. Vanom, P. Del Soldato, R. Michelettt, F Pagam and A Gtachettr, J. Med. Chem., 27 (1984) 380. 8 G. Bertaccmi, E. Poh and G. Coruzu, Agents Actions, 14 (1984) 510. 9 P. Del Soldato, A. Gmorzt, E Cereda and A. Donettt, Pharmacology, 30 (1985) 45. 10 C Scarptgnato, M. Tangwa, R. Tramacere and P. Del Soldato, Digestion, 33 (1986) 7. 11 A. Vidi, personal communication. 12 K. Patti&s, M. KaJbaf and J.W. Gorrod, Prog. Pharmacol. Chn. Pharmacol., in press. 13 J.W. Gorrod and D. Manson, Xenobrottca, 16 (1986) 933. 14 D E. Rees, Gen. Pharmacol., 10 (1979) 341 15 J.W. Gorrod and M. Chnstou, Xenobionca, 16 (1986) 575. 16 R.E. McMahon, J.C. Turner and W Whitaker, Xenobtottca, 10 (1980) 469. 17 J.W. Stanley, J. Ltq. Chromatogr., 1 (1978) 305 18 K Yamashrta, M Motohashr and T. Yashrkt, J. Chromatogr , 527 (1990) 103. 19 K.L Busch, G.L Ghsh and S.A. McLuckey, Mass Spectrometry/Mass Spectrometry: Prmctples and Apphcatrons of Tandem Mass Spectrometry, VCH, New York, 1988. and J.W Gorrod, J Pharm. 20 S.P. Lam, D J Barlow Pharmacol., 41 (1989) 373. 5 21 J.W. Gorrod, D.J. Temple and A. Beckett, Xenobiotrca, (1975) 453 22 M KaJbaf, unpubhshed results. 23 C. Fenselau and R.J. Cotter, Chem. Rev., 87 (1987) 501. 24 S. Naylor, A.F Fmders, B.W Gibson and D.H. Williams, J Am. Chem. Sot., 108 (1986) 6359 Ion 25 WV Lrgnon and S.B Dorn, Int J. Mass Spectrom Processes, 57 (1984) 75 26 J. Cushmr, S. Naylor, J H. Lamb, P.B Farmer, N. Brown and P E. Mirkes, Rapid Commun. Mass Spectrom., 4 (1990) 410 D.S. Millington, D L. Norwood and D.H 27 A.L. Burlingame, Russell, Anal. Chem., 62 (1990) 268R.
Anolytrcu Chwuca km, 247 (1991) 151-159 Elsevrer Scrence Pubhshers B.V., Amsterdam
Identification of metabolites derived from the Hz-receptor antagonist mifentidine using tandem mass spectrometry M. Kajbaf
*, J.H. Lamb and S. Naylor *
MRC Toxicology Unit, Carshalton, Surrey SM5 4EF (l_J.K)
K. Patti&is and J.W. Gorrod Chelsea Department
of Pharmacy, Kmg’s College London, Umversrty of London, London SW3 6LX (U K.)
(Received 20th August 1990)
The in vrtro metabolism of mrfentidine, a second generation lustamine Hz-receptor antagomst, IS mvestrgated after hepatic microsomal mcubatron. By employing a combinatron of both daughter and parent ran scanning tandem mass spectrometry on synthetic standards and the nucrosomal incubate, it IS revealed that mrfentrdme 1s metabohsed to at least three compounds, namely the amme, formamide and urea metabohtes. The detectron and characterisatron of the three metabohtes IS carrred out with mimmal sample punhcation. Keywords.
Mass spectrometry,
Mifentrdine
Peptic ulcers are a common malady in “Western World” populations, and one of the possible causative factors is excessive secretion of gastric acid by parietal cells in the stomach [l]. Acid secretion is stimulated by the action of histamine on the Hz-receptors of parietal cells [2,3], and hence numerous H,-antagonistic drugs such as cimetidine (trade-name Tagamet) [4] and ranitidine (trade-name Zantac) [5] have been marketed worldwide to combat the effect of histamine on such receptors. Recently, a new H,-antagonist, N’-[(Cimidazolyl)phenyl]-N2-isopropylformamidine dihydrochloride (mifentidine) (1) entered phase II clinical trials [6], and it represents the prototype of the “second generation” of histamine H,-receptor antagonists. It was originally synthesised by Donetti et al. [7], and is both a potent antisecretory agent [8] and a promising anti-ulcer drug [9,10]. Preliminary in vivo experiments by Vidi [ll] have shown that it is possibly metabolised to 0003-2670/91/$03.50
0 1991 - Elsevier Science Pubhshers B.V
the amine (2) and the formamide (3) derivative. Recently Patti&is et al. [12], using in vitro guinea pig, rat, hamster and mouse hepatic microsome preparations, have tentatively identified, by UV, liquid chromatography (LC) and thin-layer chromatography the amine (2) formamide (3) and urea (4) derivatives of mifentidine. Typically, in drug metabolism studies, metabolites that are present in low concentration have to be isolated from complex biological matrices [13,14]. Such matrices can range from urine, feces and whole blood for in vivo studies, or solution mixtures that contain high concentrations of numerous cofactors, enzymes, substrates and buffers as for in vitro microsomal incubates [15,16]. Extensive purification, followed by limited analysis [UV, gas chromatography-mass spectrometry (GC-MS) and comparison of LC retention times to synthetic standards] is then performed in order to identify the unknown metabolites [17,18]. In this work we have investigated the in vitro
M KAJBAF
152
N Formamide Scheme
ET AL
HNQN
1
microsomal metabolism of mifentidine (1) using tandem MS. In particular, by utilizing both parent and daughter ion scanning techniques [19] it is possible to determine the presence, and characterise metabolites without the need to undertake extensive purification procedures.
EXPERIMENTAL
Chemicals Mifentidine (1) and all synthetic standards (24) were synthesised and supplied by the Institute De Angeli (Milan). All other materials were obtained from British Drug Houses Ltd., FSA Laboratory Supplies and Boehringer Mannheim. All FAB matrices were purchased from Aldrich, and used without further purification. Mlcrosomal incubation Hepatic microsomes from male albino DunkinHartley guinea pigs (400-600 g) were prepared by the CaCl, precipitation method described previously (201. The final pellet was suspended in phosphate buffer (0.2 mM, pH 7.4) to a concentration of 0.5 g original liver weight per ml. The substrate, mifentidine (1) (4 pmol per flask, a total of six flasks were incubated) dissolved in 0.5 ml water; plus the cofactors NADP (disodium salt), P-nicotinamide adenine dinucleotide phosphate, glucosed-phosphate (disodium salt), glucase-6-dehydrogenase, MgCl, in 2 ml phosphate buffer (0.2 mM, pH 7.4); plus the liver microsomal preparation (equivalent to 0.5 g liver resuspended in 1 ml tris-KC1 buffer) were all in-
cubated for 20 min at 37°C in a 25-ml Erlenmeyer flask as described by Gorrod et al. [21]. After incubation, the enzymic reaction was stopped by placing the flasks on ice and rapidly adding 100 ~1 of 1% ZnSO, (w/v). The contents of the Erlenmeyer flask were transferred to a 12-ml centrifuge tube and centrifuged at 3000 rpm (2000 g) for 15 min. The six supernatants were combmed (ca. 20 ml) and immediately loaded onto a pre-washed C,, reversed-phase Sep-Pak cartridge, and then eluted with 5 ml of methanol [22]. The methanol was subsequently removed under a stream of nitrogen at 37°C. The residue was redissolved in 50 ~1 of distilled methanol and subjected to mass spectrometry. Mass spectrometry All spectra were obtained on a VG70-SEQ instrument with EBQIQz configuration, where E is the electrostatic analyser, B is the magnet, Qi is an rf-only quadrupole collision cell and Qz is a mass filter quadrupole. Samples were ionized by either: (i) fast atom bombardment (FAB) using xenon as the source of fast atoms (8.5 KeV), (ii) direct insertion (probe scan) electron ionization (EI), where the probe was heated to 260°C after 30 s in the source. In both cases the ions produced were accelerated to 8 KeV from the source region and analysed in the first mass spectrometer (EB) using a scan speed of 10 s per decade over the mass range 400-60 daltons at a resolution of ca. 1000. Parent ions were selected using EB (equivalent to MS,) and subjected to collision activated dissociation (CAD) in Qi. The collision gas used was argon and the gas pressure was typically ca. 5 X
MS-MS
OF Hz-RECEPTOR
ANTAGONIST
MIFENTIDINE
153
METABOLITES
10e6 mbar. The collision energies used were lo-30 eV for both EI and FAB. Daughter ion spectra were acquired by selecting a specific ion using EB (MS,), subjecting that ion to CAD and scarming the mass filter quadrupole Q2 over a mass range of 300-30 daltons and ten scans were obtained in the multi-channel analysis (MCA) mode. Parent ion spectra were obtained using the MCA mode by selecting a specific ion to be passed through Q2 (MS,) and scanning the magnet over the mass range 400-60 daltons. MS sample preparation
Usually 1 ~1 of the 50 ~1 combined microsomal mixture in methanol was removed by a Hamilton syringe. In FAB-MS the l-p1 methanol was thoroughly mixed with 1.5 ~1 NBA on a stainless-steel probe tip and inserted into the source. In EI-MS, the l-p1 methanol 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 investigations focused on determining optimum matrix conditions, for detecting mifentidine (1) and a series of synthetic derivatives (2-4), using positive ion FAB-MS. A variety of FAB matrices were investigated including dithiothreitol-dithioerythreitol (3 : 1) (magic bullet), glycerol-thioglycerol (1: l), thiodiglycol, 0.2 M p-toluenesulfonic acid in glycerol, glycerol, 10 mM trifluoroacetic acid in glycerol, thioglycerol and 3-nitrobenzylalcohol (NBA). In all cases for each compound (l-4), NBA afforded the most abundant signal-to-noise ratio for the molecular ion (MH)+. A positive ion FAB spectrum (NBA as matrix) of an approximately equimolar mixture of pure synthetic derivatives, including the amine (2) (MH+= 160), formamide (3) (MH+= 188), and urea (4) (MH+= 245), plus mifentidine (1) (MH+ = 229) revealed molecular ions (MH)+ for all compounds, as shown in Fig. 1. The detection limit was G 2 nmol for each compound. However, a positive ion FAB spectrum of a guinea pig
m/z Fig. 1 Positive ion FAB-MS (matnx NBA) of an eqmmolar rmxture (2 nmol of each compound on the probe tip) of mifenkdine (1) and the synthetic derivatives (2,3,4). Note that “m” corresponds to matrix ions
hepatic microsomal incubate from mifentidine revealed only a distinct molecular ion (MH)+ for the substrate (l), with no indication of other molecular ions (MH)+ corresponding to expected metabolites (2-4). The failure to detect other metabolites in the microsomal incubate could be a result of two factors: (i) sensitivity of the FAB-MS method due to the abundant “chemical noise” background [23]. (ii) suppression effects on molecular ion abundance caused by the presence of salts [23] and other hydrophobic components in the mixture [24,25]. To overcome both of these problems we reverted to tandem MS analysis and used a C,, reversed-phase Sep-Pak cartridge to remove salts and any very hydrophobic contaminants. Recently, tandem MS has found increased use in the analysis of complex biological matrices with only minimal sample purification required [26,27]. In this instance the strategy developed was firstly to acquire daughter ion spectra of mifentidine (1) and a series of synthetic derivatives of (1). Secondly, using the most abundant fragment ion m/z value (obtained from the daughter ion spectra), individual parent ion scans were carried out on the microsomal incubate to determine the presence of metabolites in this complex biological
154
.
M. KAJBAF
ET AL
Fig. 2. FAB daughter 1011spectra (matrix NBA, colhsion energy 20 eV) of synthetic standards: (a) mifentldine (l), MH+= 229, (b) amine (2), MH+= 160, (c) form-de (3), MH+= 188, (d) urea (4), MH+= 245.
MS-MS
OF Hz-RECEPTOR
TABLE
ANTAGONIST
MIFENTIDINE
METABOLITES
I
Fragment ions observed synthettc standards (l-4) Fragment ton (m/z) Mifentidme 187
m the daughter
Fragment lost
of the
Possible structure of ton detected ’
(m/r) (1) 42, ]CH(CH,), i
ion spectra
-HI
170
59, [(NH-CH(CH,),)
160
69, [(CH=N-CH(CH,),)
[HIPh-N=CH -NH*]+ [HIPh-N=C]+
+Hl [HIPh-NH,]+
-HI Amme (2) 143 133
17, [NH,] 27, [HCN]
Formarmde (3) 170 18, [Hz01 160 28, WI 143 45, [CO + NH,] or [HCN + H,O] 133 55, [CO + HCN] Urea (4) 186
59, [(NH-CH(CH,),)
[HIPh-N=C]+ [HIPh-NHJ+
[HIPh-N=C=O]+
+Hl 160
85, [(C(O)-NH
[HIPh-NH,]
+
-CW’=,),)-HI ’ I = Irmdazolyl;
Ph = phenyl
mixture. Lastly, to unequivocally confirm the presence and structurally characterise ions corresponding to mifentidine metabolites in the incubate, daughter ion spectra of such ions were acquired and compared to the daughter ion spectra of synthetic standards. Initiahy FAB daughter ion spectra were obtained on mifentidine (l), plus a series of synthetic derivatives including the amine (2), formamide (3) and urea (4), which have been reported to be tentatively present in a microsomal incubate from mifentidine [12]. As shown in Fig. 2a, the daughter ion spectrum of mifentidine (1) (MH+= 229) afforded abundant fragment ions at m/z 187, 170 and 160 corresponding to loss of [CH(CH,), - H], and [(CH=N-CH [(NH-CH(CH,),) + HI (CH,),) - H] respectively (as outlined in Table 1). A daughter ion spectrum of the synthetic standard amine (2) exhibited fragment ions at m/z 143 (loss of NH,) and 133 (loss of HCkN) (see Fig. 2b
155
and Table 1); whereas the formamide (3) derivative contained daughter ions at m/z 170 [loss of H,O], 160 (loss of CO), 143 (see Table 1) and 133 (see Table 1) as shown in Fig. 2c. The synthetic urea (4) daughter ion spectrum showed fragment ions at m/z 186 {loss of (NH-CH(CH,),) + H} and at m/z 160 {loss of (-C(O)-NH-CH(CH,), - H)} (as detailed in Fig. 2d and Table 1). One of the principal factors in determining the sensitivity of parent ion scanning is the abundance of the selected daughter ion detected at the second collector [19]. Therefore, as can be seen from inspection of Fig. 2a-d, the most abundant daughter ion for mifentidine (1) at m/z 187, amine (2) at m/z 133, formamide (3) at m/z 160, and urea (4) at m/z 186 was selected to scan for parent ions using the microsomal incubate from mifentidine (1). Individual FAB parent ion scans of m/z 187, 133, 160 and 186 revealed ions at m/z 229, 160, 188 and 245 respectively, as shown in Fig. 3a-d. The parent ion at m/z 229 of m/z 187 (Fig. 3a) confirms the presence of the unmetabolised substrate, mifentidine (1). However, parent ions at m/z 160 (Fig. 3b), 188 (Fig. 3c) and 245 (Fig. 3d) correspond to the presence of the amine (2), formamide (3) and urea (4) respectively, in the microsomal mixture. It should be noted that when a control mixture (identical to the rnicrosomal mixture, but containing dead microsomes) was subjected to parent scanning of the same m/z values at m/z 133, 160 and 186, no parent ions at m/z 160, 188 and 245, respectively, were detected (results not shown). The parent ion spectrum of m/z 160, derived from the microsomal incubate (Fig. 3c) not only exhibits an ion at m/z 188 (corresponding to MH+ of the formamide (3) metabolite) but also contains abundant ions at m/z 229 {corresponding to mifentidine (l)}, and m/z 187 a corresponding to [(l) - CH(CH,), + 2H]+). In this instance a daughter ion (m/z 160) common to mifentidine (l), a mifentidine fragment ion (m/z 187) and the formamide (3) derivative in FAB-MS-MS tends a This ion is also observed m the positive ton FAB-MS
of nnfentidme (1) and ts formed durmg the fast atom bombardment process, due to fragmentation of (1).
156
M KAJBAF
ET AL.
hg. 3. FAB parent ion spectra (matnx NBA, collision energy 25 ev), of ions derived from a rmcrosomal Incubate. (a) parents of m/r 187; Ion at m/z 229 corresponds to (MH)+ of rmfentidine (I), (b) parents of m/z 133; ion at m/z 160 corresponds to (MH)+ of amme (2), (c) parents of m/z 160; ion at m/z 188 corresponds to (MH)+ of formamide (3), (d) parents of m/z 186, Ion at m/z 245 corresponds to (MH)+ of urea (4).
MS-MS
OF Hz-RECEPTOR
ANTAGONIST
MIFENTIDINE
157
METABOLITES
159
‘a (4
184
I*.
196
171
48
n.
213
1.28.
ICI 61
. ..III...I,I, *
l..llt
I II, I..,.,...I....~I,.., Is( El
I,,ll,,,,., 141
,,,,I 161
,
,
,.,. w
9
Fig. 4. EI daughter ton spectra of synthetic standards:
(a) mifentldine
to obscure the presence of (3) (MH+= 188) in the parent ion spectrum. Inspection of a daughter ion spectrum of mifentidine (l), (M’= 228), using EI as the primary ionization technique, revealed fragment ions at m/z 213, 186, 184, 171 and 159, whereas for the synthetic formamide (3) derivative (Mt= 187) only ions at m/z 160 and 159 were observed,
(l), Mt=
228; (b) formarmde (3), Mt=
as shown in Fig. 4. Subsequently an EI parent ion scan of m/z 160 (which is only present in the formamide (3) spectrum), derived from the microsomal mixture, revealed only a parent ion at m/z 187, assignable to the (Mf) of the formamide (3) metabolite (Fig. 5). To conclusively confirm the presence of unmetabolised substrate (1) and the three metabo-
10
I
1
I
6l
u
a
1
Fig. 5. EI parent Ion spectrum of ion m/z
160 dewed
187.
from a rmcrosomal
Incubate.
M
158
lites (2-4) in the microsomal mixture, individual FAB daughter ion spectra were obtained on the ions m/z 229, 160,188 and 245, derived from the
KAJBAF
ET AL
microsomal incubate and these spectra are shown in Fig. 6. With the exception of the daughter ion spectrum of mifentidine (1) (Fig. 6a), all the other 181
Im(a) 81.
170
‘7 (cl
-i&’ (d)
7
Fig. 6 Daughter nufentldine
eon spectra obtained
(1): (a) m/z
229, (b) m/z
from Ions correspondmg 160; (c) m/z
188; (d) m/z
to metabolites 245.
present
following
the rmcrosomal
metabohsm
of
MS-MS
OF H,-RECEF-TOR
ANTAGONIST
MIFENTIDINE
METABOLITES
daughter ion spectra derived from analysis of the rmcrosomal incubate, contained additional fragment ions (Fig. 6b-d) as compared to the daughter ion spectra of pure synthetic standards (2-4) shown in Fig. 2b-d. However, by comparing the daughter ion spectra obtained from ions derived from the microsomal incubate (Fig. 6) with synthetic standard spectra (Fig. 2), it is clear that all four compounds (l-4) are present in the microsomal incubate. Conclu.sion Using a combination of daughter and parent ion scanning of both synthetic standards and a microsomal incubate from the H,-antagonist drug mifentidine, three metabolites have been conclusively demonstrated to be present in the mixture. They are the amine (2) formamide (3) and urea (4) In the case of the urea (4) metabolite, it is interesting to note that this represents a rare example of the metabolism of an amidine to a urea. The method developed allows rapid determination of the presence of metabolites in a complex biological matrix such as a microsomal incubate with minimal sample purification.
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