Anal. Chem. 2002, 74, 3449-3457
Comparison of Electrospray, Atmospheric Pressure Chemical Ionization, and Atmospheric Pressure Photoionization in the Identification of Apomorphine, Dobutamine, and Entacapone Phase II Metabolites in Biological Samples Helena Keski-Hynnila 1 ,† Mika Kurkela,‡ Eivor Elovaara,§ Laurence Antonio,⊥ Jacques Magdalou,⊥ †,‡ Leena Luukkanen, Jyrki Taskinen,†,‡ and Risto Kostiainen*,†,‡
Department of Pharmacy, Division of Pharmaceutical Chemistry, P.O. Box 56, FIN-00014 University of Helsinki, Finland, Viikki Drug Discovery Technology Center, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Finland, Laboratory of Toxicokinetics and Metabolism, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland, and UMR 7561 CNRS-University Henri Poincare Nancy, Faculty of Medicine, P.O. Box 184, 54505 Vandoeuvre Nancy, France
The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no sitecharacteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism. * Author for correspondence. Tel: +358-9-19159134. Fax: +358-9-19159556. E-mail:
[email protected]. † Department of Pharmacy, University of Helsinki. ‡ Viikki Drug Discovery Technology Center, Department of Pharmacy, University of Helsinki. § Finnish Institute of Occupational Health. ⊥ CNRS-University Henri Poincare Nancy. 10.1021/ac011239g CCC: $22.00 Published on Web 06/14/2002
© 2002 American Chemical Society
Liquid chromatography-mass spectrometry (LC-MS) allows the determination of polar compounds without derivatization and has, therefore, been widely utilized in the analysis of drug metabolites since its introduction. Nowadays, the most commonly used ionization techniques in LC-MS are atmospheric pressure ionization (API) methods, such as electrospray ionization (ESI), and atmospheric pressure chemical ionization (APCI). Additionally, a new atmospheric pressure photoionization method (APPI) has been recently introduced.1 These techniques provide a very soft ionization process and are, therefore, highly suitable for the direct analysis of thermolabile drug conjugates. The preliminary identification of metabolites is based on the measurement of molecular weights by LC-MS, and the final identification can be achieved by tandem mass spectrometry (MS/MS). To date, only a few studies can be found in which the suitability of LC-MS methods coupled to different ionization interfaces has been compared for the analysis of drug metabolites.2,3 The present work is, to the best of our knowledge, the first investigation in which the suitability of conventional ionization techniques (ESI, APCI) is actually compared with an APPI source for detection of drug conjugates in matrixes of real samples of drug modeling studies. APPI is a newly introduced high-sensitivity ionization method meant for LC-MS, providing an 8-times-higher signal than the signal obtained with an APCI source for the drugs (carbamazepine and reserpine, in methanol) analyzed by Robb and co-workers.1 In our work, we compare the capability of three API techniques to identify conjugation products of apomorphine, dobutamine, and entacapone in different biological models. The catecholic structures of these compounds and their pKa values are illustrated in Figure 1. The selected compounds offer a good platform for this study, since a catechol group (benzene ring with (1) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 3653-3659. (2) Keski-Hynnila¨, H.; Luukkanen, L.; Taskinen, J.; Kostiainen, R. J. Am. Soc. Mass Spectrom. 1999, 10, 537-545. (3) Haruo, I.; Eiichi, K.; Nobuhiro, K.; Hidetoshi, W.; Michiko, K.; Kan-ichi, N. Biol. Mass Spectrom. 1994, 23, 540-546.
Analytical Chemistry, Vol. 74, No. 14, July 15, 2002 3449
considered in this work7-19 only a few assays were found for their conjugates.20-23 The method for quantification of glucuronides and sulfates of apomorphine is generally performed upon hydrolysis of the conjugate by β-glucuronidases and sulfatases.20 This indirect technique, however, measures the total apomorphine freed from its conjugates but does not distinguish between different isomers. With direct methods based on synthesized reference compounds, a stereospecific analysis, for example, of (E)- and (Z)-entacapone glucuronides is possible.21-23 Mass spectrometric detection of the phase II metabolites considered in this work has not been extensively used, although it makes the identification of different conjugates simple. Only electrospray ionization has been utilized in the LC-MS studies of apomorphine24 and entacapone glucuronides.21-23 In this paper, the applicability of different ionization methods, ESI, APCI, and APPI, for the identification of catechol conjugates in biological samples is described. The conjugates found in rat urine, rat liver cell cultures, and human liver microsomes were identified by LC-MS/MS, and the formation of the conjugates in such medium were compared for analytical and metabolic evaluations. Figure 1. Structures of the studied compounds.
two neighbor hydroxyl groups) is a substrate of several drugmetabolizing enzymes. The catechol is a central pharmacophore that is found in many physiological compounds as well as in various drugs used for the treatment of neurological and mental illnesses (disorders). The metabolism of catechols is usually dominated by three competing conjugation reactions: glucuronidation, sulfation, and O-methylation. The selected compounds are of special interest also because of their complex acid-base properties. Apomorphine and dobutamine are ampholytes, whereas entacapone is an acid. The acidic character of these compounds is increased by glucuronidation or sulfation on a phenolic hydroxyl, since the pKa values of the ionized carboxylate (pKa ) 3.2) in the glucuronyl moiety4 and that of the sulfate group (pKa < 1) are significantly lower than the pKa value of the phenolic hydroxyls. The basic character of an amino group may decrease slightly in N-glucuronidation as a result of the steric effect of the glucuronide moiety. Methylation of a phenolic hydroxyl neutralizes the acidity of the compound. If the other hydroxyl remains free, the conjugation product still has weak acidic properties. The knowledge of the metabolic fate of a drug is highly important, since the metabolism can lead to the loss of pharmacological activity, generate toxic species, or activate the drug.5 Biotransformation pathways leading to excretion of a drug are usually divided into phase I and phase II reactions.6 Phase I reactions (functionalization) prepare the molecule for phase II reactions (conjugation) producing glucuronidated, sulfated, methylated, or other conjugated drug molecules.6 Though multiple methods have been developed for the analysis of the compounds (4) Perel, J. M.; McMillan Snell, M.; Chen, W.; Dayton, P. G. Biochem. Pharmacol. 1964, 13, 1305-1317. (5) Clarke, D. J.; Burchell, B. Conjugation-Deconjugation Reactions in Drug Metabolism and Toxicity; Springer-Verlag: Berlin-Heidelberg, 1994; Chapter 1. (6) Gibson, G. C.; Skett, P. Introduction to Drug Metabolism, 2nd ed.; Blackie Academic and Professional: London, 1994; Chapter 1.
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EXPERIMENTAL SECTION Chemicals. Entacapone was kindly donated by Orion Pharma (Espoo, Finland). Apomorphine hydrochloride was from Sigma Chemical Co. (St. Louis, MO), and dobutamine hydrochloride, from Eli Lilly Company (Indianapolis, IN). HPLC-grade solvents were used for all LC-MS experiments. Water was purified in a Milli-Q water purification system (Millipore, Molsheim, France). Sample Preparation. Metabolism in the Rat. Animal experiments were approved by the local Ethical Committee for Animal Studies. Urine samples were collected from six male Wistar rats (7) Husseini, H.; Mitrovic, V.; Schlepper, M. J. Chromatogr. 1993, 620, 164168. (8) Rouru, J.; Gordin, A.; Huupponen, R.; Huhtala, S.; Savontaus, E.; Korpela, K.; Reinikainen, K.; Scheinin, M. Eur. J. Clin. Pharmacol. 1999, 55, 461467. (9) Nicolle, E.; Pollak, P.; Serre-Debeauvais, F.; Richard, P.; Gervason, C. L.; Broussolle, E.; Gavend, M. Fundam. Clin. Pharmacol. 1993, 7, 245-252. (10) Przedborski, S.; Levivier, M.; Raftopoulos, C.; Naini, A. B.; Hildebrand, J. Movement Disord. 1995, 10, 28-36. (11) Sam, E.; Augustijns, P.; Verbeke, N. J. Chromatogr. B 1994, 658, 311317. (12) Sam, E.; Sarre, S.; Michotte, Y.; Verbeke, N. Eur. J. Pharmacol. 1997, 329, 9-15. (13) Alberts, G.; Boosma, F.; Man in’t Veld, A. J.; Schalekamp, M. A. D. H. J. Chromatogr. 1992, 583, 236-240. (14) Priston, M. J.; Sewell, G. J. J. Chromatogr. B 1996, 681, 161-167. (15) Smith, R. V.; De Moreno, M. R. J. Chromatogr. 1983, 274, 376-380. (16) Smith, R. V.; Humphrey, D. W.; Szeinbach, S.; Glade, J. C. Anal. Lett. 1979, 12, 371-379. (17) Wikberg, T.; Vuorela, A.; Ottoila, P.; Taskinen, J. Drug Metab. Dispos. 1993, 21, 81-92. (18) Wikberg, T.; Ottoila, P.; Taskinen, J. Eur. J. Drug Metab. Pharmacokinet. 1993, 18, 359-367. (19) Wikberg, T.; Vuorela, A. Eur. J. Drug Metab. Pharmacokinet. 1994, 19, 125135. (20) van der Geest, R.; Kruger, P.; Gubbens-Stibbe, J. M.; van Laar, T.; Bodde, H. E.; Danhof, M. J. Chromatogr. B 1997, 702, 131-141. (21) Keski-Hynnila¨, H.; Luukkanen, L.; Andersin, R.; Taskinen, J.; Kostiainen, R. J. Chromatogr. A 1998, 794, 75-83. (22) Keski-Hynnila¨, H.; Raanaa, K.; Forsberg, M.; Ma¨nnisto¨, P.; Taskinen, J.; Kostiainen, R. J. Chromatogr. B 2001, 759, 227-236. (23) Keski-Hynnila¨, H.; Raanaa, K.; Taskinen, J.; Kostiainen, R. J. Chromatogr. B 2000, 749, 253-263. (24) El-Bacha´, R. S.; Leclerc, S.; Netter, P.; Magdalou J.; Minn, A. Life Sci. 2000, 67, 1735-1745.
(284-370 g, from Helsinki University Breeding Centre) after administration of apomorphine (10 mg/kg bw), dobutamine (50 mg/kg bw), and entacapone (200 mg/kg bw) by oral gavage (two rats in each metabolism cage, kept in an air-conditioned room with artificial lighting from 0700 to 1900 h). Rats were allowed to drink tap water ad libitum but were not fed during the treatments. Control urine was obtained from two rats treated with vehicle (olive oil). The samples were collected for 8 h (entacapone) or 24 h (apomorphine and dobutamine) in ice-cold glass bottles containing 10 mg sodium metabisulfite in 1% aqueous EDTANa2 solution. The urine samples (collected quantitatively by rinsing the cage bottoms with 10-15 mL dist. water) were weighed and stored frozen (-20 °C). Urine samples (1.0 mL) were acidified by adding 100 µL of 1 M hydrochloric acid before purification by solid-phase extraction (SPE) with Sep-Pak C18 cartridges (Waters, Milford, MA). The cartridges were conditioned with methanol and 50 mM hydrochloric acid, then loaded with the samples and washed once with 2 mM HCl (1 mL) and water (1 mL). The analytes were eluted with methanol (1.5 mL) and evaporated to dryness under a flow of nitrogen. The residues were dissolved in 1.0 mL of 0.1% (v/v) formic acid. Metabolism in Rat Hepatocytes. Hepatocytes from adult male Sprague-Dawley rats weighing 180-200 g were isolated by the two-step collagenase perfusion method.25 They were seeded at a density of 105 cells/cm2 on plastic dishes in a medium consisting of 75% minimal essential medium and 25% medium 199, supplemented with 0.2 mg/mL bovine serum albumin, 10% fetal calf serum, 2 mM glutamine, 10 IU/mL penicillin, 10 µg/mL streptomycin, 10 µM hydrocortisone hemisuccinate, and 0.1 IU/mL insulin at 37 °C in an atmosphere of 95% air and 5% CO2. The pH of the medium was adjusted to 7 with sodium carbonate. The medium was renewed 4 h after seeding. Drugs were added in the new medium, at two final concentrations of 50 and 500 µM, and they were diluted in a constant volume of DMSO (2% v/v) for two different incubation times of 2 and 20 h. Proteins were removed by centrifugation (5000g, 10 min) and the samples (1.0 mL) were evaporated to dryness under nitrogen. The residues were dissolved in 1.0 mL of 0.1% (v/v) formic acid. Metabolism by Human Microsomes. The drugs were incubated also with pooled human liver microsomes (2 mg protein/mL, pooled livers, Human Biologics, Scottsdale, AZ). The catechols (5 mM) were added to 125 µL of 50 mM phosphate buffer (pH 7.4) containing 5 mM magnesium chloride. The reaction was started with 25 µL of 10 mM NADPH (tetrasodium salt, Roche, Mannheim, Germany), and the mixtures were incubated for 30 min at 37 °C. A 25-µL portion of 50 mM UDP-glucuronic acid (disodium salt, Boehringer, Ingelheim, Germany) was added, and the reaction was allowed to continue for 30 min. The reaction was stopped by ice-cold 4 M perchloric acid. The samples were centrifuged (4000g, 10 min) to remove the proteins, and the supernatants were analyzed. HPLC. The HP 1100 series (Hewlett-Packard, Germany) HPLC system was used for chromatography. A Luna C18 reversedphase column (150 × 1 mm i.d.; particle size, 5µm) Phenomenex (Phenomenex, Torrance, CA) and a Luna C18 precolumn of the (25) Yamazoe, Y.; Shimada, M.; Murayama, N.; Kato, R. J. Biol. Chem. 1987, 262, 7423-7428.
Table 1. Major Operating Parameters for Different Interfaces electrospray parameter (ESI)
setting
electrospray voltage declustering potential curtain gas flow rate nebulizer gas flow rate vaporizer temperature
5.5 kV 20 V 1.8 L min-1 1.6 L min-1 400 °C
heated nebulizer parameter (APCI)
setting
discharge current declustering potential curtain gas flow rate nebulizer gas pressure auxiliary gas flow rate vaporizer temperature
3 kV 20 V 1.8 L min-1 75 psi 1.2 L min-1 400 °C
atmospheric pressure photoionization parameter (APPI)
setting
lamp current declustering potential curtain gas flow rate nebulizer pressure auxiliary gas flow rate vaporizer temperature lamp gas flow
0.70 mA 20 V 2 L min-1 75 psi 2 L min-1 350 °C 1 L min-1
same material (30 × 1 mm i.d.) were used for separation for ESI or APPI applications. A Waters Symmetry C18 (150 × 2.1 mm i.d.) (Waters, Milford, MA) reversed-phase column was used with APCI ionization. The mobile phases were (A) 0.1% formic acid and (B) 0.1% formic acid made in 80% aqueous acetonitrile (HCOOH/ACN/H2O, 1:800:200, v/v/v) with the following linear B gradient: 7.5 f 50% in 45 min, 50 f 100% in 1 min, 100% 2 min, and then back to B 7.5% in 1 min and reequilibrated for 35 min. The injection volumes were 2 (APPI, ESI) and 10 µL (APCI) and flow rates 0.1 and 0.5 mL/min, respectively. Water was added to the organic solvent (B) in order to diminish bubble formation on mixing. All samples were filtered before analysis with MillexHV 0.45-µm filters (Nikon Millipore, Yonezawa, Japan). Mass Spectrometry. The mass spectrometer that was operated in a positive ion mode in LC-MS analysis was a Sciex API3000 triple quadrupole equipped with a commercial turboionspray and heated nebulizer interfaces (Sciex, Concord, Canada). An atmospheric pressure photoionization interface, APPI, (Machine Shop, University of Groeningen, The Netherlands) was used. It is described in detail by Robb et al.1 The major operating parameters for the different interfaces are listed in Table 1. A microsyringe pump (Harvard Apparatus Inc., Holliston, MA) was used in the tuning of the instrument and for dopant delivery in APPI ionization. High-purity air (99.998%, Woikoski, Helsinki, Finland) was used as the nebulizing gas and nitrogen produced by a Whatman 75-720 (Whatman Inc., Haverhill, MA) nitrogen generator was used as the curtain and auxiliary gas. The lamp gas in APPI interface was 99.999% nitrogen (Woikoski). The operating parameters for the LC-MS analysis were optimized for obtaining a maximum absolute abundance of [M + H]+ using the infusion of entacapone glucuronide, apomorphine, and dobutamine solutions (5 µg/mL in 0.1% formic acid/acetonitrile, 50:50). Optimized parameters were declustering potential, the temperaAnalytical Chemistry, Vol. 74, No. 14, July 15, 2002
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Table 2. Conjugation Products of the Compounds in Different Matrixes Detected with ESI, APCI, and APPI ESI
m/z
APCI
APPI
compd
[M + H]+
RHa
RUb
HMc
RH
RU
HM
RH
RU
HM
apomorphine (Apo) Apo(glu) Apo(glu) Apo(sulf) Apo(sulf) dobutamine (Dobu) Dobu(meth) Dobu(meth) Dobu(meth) Dobu(glu) Dobu(glu) Dobu(glu) Dobu(glu) Dobu(glu + meth) Dobu(glu + meth) Dobu(glu + meth) Dobu(glu + meth) Dobu(sulf) Dobu(sulf) Dobu(sulf + meth) Dobu(sulf + meth) Dobu(sulf + meth) (E)-entacapone ((E)-Enta) (Z)-entacapone ((Z)-Enta) (E)-Enta(glu) (Z)-Enta(glu)
268 444 444 348 348 302 316 316 316 478 478 478 478 492 492 492 492 382 382 396 396 396 306 306 482 482
++++ ++ +++ ++ +++ ++++ +++ +++ ++ +++ ++ + ++ ++ + + ++ + ++++ ++ ++
++++ +++ ++++ ++ ++++ ++++ ++++ +++ ++ ++++ +++ ++ ++++ +++ +++ ++ ++ +++ ++ + ++ +++ +++ +++
+++ +++ ++++ N.T N.T. ++++ N.T N.T N.T. ++ ++ +++ N.T. N.T. N.T. N.T. N.T. N.T N.T N.T. N.T. ++++ ++ ++++
+++ + +++ ++ ++ ++++ -
+++ +++ ++++ +++ +++ +++ ++ ++ +++ ++++
++++ ++ ++ N.T. N.T. ++++ N.T. N.T. N.T. + +++ N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. ++++ +++ ++++
+++ +++ ++ ++ + ++++ ++ -
++++ + +++ +++ +++ +++ +++ ++ +++ ++ ++ + -+++ +++
++++ ++ ++ N.T. N.T. ++++ N.T. N.T. N.T. ++ N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. N.T. ++++ ++++
a
Rat hepatocytes 500 µM/2h. v Rat urine. c Human microsomes. d Not tested.
tures used for solvent evaporation in different interfaces, and the amount of the dopant solution (between 5 and 20%) in APPI. Toluene (IP ) 8.83 eV) was chosen as a dopant in APPI experiments. The optimal flow rate of toluene in our experiments was 20 µL/min (17%), confirming a sufficient amount of dopant ions in the solvent vapor. The scan range in all experiments was m/z 100-700 (1s/scan). The identities of detected compounds were confirmed in LCESI-MS/MS analysis with an Esquire LC-ion trap LC/MS(n) system (Bruker Daltonics GmbH, Bremen, Germany). For this purpose, all of the samples were analyzed by using auto MS/MS with a fragmentation amplitude of 0.6 V. RESULTS AND DISCUSSION HPLC. The development of an LC-MS analysis method for drug conjugation metabolites, as demonstrated for apomorphine, dobutamine, and entacapone, is not a straightforward task, since the glucuronidated and sulfated metabolites have acid-base properties that differ greatly from those of their parent molecules. Hence, careful choosing of a suitable eluent system for our LCMS method proved to be of critical importance for achieving high sensitivity and selectivity. Glucuronide and sulfate conjugates are often known to have better responses in the negative ion mode. However, one purpose of this study was to study the possibility of analyzing the parent compounds as well as all possible metabolites by one chromatographic run using positive ionization. An acidic eluent system consisting of (A) 0.1% formic acid and (B) 80% of acetonitrile in water (0.1% formic acid) was chosen for chromatography. Usage of this composition provided baseline separation of all of the standard compounds and metabolites available for optimization. Moreover, acidic eluent systems (pH 3452 Analytical Chemistry, Vol. 74, No. 14, July 15, 2002
1.8-4.0) have been commonly used, as reported for the compounds of this work.7,8,11,14-19 Apomorphine and dobutamine, like many other drugs, have a basic amino group in their structures. This is positively charged in acidic conditions, and good ionization efficiency can be expected with positive ion ESI. According to previous experiments, entacapone glucuronide also can be ionized in the positive ion mode despite having no basic groups in its structure.2 In APCI and APPI, the ionization most often occurs by a proton-transfer reaction in the gas phase. Since all the compounds studied include a group with a high proton affinity (amine or amide), the proton-transfer reaction in positive ion mode is possible with the chosen eluent system. Mass Spectrometry. Comparison of Different Ionization Methods. Formation of conjugation products in different specimens was followed by LC-MS, and the applicability of three different ionization methods was tested for this purpose. The highest number of metabolites (22) was found by electrospray ionization, whereas APCI and APPI revealed only 12 and 14 conjugates, respectively (Table 2, Figure 2). The protonated drug molecules and their methyl conjugates were detected with all of the ionization methods. Glucuronides were ionized most efficiently in ESI, although they were detected also with APCI and APPI. Sulfate conjugates were detected only with ESI. Positive ion ESI was able to detect methylated, glucuronidated, and sulfated drug products as their protonated molecules with a good sensitivity (Figure 2, Table 2). With apomorphine and dobutamine, the negatively charged groups are neutralized, whereas the amino group retains a proton during the ionization process. Neutralization of the negative charge during the ESI process is possible, since the pH can decrease significantly as a
Figure 2. Application of the LC-ESI-MS method for the detection of drug conjugates. Apomorphine, dobutamine, and entacapone conjugates were biosynthesized upon incubation with rat hepatocytes or recovered in urine after administration.
result of the oxidation reactions of the eluent components at the tip of the sprayer. The difference in pH between the solution and a charged droplet can be as high as four pH units.26,27 For acidic entacapone glucuronides, the negative charge is neutralized in the ESI process, and protonation most likely takes place in the gas phase. In APCI, the eluent components and analytes are vaporized before the ionization process is initialized by a corona discharge needle. In this process, protonated eluent molecules are produced, which can transfer a proton to an analyte if its proton affinity (PA) is higher than that of the eluent molecules. The proton transfer reaction should be efficient with the drug compounds studied here, since the PAs of the amine or amide groups in their structures are higher than PAs of the eluent molecules; however, none of the sulfates and only some of the glucuronides and methylated metabolites identified by ESI were detected by APCI (Table 2). In addition to thermodynamic reasons, the sensitivity in APCI is dependent on the volatility of the compounds. Apomorphine and dobutamine sulfates and glucuronides were ionized in the eluent used as a result of the acidic groups and the (26) van Berkel, G. J.; Zhou, F.; Aronson, J. T. Int. J. Mass Spectrom. Ion Processes 1997, 162, 55-67. (27) Kelly, M. A.; Vestling, M. M.; Fenselau C. C.; Smith, P. B. Org. Mass Spectrom. 1992, 27, 1143-1147.
basic amine groups (see pKa values in Figure 1). These molecules were poorly vaporized in APCI and not detected in small amounts, because charged species are less volatile than the neutral forms.28 Entacapone glucuronide contains only one acidic group with a pKa value of ∼3.5-4.0. Under the present acidic eluent conditions, entacapone glucuronide at least partly exists in its neutral form and was, therefore, detected with reasonable sensitivity also with APCI. Furthermore, strong fragmentation or thermal degradation detected as an intense [M + H - glu]+ ion decreased sensitivity with glucuronides when the detection of the metabolites was based on the recognition of a protonated molecule. The initial reaction in APPI is a formation of a radical cation of the dopant molecule due to the photons emitted by the photoionization lamp.1,29 The radical cation reacts with eluent molecules, producing protonated eluent molecules if the PA of the eluent molecule is higher than that of benzyl radical (831.4 kJ/mol).29-31 The analyte is ionized by the proton transfer reaction if its PA is higher than that of the eluent molecule. When the PA of the eluent molecules is lower than that of the benzyl radical, (28) Willoughby, R.; Sheehan, E.; Mitrovich, S. A Global View of LC/MS; Global View Publishing: Pittsburgh, 1998; p 417. (29) Koster G.; Bruins, A. P. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics (CD-ROM), Chicago, Illinois, May 27-31, 2001. (30) http://webbook.nist.gov/chemistry/
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Figure 3. Total ion chromatograms (TIC; m/z 100-700) obtained in the LC-ESI-MS analysis of apomorphine conjugates formed in different matrixes.
the analyte can be ionized directly by a charge exchange reaction, provided that the ionization energy of the analyte is lower than that of the radical cation.1,29,31 Since the drug aglycones and their glucuronide conjugates were detected as their protonated molecules, the ionization with APPI occurs via proton transfer reaction under the chosen conditions. The results obtained by using APPI were quite similar to those for APCI (Table 2). Notably, sulfate conjugates could not be detected by using these ionization sources. The sensitivity of APPI was 1-3 orders of magnitudes lower than that of ESI for the detection of conjugates of the drug glucuronide. The reasons are probably the same as for APCI, that is, the high vaporizing temperatures of the charged glucuronide and sulfate conjugates and the strong fragmentation or thermal degradation of glucuronides. The total ion chromatograms (TIC) from the LC-ESI-MS analysis of apomorphine samples are shown in Figure 3. It can be seen that the background current was high, especially with urine samples. Because the masses of the conjugates of interest were known, these were extracted from TIC, which led to a noticeably clarified chromatogram (Figure 4). Because of a strong background, tandem mass spectrometry was required for the confirmation of the metabolites detected primarily by LC-MS. Tandem Mass Spectrometry of the Compounds. The MS/MS spectra of the identified metabolites are presented in Tables 3 and 4. The MS and MS/MS data (not shown) acquired for entacapone and its glucuronide in this study were comparable with the spectral behavior thoroughly studied and reported previously.2 (31) Kauppila, T.; Kuuranne, T.; Kotiaho, T.; Kostiainen, R. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics (CD-ROM), Chicago, Illinois, May 27-31, 2001.
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Figure 4. Extracted ion chromatograms obtained in the LC-ESIMS analysis of apomorphine conjugates formed in different matrixes. The extracted ions are m/z 267.3, 348.3, and 444.3 ((0.5 amu) for apomorphine, apomorphine sulfate, and apomorphine glucuronide, respectively.
Apomorphine. Apomorphine bears two hydroxyl groups able to form O-conjugates. Glucuronidation of tertiary amine is unlikely to occur, because quaternary N-glucuronides are not usually formed in the rat.32 The glucuronide conjugates of apomorphine [Apo(glu) + H]+ (m/z 444) were detected at 6.8 and 8.7 min, suggesting that two different monoglucuronides were formed. Despite the possibility that glucuronidation occurs at both hydroxyls, no diglucuronide was detected. The most abundant ions in the MS/MS spectra were the ions at m/z 268 and 237, corresponding to the loss of the glucuronide moiety (176 amu) and subsequent loss of methylamine (31 amu), respectively (Table 3). The ion at m/z 219 is formed by the cleavage of water, and m/z 191, obviously by the loss of water and ethene from m/z 237. Although the spectra of different glucuronides were not identical, it was not possible to determine which of the two hydroxyls was conjugated. Two sulfate conjugates of apomorphine [Apo(sulf) + H]+ at m/z 348 were detected at 20.1 and 22.2 min. The most abundant ion in the MS/MS spectra of these compounds was the ion at m/z 268 formed by the loss of SO3. The ions at m/z 237 and 219 were formed by the loss of methylamine and methylamine + water, respectively, in a manner similar to the glucuronides. The (32) Chiu, S. H. L.; Huskey, S. E. W. Drug Metab. Dispos. 1998, 26, 838-847.
Table 3. MS/MS Spectra of Apomorphine Conjugates Formed with Rat Hepatocytesa compound
rt (min)
precursor m/z (rel abundance)
15.3
H]+ 268
apomorphine (Apo)
a
[M +
ion species, m/z (rel abundance)
(11)
Apo(glu)
6.8
[M + H]+ 444 (6)
Apo(glu)
8.7
[M + H]+ 444 (6)
Apo(sulf)
20.1
[M + H]+ 348 (14)
Apo(sulf)
22.2
[M + H]+ 348 (67)
[M + H - NH2CH3]+ 237 (100) [M + H - NH2CH3 - H2O]+ 219 (17) [M + H - H2O]+ 426 (3) [M + H - glu]+ 268 (100) [M +H - glu - NH2CH3]+ 237 (97) [M + H - glu - NH2CH3 - H2O]+ 219 (39) [M + H - glu - NH2CH3 - C2H4 - H2O]+ 191 (22) [M + H - NH2CH3]+ 413 (33) [M + H - glu]+ 268 (22) [M + H - glu - NH2CH3]+ 237 (100) [M + H - glu - NH2CH3 - H2O]+ 219 (53) [M + H - glu - NH2CH3 - C2H4 - H2O]+ 191 (14) [M + H - SO3]+ 268 (100) [M + H - SO3 - NH2CH3]+ 237 (22) [M + H - SO3 - NH2CH3 - H2O]+ 219 (6) [M + H -SO3]+ 268 (100) [M + H - NH2CH3]+ 317 (28) [M + H - SO3 - NH2CH3]+ 237 (39) [M + H - SO3 - NH2CH3 - H2O]+ 219 (6)
The hepatocytes were incubated with 500 µM apomorphine for 20 h. rt, retention time.
Table 4. MS/MS Spectra of Dobutamine Conjugates Formed with Rat Hepatocytesa rt (min)
precursor m/z (rel abundance)
dobutamine (Dobu)
18.9
[M +
H]+ 302
Dobu(glu)
12.8
[M + H]+ 478 (6)
Dobu(glu)
17.0
[M + H]+ 478 (14)
Dobu(glu)
19.6
[M + H]+ 478 (8)
Dobu(glu)
20.6
[M + H]+ 478 (8)
Dobu(glu + meth)
18.0
[M + H]+ 492 (14)
Dobu(glu + meth)
19.9
[M + H]+ 492 (14)
Dobu(glu + meth)
22.6
[M + H]+ 492 (-)
Dobu(meth)
22.9
[M + H]+ 316 (100)
Dobu(meth)
24.5
[M + H]+ 316 (100)
Dobu(sulf)
26.8
[M + H]+ 382 (-)
Dobu(sulf)
29.9
[M + H]+ 382 (-)
Dobu(sulf + meth)
28.3
[M + H]+ 396 (-)
compd
a
(100)
ion species, m/z (rel abundance) [M + H - (OH)2PhCHCH2]+ 166 (25) [M + H - NH3CH(CH3)CH2CH2PhOH]+ 137 (25) [M + H - NH3CH(CH3)CH2CH2PhOH - NH2CH3]+ 106 (17) [M + H - glu]+ 302 (100) [M + H - glu - (OH)2PhCHCH2]+ 166 (6) [M + H - glu - NH3CH(CH3)CH2CH2PhOH]+ 137 (3) [M + H - glu]+ 302 (100) [M + H - glu - (OH)2PhCHCH2]+ 166 (3) [M + H - glu - NH3CH(CH3)CH2CH2PhOH]+ 137 (3) [M + H - glu]+ 302 (100) [M + H - glu - (OH)2PhCHCH2]+ 166 (3) [M + H - glu - NH3CH(CH3)CH2CH2PhOH]+ 137 (3) [M + H - glu]+ 302 (100) [M + H - glu - (OH)2PhCHCH2]+ 166 (3) [M + H - glu - NH3CH(CH3)CH2CH2PhOH - NH2CH3]+ 106 (2) [M + H - C9H10O2]+ 342 (3) [M + H - glu]+ 316 (100) [M + H - glu - CH3 - (OH)2PhCHCH2]+ 166 (3) [M + H + CH3 - glu - NH3CH(CH3)CH2CH2PhOH]+ 151 (6) [M + H - glu]+ 316 (100) [M + H - glu - NH3CH(CH3)CH2CH2PhOH]+ 151 (11) [M + H - glu]+ 316 (100) [M + H - glu - NH3CH(CH3)CH2CH2PhOH]+ 151 (8) [M + H - CH3 - (OH)2PhCHCH2]+ 166 (8) [M + H - NH3CH(CH3)CH2CH2PhOH]+ 151 (67) [M + H - NH3CH(CH3)CH2CH2PhOH - NH2CH3]+ 106 (2) [M + H - NH3CH(CH3)CH2CH2PhOH]+ 151 (51) [M + H - NH3CH(CH3)CH2CH2PhOH - 32]+ 119 (5) [M + H - SO3]+ 302 (100) [M + H - SO3 - NH3CH(CH3)CH2CH2PhOH]+ 137 (2) [M + H - SO3]+ 302 (100) [M + H - SO3 - NH3CH(CH3)CH2CH2PhOH]+ 137 (2) [M + H - SO3]+ 316 (100) [M + H - SO3 - NH3CH(CH3)CH2CH2PhOH]+ 151 (2)
The hepatocytes were incubated with 500 µM apomorphine for 20 h. rt, retention time.
only difference in the spectra of the two sulfate conjugates was the ion at m/z 317, which corresponds to the loss of methylamine from the protonated molecule that was detected only in the spectrum of the later eluting sulfate. Again, the spectra were too similar for the determination of the site of sulfation.
The compound eluting at 15.3 min was confirmed to be the protonated apomorphine [M + H]+ at m/z 268. The product ions at m/z 237 and 219 were the same as recorded with the glucuronides and sulfates. No peaks corresponding to methylated apomorphine were detected. Analytical Chemistry, Vol. 74, No. 14, July 15, 2002
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Dobutamine. In this study, altogether 16 metabolites of dobutamine were found (Table 2). The MS/MS spectra of dobutamine conjugates are presented in Table 4. Dobutamine bears three phenolic hydroxyl groups and one secondary aliphatic amine group, all known as possible sites for glucuronidation (Figure 1). Four of the 16 peaks detected in the dobutamine chromatograms were monoglucuronides [Dobu(glu) + H]+ (m/z 478). The most intensive ion in the MS/MS spectra of all glucuronides was the ion at m/z 302, corresponding to protonated dobutamine formed after the loss of the glucuronide moiety (176 amu). The fragment ion at m/z 166 [M + H - glu NH3CH(CH3)CH2CH2PhOH]+ was generated by protonation of the amine group followed by the cleavage of the C-N bond with hydrogen transfer to the amine group from the fragmented neutral catecholic side group. The ion seen at m/z 137 [M + H - glu (OH)2PhCHCH2]+ is a result of the same cleavage but leaving the proton on the catecholic side of the molecule. The spectra of dobutamine glucuronides were too similar to allow the determination of the site of glucuronidation. Evidently, the explanation here is the same as for apomorphine: the glycoside bond was the first one to fragment. In view of the present chromatographic and mass spectrometric results, it is also evident that four different monoglucuronides were formed. Hence, the glucuronidation can take place in all of the hydroxyl groups as well as in the secondary amine group to form nonquaternary N-glucuronide. Formation of N-glucuronide is more probable with dobutamine than with apomorphine, because secondary amines also form nonquaternary glucuronides in the rat.33,34 Two sulfate conjugates [Dobu(sulf) + H]+ (m/z 382) of dobutamine were detected at 26.8 and 29.9 min. The main ion in the MS/MS spectra of the sulfates was the protonated drug (m/z 302) formed after the cleavage of SO3. The spectra of the two sulfate conjugates were identical, providing no information about the site of sulfation. The MS/MS spectra of protonated methyl conjugates of dobutamine (m/z 316) at 22.9 and 24.5 min exhibited an intense product ion at m/z 151 that corresponded to the catecholic end of the molecule after the cleavage of the C-N bond. A methyl group was still attached to the fragment, indicating that the site of methylation is one of the catecholic hydroxyl groups. The spectrum of the methyl conjugate (eluting at 22.9 min) exhibited a peak at m/z 166 that corresponded to the phenolic end of the molecule after the cleavage of the C-N bond. This supports the conclusion that the site of methylation is one of the catecholic hydroxyls and not the phenolic hydroxyl, indicating that the methylation is catalyzed by catechol-O-methyltransferases. MS/MS spectra of dobutamine exhibiting two different conjugates (glucronide + methyl, or sulfate + methyl) were very similar to the singly conjugated species; glucuronide and SO3 moieties were lost first, whereas methyl was attached more tightly to the drug. Three conjugation products at m/z 492 (eluting at 18.0, 19.9, and 22.6 min) corresponded to a protonated molecule of dobutamine with glucuronide and methyl moieties attached, [Dobu(glu + meth) + H]+. The main fragment ion at m/z 316 corresponded to a protonated molecule of methylated dobutamine formed by deglucuronidation. The ion at m/z 151 was formed in (33) Tukey, R. H.; Strassburg, C. P. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 581-616. (34) Green, M. D.; Tephly, T. R. Drug Metab. Dispos. 1998, 26, 860-867.
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a way similar to that observed for methylated dobutamine, indicating that the site of methylation was a catecholic hydroxyl. A compound eluting at 28.3 min was identified as sulfated and methylated dobutamine [Dobu(sulf + meth) + H]+ at m/z 396. The main ion in the MS/MS spectrum of this compound was the ion formed by the loss of SO3 at m/z 316. The ion at m/z 151 was again formed in the same way as with the methyl conjugates. Entacapone. Entacapone led to only two conjugation products, both glucuronides, at m/z value 482 corresponding to [Enta(glu) + H]+. The site proposed for glucuronidation is the position meta to the nitro group, because the phenolic hydroxyl group in ortho position to a nitro group is more acidic than that in the meta position.18 Isomerization of entacapone to the Z isomer is an important enzymatic reaction, which explains the detection of two glucuronides, (E)- and (Z)-entacapone, at the same m/z value.17-19 The only product ion observed in the MS/MS spectra of both entacapone glucuronide isomers was the protonated aglycone ion (m/z 302) formed by the loss of the glucuronide moiety. Thus, no further information of conjugation could be obtained from this reaction.2 The use of the positive ion LC-ESI-MS and LC-ESI-MS/MS methods provided powerful techniques in the identification of the unknown metabolites. Unfortunately, the site of glucuronidation or sulfation could not be determined solely by the MS and MS/ MS data. This is because the glycoside bond in glucuronide conjugates as well as the bond between sulfate and the sulfateaccepting moiety are weak. The first fragment formed of these conjugates was always produced by the loss of the conjugate moiety, leading to similar spectra. The bond between the methyl group and the acceptor drug, however, is a relatively strong one and enables the formation of characteristic fragment ions in the case of methyl conjugates. Interestingly, some of the metabolites had longer retention times than the dosed compounds in the reversed-phase HPLC system used, although the phase II metabolites are often more polar than the aglycones. It is suggested that this unusual behavior may reflect changes in the charged states of the compounds studied. Comparison of the Models. The metabolite pattern and the relative amounts of the different drug conjugates that were detected by LC-ESI-MS in samples indicative of the drug metabolism both in vivo (urine) and in vitro (hepatocytes) correlated well with each other (Figure 2). Metabolism of apomorphine, dobutamine, and entacapone as tested here with human liver microsomes produced only glucuronide conjugates (Table 2). In the urine of rats treated with apomorphine, two glucuronidated and two sulfated metabolites were found. The same conjugation products with similar relative proportions were also found in rat hepatocytes. No methyl conjugates of apomorphine were detected. This is in good agreement with negative data reported for mice35 and humans: apomorphine is not methylated by the human recombinant enzyme catechol-O-methyltransferase,36 and no methyl conjugates have been found in human plasma or urine of patients with medication for Parkinson’s disease.20 (35) Smith, R. V.; Klein, A. E.; Wilcox, R. E.; Riffee, W. E. J. Pharm. Sci. 1981, 70, 1144-1147. (36) Lautala, P.; Ulmanen, I.; Taskinen, J. J. Chromatogr. B 1999, 736, 143151.
Dobutamine biotransformation mediated by catechol-O-methyltransferase, sulfotransferase, and glucuronosyltransferase enzymes yielded methylated, sulfated, and glucuronidated drug products. All of the conjugates were detected both in urine and in rat hepatocytes, whereas in the microsomal incubations, only glucuronides were formed. Dobutamine metabolites bearing both methyl and sulfate groups were detected in rat urine but not in rat hepatocytes. In the urine of rats treated with entacapone, only glucuronides of entacapone and its Z isomer were found. The same products were also detected in rat hepatocytes. Metabolism studies with human recombinant enzymes have shown that entacapone is a good substrate for glucuronidation (e.g., by the human UGT1A9 form) but a poor substrate for SULT and COMT enzymes.37 CONCLUSION The results show that positive ion ESI is better suited than positive ion APCI or APPI for the identification of the thermolabile phase II conjugates of the compounds studied here. This is because ESI is a more gentle ionization technique than APCI and APPI and is, thus, able to detect protonated molecules of the (37) Lautala, P.; Ethell, B.; Taskinen, J.; Burchell, B. Drug Metab. Dispos. 2000, 28, 1385-1389.
conjugates with higher relative abundance. Furthermore, with APCI or APPI, no additional conjugates were detected, as compared with those found with ESI. Tandem mass spectrometry is required for confirmation of the metabolites. The sites of glucuronidation or sulfatation cannot be determined by means of MS/MS only, since the primary cleavage is the loss of a conjugate group (glu or SO3), and no site-characteristic product ions are formed. The site of methylation can, however, be determined in some cases, since methylated products are more stable than glucuronides or sulfates, and the loss of methyl is not necessarily the primary cleavage, and site-characteristic products can be formed as shown with Dobu(meth) and Dobu(glu + meth). The results obtained also show good correlation between the in vivo and in vitro models used in this study. ACKNOWLEDGMENT The E.U. Biomed 2 project BMH4-CT97-2621 and the National Technology Agency (TEKES) are acknowledged for financial support. Dr. S. Ratanasavanh (University of Brest, France) is also acknowledged for his help for the culture of rat hepatocytes. Received for review December 6, 2001. Accepted April 24, 2002. AC011239G
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