Analysis of Intact Glucuronides and Sulfates of Serotonin, Dopamine

Sep 22, 2009 - of phase II metabolism in the human brain in more detail. Since .... of 5-HIAA-S. A linear gradient 5-35% organic solvent for 0-10 min ...
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Anal. Chem. 2009, 81, 8417–8425

Analysis of Intact Glucuronides and Sulfates of Serotonin, Dopamine, and Their Phase I Metabolites in Rat Brain Microdialysates by Liquid Chromatography-Tandem Mass Spectrometry Pa¨ivi Uutela,† Ruut Reinila¨,† Kirsi Harju,† Petteri Piepponen,‡ Raimo A. Ketola,§ and Risto Kostiainen*,† Division of Pharmaceutical Chemistry, Division of Pharmacology and Toxicology, and Centre for Drug Research (CDR), Faculty of Pharmacy, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland A method for the analysis of intact glucuronides and sulfates of common neurotransmitters serotonin (5-HT) and dopamine (DA) as well as of 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat brain microdialysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Enzyme-assisted synthesis using rat liver microsomes as a biocatalyst was employed for the production of 5-HT-, 5-HIAA-, DOPAC-, and HVAglucuronides for reference compounds. The sulfate conjugates were synthesized either chemically or enzymatically using a rat liver S9 fraction. The LC-MS/MS method was validated by determining the limits of detection and quantitation, linearity, and repeatability for the quantitative analysis of 5-HT and DA and their glucuronides, as well as of 5-HIAA, DOPAC, and HVA and their sulfateconjugates. In this study, 5-HT-glucuronide was for the first time detected in rat brain. The concentration of 5-HTglucuronide (1.0-1.7 nM) was up to 2.5 times higher than that of free 5-HT (0.4-2.1 nM) in rat brain microdialysates, whereas the concentration of DA-glucuronide (1.0-1.4 nM) was at the same level or lower than the free DA (1.2-2.4 nM). The acidic metabolites of neurotransmitters, 5-HIAA, HVA, and DOPAC, were found in free and sulfated form, whereas their glucuronidation was not observed. Dopamine (DA) and serotonin (5-HT) are important neurotransmitters, which chemically transfer the electrical impulse from one neuron to another in brain. After transmission DA and 5-HT are metabolized through monoamine oxidase (MAO)- and aldehyde dehydrogenase-catalyzed reactions to 3,4-dihydroxyphenylacetic acid (DOPAC) and to 5-hydroxyindoleacetic acid (5HIAA), respectively.1 DOPAC can be further metabolized to homovanillic acid (HVA) by catechol-O-methyltransferase (COMT). * Corresponding author. E-mail: [email protected]. Fax: +358 9 191 59556. † Division of Pharmaceutical Chemistry. ‡ Division of Pharmacology and Toxicology. § Centre for Drug Research (CDR). (1) Cooper, J. R., Bloom, F. E. Roth, R. H. The Biochemical Basis of Neuropharmacology, 8th ed.; Oxford University Press: New York, 2003. 10.1021/ac901320z CCC: $40.75  2009 American Chemical Society Published on Web 09/22/2009

The glucuronidation of DA2 and the sulfation of DA, HVA, DOPAC, and 5-HIAA have been observed in brain.3-5 Sulfation and glucuronidation are common phase II metabolic reactions catalyzed by cytosolic sulfotransferases (SULT)6 and membranebound uridine diphosphoglucuronosyltransferases (UGTs),7 respectively. SULT1A1, 1A2, and 1A3,8 responsible for the sulfation of phenols, and isoforms UGT1A6, 2A1, and 2B79 have been found in human brain. In rat brain, the activity of UGT1A610 and phenolsulfating SULT11 has been observed. UGT1A6 and 2B7 catalyze the glucuronidation of 5-HT, the former enzyme having a much higher glucuronidation rate than the latter.12 These research results suggest that phase II metabolism can affect the concentration levels of neurotransmitters in the brain. For example, the altered concentrations of DA and 5-HT as well as their metabolites are connected to various neurological disorders, such as schizophrenia13 and Parkinson’s disease,14 and therefore reliable and highly sensitive analytical methods are needed to study the role of phase II metabolism in the human brain in more detail. Since the availability of human microdialysis samples is limited, test animals are commonly used in brain research. (2) Uutela, P.; Karhu, L.; Piepponen, P.; Kaenmaki, M.; Ketola, R. A.; Kostiainen, R. Anal. Chem. 2009, 81, 427–434. (3) Swahn, C. G.; Wiesel, F. A. J. Neural Transm. (1972-1989) 1976, 39, 281–290. (4) Gordon, E. K.; Markey, S. P.; Sherman, R. L.; Kopin, I. J. Life Sci. 1976, 18, 1285–1292. (5) Buu, N. T.; Duhaime, J.; Savard, C.; Truong, L.; Kuchel, O. J. Neurochem. 1981, 36, 769–772. (6) Weinshilboum, R. M.; Otterness, D. M.; Aksoy, I. A.; Wood, T. C.; Her, C.; Raftogianis, R. B. FASEB J. 1997, 11, 3–14. (7) Mackenzie, P. I.; Bock, K. W.; Burchell, B.; Guillemette, C.; Ikushiro, S.; Iyanagi, T.; Miners, J. O.; Owens, I. S.; Nebert, D. W. Pharmacogenet. Genomics 2005, 15, 677–685. (8) Eisenhofer, G.; Coughtrie, M. W. H.; Goldstein, D. S. Clin. Exp. Pharmacol. Physiol. 1999, 26, S41–S53. (9) Tukey, R. H.; Strassburg, C. P. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 581–616, 2 plates. (10) Suleman, F. G.; Abid, A.; Gradinaru, D.; Daval, J. L.; Magdalou, J.; Minn, A. Arch. Biochem. Biophys. 1998, 358, 63–67. (11) Rivett, A. J.; Francis, A.; Whittemore, R.; Roth, J. A. J. Neurochem. 1984, 42, 1444–1449. (12) King, C. D.; Rios, G. R.; Assouline, J. A.; Tephly, T. R. Arch. Biochem. Biophys. 1999, 365, 156–162. (13) Filip, M.; Frankowska, M.; Zaniewska, M.; Golda, A.; Przegalinski, E. Pharmacol. Rep. 2005, 57, 685–700. (14) Jacintho, J. D.; Kovacic, P. Curr. Med. Chem. 2003, 10, 2693–2703.

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Glucuronide and sulfate conjugates in rat brain or cerebrospinal fluid (CSF) have commonly been analyzed after acid or enzymatic hydrolysis of the conjugate by liquid chromatography electrochemical detection (LC-EC),15-18 LC fluorescence (FL) detection,19 or gas chromatography/mass spectrometry (GC/MS).3,4 However, the identity of the conjugate cannot be confirmed using acid hydrolysis.15,16,18,19 The glucuronides and sulfates can be identified with higher specificity by hydrolyzing the conjugate with specific enzymes, β-glucuronidase, or sulfatase, respectively. The concentrations of DOPAC, HVA, and 5-HIAA in rat brain samples clearly increased after sulfatase hydrolysis, indicating that they are conjugated by sulfation.3,17 5-HT-S has not been detected in rat brain with indirect methods using sulfatase17 or acid hydrolysis.20 However, conjugates of 5-HT have been detected in rat CSF after acid hydrolysis.15 Swahn et al.3 studied the glucuronidation of DOPAC, HVA, and 5-HIAA in brain with an indirect method by hydrolyzing the conjugate with β-glucuronidase. However, the increase in the concentration of aglycone after hydrolysis was very small, and the presence of glucuronides was ambiguously confirmed. In a recent study, the glucuronidation of DA in rat and mouse brain was observed when intact DA-glucuronide (DA-G), without hydrolysis, was detected in brain microdialysates using LC-MS/ MS.2 Also neurosteroid glucuronides have been directly detected in mouse brain by using an LC-MS/MS method.21 The metabolism of neurotransmitters in the brain can be studied by microdialysis, where physiological perfusion fluid is pumped through a dialysis membrane, which is surgically implanted into a region of interest in an animal’s brain.22-24 The extracellular fluid of brain contains synaptically released neurotransmitters and their metabolites, as well as compounds from nonsynaptic sources.24 These low-molecular-weight compounds in the extracellular fluid are extracted to the perfusion fluid by passive diffusion, and the perfusion fluid is subsequently analyzed. So far, indirect analytical methods employing either acid or enzymatic hydrolysis of the 5-HT-, 5-HIAA-, DOPAC-, and HVAconjugates have been used, since glucuronide and sulfate standards are commercially unavailable. Indirect analysis methods, however, are prone to errors due to a hydrolysis step that also complicates the analysis. Even though the identity of the conjugate can be investigated with greater certainty after enzymatic hydrolysis as opposed to acid hydrolysis, the presence of the conjugate remains difficult to confirm with indirect methods, especially if the concentration of the conjugate is low compared to that of free aglycone. In this study, a specific LC-electrospray (ESI)/MS/MS method for the quantification of intact sulfate and (15) Hammond, D. L.; Yaksh, T. L.; Tyce, G. M. J. Neurochem. 1981, 37, 1068– 1071. (16) Sarna, G. S.; Hutson, P. H.; Curzon, G. Eur. J. Pharmacol. 1984, 100, 343– 350. (17) Warnhoff, M. J. Chromatogr. 1984, 307, 271–281. (18) Curzon, G.; Hutson, P. H.; Kantamaneni, B. D.; Sahakian, B. J.; Sarna, G. S. J. Neurochem. 1985, 45, 508–513. (19) Dedek, J.; Baumes, R.; Tien-Duc, N.; Gomeni, R.; Korf, J. J. Neurochem. 1979, 33, 687–695. (20) Korf, J.; Sebens, J. B. J. Neurochem. 1970, 17, 447–448. (21) Kallonen, S. E.; Tammima¨ki, A.; Piepponen, P.; Raattamaa, H.; Ketola, R. A.; Kostiainen, R. Anal. Chim. Acta 2009, 651, 69–74. (22) Westerink, B. H. C. J. Chromatogr., B: Biomed. Sci. Appl. 2000, 747, 21– 32. (23) Plock, N.; Kloft, C. Eur. J. Pharm. Sci 2005, 25, 1–24. (24) Bourne, J. A. Clin. Exp. Pharmacol. Physiol. 2003, 30, 16–24.

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glucuronide conjugates of 5-HT, 5-HIAA, DA, HVA, and DOPAC was developed. For the first time, 5-HT-G was detected in rat brain microdialysates. The direct LC-MS/MS method also allows the detection of different regioisomers of sulfated and glucuronidated compounds, which is impossible with indirect methods that use hydrolysis.

EXPERIMENTAL SECTION Reagents and Standards. 3,4-Dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid (HVA), 5-hydroxytryptamine (serotonin, 5-HT), and saccharic acid 1,4-lactone were purchased from Sigma-Aldrich (St. Louis, MO). 5-Hydroxyindole-3-acetic acid (5-HIAA), 3,4-dihydroxyphenethylamine hydrochloride (DA), and uridine-5′-diphosphoglucuronic acid (UDPGA, trisodium salt) were purchased from Sigma-Aldrich (Steinheim, Germany), potassium dihydrogen phosphate from Merck (Darmstadt, Germany), and ammonium formate and disodium hydrogen phosphate dihydrate from Fluka (Buchs, Switzerland). Acetonitrile (ACN) was purchased from Rathburn (Walkerburn, Scotland), and methanol (MeOH) from J.T. Baker (Deventer, Holland). Ringer’s solution, used for microdialysis and dilution of the standards, contained 147 mM NaCl, 1.2 mM CaCl2 · 2 H2O (Merck, Darmstadt, Germany), 2.7 mM KCl (Riedel de Hae¨n, Seelze, Germany), 1.0 mM MgCl2 · 6 H2O (Merck, Darmstadt, Germany), and 0.04 mM ascorbic acid (University Pharmacy, Helsinki, Finland). Sulfuric acid (H2SO4, BDH, 98%) was used in the chemical synthesis of sulfate conjugates. Chemical Synthesis of Sulfates. Cold concentrated H2SO4 (200 µL) was added to 20.5 mg of HVA, 20.4 mg of DOPAC, 25.2 mg of 5-HIAA, or 24.3 mg of 5-HT. The reaction mixture was kept in ice for 2 h and then pipetted over 1 mL of frozen water. The reaction mixtures were neutralized with 5 M NaOH. Sulfates were fractionated using an Agilent HP 1100 liquid chromatograph (Hewlett-Packard GmbH, Waldbronn, Germany) equipped with a binary pump, an autosampler, a column compartment, UV diode array detector, and fraction collector. A Discovery HS F5 column (150 mm × 4 mm, 3 µm, SigmaAldrich, Bellefonte, PA) was used in the purification. MeOH and 5 mM ammonium formate (pH 3.4) were used as eluents in the separation of 5-HT-S, HVA-S, and DOPAC-S and MeOH/ ACN (1:2 (v/v)) and aqueous 0.1% formic acid in the separation of 5-HIAA-S. A linear gradient 5-35% organic solvent for 0-10 min was used for the separation of 5-HT-S and 5-HIAA-S. Similar linear gradients were used for HVA-S and DOPAC-S, except the first 1 min was isocratic at 5% of the organic solvent. The flow rate was 0.9 mL/min, and a wavelength of 210 nm was used for peak detection. The sulfate fractions were evaporated to dryness and lyophilized. 5-HIAA-S and 5-HT-S were used as reference standards in the LC-MS/MS analysis of brain microdialysates. Enzymatic Synthesis of Sulfates. The incubation mixture contained 2 mM 5-HT, 5-HIAA, DOPAC, or HVA, 100 µM 3-phosphoadenosine-5-phosphosulfate (PAPS, 99% H. Glatt, German Institute for Human Nutrition, Potsdam, Germany), 10 mM phosphate buffer (pH 7.4), and 30% v/v rat liver S9 fraction (male Sprague-Dawley rats induced by Aroclor 1254, a mixture of

Figure 1. Structures of 5-HT, 5-HIAA, DA, HVA, and DOPAC. The possible glucuronidation sites are phenol, amine, and carboxylic acid groups, whereas the phenolic hydroxyl group is most favorably sulfated in the reaction catalyzed by SULT.

polychlorinated biphenyls) as described previously.25 The treatment of the rats was approved by the local Ethics Committee for Animal Studies. The reaction mixture was incubated (37 °C) for 2 h and centrifuged for 15 min (16 100g). To the supernatant, 5% aqueous ZnSO4 (Sigma-Aldrich, Steinheim, Germany) was added (1:1, v/v), and then MeOH was added to the final concentration of 50%. The mixture was centrifuged for 15 min (16 100g), and the supernatant was filtered (0.45 µm). The filtrate was diluted with 0.1% aqueous formic acid until the concentration of MeOH was 10% (v/v) and then purified by solid phase extraction using mixed-mode strong cation exchange and reversed-phase cartridges (Oasis MCX, Waters, MA). The cartridges were conditioned with MeOH and 0.1% aqueous formic acid. After the sample load, the sulfates were eluted from the cartridge with 0.1% aqueous formic acid and a mixture of MeOH/0.1% aqueous formic acid (20:80). The solvent was evaporated by lyophilization. The DOPAC-S and HVA-S were fractionated by LC as described for chemically synthesized 5-HIAA-S. The concentrations of enzymatically synthesized DOPAC-S and HVA-S solutions were determined indirectly by hydrolyzing the conjugate with 0.5 or 0.75 M HCl at 90 °C for 20 min and analyzing the concentration of free aglycone by LC-MS/MS (method described later). The recovery for 800 nM free HVA and DOPAC, heated in acid similarly to sulfates, was 87% and 90%, respectively. The solutions containing HVA-S and DOPAC-S were used for the preparation of calibration standards in the LC-MS/MS analysis. The quantification of both DOPAC-S regioisomers was done using the calibration curve of the first eluting DOPAC-S (retention time (rt) ) 6.6 min, SRM pair 247 f 123). Enzymatic Synthesis of Glucuronides. The enzymatic synthesis of 5-HT-G, 5-HIAA-G, DOPAC-G, and HVA-G were performed using microsomes prepared from rat liver (male Sprague-Dawley rats induced by Aroclor 1254; a mixture of polychlorinated biphenyls) as described previously.25 The synthesis of DA-G is described elsewhere.2 Protein concentrations of the microsomes were determined with the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL). In addition to rat liver microsomes (protein concentration 1 mg/mL), the incubation mixture contained 2 mM 5-HT, 5-HIAA, DOPAC, or HVA, 5 mM saccharic acid 1,4-lactone, 5 mM UDPGA, 5 mM MgCl2, 50 mM (25) Luukkanen, L.; Elovaara, E.; Lautala, P.; Taskinen, J.; Vainio, H. Pharmacol. Toxicol. 1997, 80, 152–158.

phosphate buffer (pH 7.4), and 2% ACN in a total volume of 30 mL. After 3 h of incubation (37 °C), an additional 6 mg of 5-HT, 5-HIAA, DOPAC, or HVA and 50 mg of UDPGA was added. The incubation was continued for an additional 4 h with 5-HT and for 19 h with the other compounds. The reaction mixture was acidified with formic acid (98-100%, Riedel de Hae¨n, Seelze, Germany) to pH 3 and centrifuged for 14 min (26 700g, 6 °C). The supernatant containing glucuronides was filtrated (0.45 µm). Filtrates containing 5-HT-G or 5-HIAA-Gs were purified by solid phase extraction using mixed-mode strong cation exchange and reversed-phase cartridges (Oasis MCX, 20 cc, Waters, MA). For HVA-Gs and DOPAC-Gs, reversed-phase cartridges (Oasis HLB, 20 cc, Waters, Milford, MAA) were used. The cartridges were conditioned with 15 mL of MeOH and 15 mL of 0.1% aqueous formic acid. After the addition of 10 mL of filtrate containing glucuronides, the cartridge was washed with 15 mL of 2% (MCX) or 0.1% (HLB) aqueous formic acid. For 5-HT-G, the wash was continued with 10 mL of MeOH, and the glucuronides were eluted from the cartridge with 10 mL of MeOH containing 5% ammonia. 5-HIAA-G was eluted with MeOH (2 × 10 mL) and MeOH containing 2% ammonia (1 × 10 mL). DOPAC-G and HVA-G were eluted with 50% or 25% MeOH in water, respectively. The solvent was evaporated with a rotary evaporator, and glucuronides were fractionated from the residue as described for sulfates; 0.1% formic acid and ACN (5-HIAA-G and DOPAC-G) or ACN/MeOH (2:1) (HVA-G and 5-HT-G) were used as eluents. A linear gradient 5-35% organic solvent for 0-10 min was used to separate the glucuronides and aglycones. The collected glucuronide fractions were evaporated to dryness and lyophilized. Liquid Chromatography-Mass Spectrometry. The Discovery HS F5 column (150 mm × 4 mm, 3 µm, Sigma-Aldrich, Bellefonte, PA) was used for the separation of DA, DOPAC, HVA, 5-HT, and 5-HIAA as well as their sulfates and glucuronides (Figure 1) in brain microdialysates. Aqueous 0.1% formic acid and ACN/MeOH (1:1, quantitative analysis) or (2:1, qualitative analysis) were used as eluents. Different linear gradients were used for basic (DA, 5-HT, DA-G, 5-HT-G) and acidic compounds (phase I metabolites and their sulfates). Gradient 1 (acidic compounds): 5% organic solvent for 0-1 min, 5-20% organic solvent for 1-11 min, 20-85% organic solvent for 11-13 min, 85% organic solvent for 13-15 min, 85-5% organic solvent for 15-15.1 min, and 5% Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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organic solvent for 15.1-19 min. Gradient 2 (basic compounds): similar to gradient 1 but 20-65% organic solvent for 11-13 min and 65% organic solvent for 13-15 min. The flow was directed to waste for the first 3.8 min to prevent the inorganic ions of Ringer’s solution to enter the mass spectrometer. The flow was split with a ratio of 1:10, 10% entering the mass spectrometer. The brain microdialysates and standards diluted to Ringer’s solution were injected as such without sample pretreatment, and the injection volume was 100 µL. An Agilent 6410 triple-quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) was used in the quantitative analysis of brain microdialysates. Nitrogen (Parker Balston N222 nitrogen generator, Parker Hannifin Corporation, Haverhill) was used as a nebulizer (40 psi), curtain (12 L/min, 350 °C), and collision gas. The ESI needle and fragmentor voltages as well as the collision energy were optimized for each compound. Agilent Mass Hunter software version B.01.03 was used for data acquisition and processing. An API3000 triple-quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Canada) with a turbo ion spray source was used in the qualitative analysis of rat brain microdialysates. Purified air (Atlas Copco CD 2, Wilrijk, Belgium) was used as a nebulizing gas, and nitrogen generated with a Whatman 75-72 generator (Haverhill, MA) was used as a turbo, curtain, and collision gas. The turbo gas flow rate was 6 L/min and was heated to 280 °C. The ion spray and orifice (declustering) voltages, the collision energy, and the collision cell exit potentials for different selected reaction monitoring (SRM) pairs were individually optimized for each compound. The data were collected and processed by Analyst 1.4.2 software. Mass spectrometric detection in an ESI positive ion mode was carried out using SRM with the following reactions: 5-HT (m/z 177 f 115, 117, 132, 160), 5-HT-G (m/z 353 f 160, 177, 336), 5-HT-S (m/z 257 f 115, 160, 240), DA (m/z 154 f 91, 137), and DA-G (m/z 330 f 91, 137, 154). A negative ion mode was used in the analysis of HVA (m/z 181 f 122, 137), HVA-G (m/z 357 f 113, 193), HVA-S (m/z 261 f 80, 181, 217), DOPAC (m/z 167 f 93, 95, 108, 123), DOPAC-G (m/z 343 f 113, 123), DOPAC-S (m/z 247 f 80, 123, 167, 203), 5-HIAA (m/z 190 f 116, 144, 146), 5-HIAA-G (m/z 366 f 113, 146), and 5-HIAA-S (m/z 270 f 80, 131, 146, 226). Data in the positive and negative ion modes were acquired in separate runs. The SRM pairs used for the quantitative analysis are shown in Table 3. NMR Spectroscopy. The nuclear magnetic resonance (NMR) spectra of 5-HT and synthesized and purified 5-HT-G and 5-HT-S were measured in D2O with a Varian Mercury Plus 300 spectrometer (Varian, Palo Alto, CA) at 24 °C; 5-HIAA and synthesized and purified 5-HIAA-S were dissolved in CD3OD. Chemical shifts (δ) are denoted in parts per million (ppm) relative to the internal standard acetone added to the samples (1H NMR 2.2 ppm and 13C NMR 30.2 ppm). The protons and carbons were assigned with heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments. Animals. Male Wistar rats were used at 8-16 weeks of age. All procedures with the animals were performed according to the European Community Guidelines for the use of experimental animals (European Communities Council Directive 86/609/EEC) and reviewed by the State Provincial Office of Southern Finland 8420

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and approved by the Animal Experiment Board in conformance with current legislation. The rats were housed in groups of four to five per cage and had free access to chow and water. They were maintained under a 12:12 h light/dark cycle with lights on from 06:00 to 18:00 at an ambient temperature of 20-22 °C. Microdialysis. The animals were implanted with a guide cannula (BAS MD-2250, Bioanalytical Systems Inc., IN) using a stereotaxic device (Stoelting, Wood Dale, IL) under isoflurane anesthesia (4.5% during induction for 5 min and then 3.5% during surgery). The guide cannula was aimed above the rat dorsal striatum (A/P ) +1.0, L/M ) 2.7, D/V ) -6.0) according to the atlas by Paxinos and Watson.26 The cannula was fastened to the skull with dental cement (Aqualox, Voco, Germany) and three stainless steel screws. Buprenorphine (0.05 mg/kg subcutaneously) was given for postoperative pain. The animals were placed into individual test cages and allowed to recover for 5-7 days before the experiment. A microdialysis probe was inserted into the striatum (BAS MD2204, 4 mm membrane, Bioanalytical Systems Inc., IN) through the guide cannula on the morning of the experimental day. The collection of microdialysis samples (2.5 µL/min) began 2.5-3 h after insertion of the probe. The microdialysis samples were stored in a freezer (-70 °C) before analysis with LC-MS/MS. The microdialysates (100 µL) were injected as such without sample pretreatment. After completion of the experiments, the positions of the probes were verified histologically by fixing coronal brain sections on gelatin/chrome-coated slides and confirming the respective placements of the implanted probes in the striatum. RESULTS AND DISCUSSION Sulfation and glucuronidation in the brain have usually been studied using indirect analytical methods employing acid or enzymatic hydrolysis. One reason for this is the lack of commercial sulfate and glucuronide standards. In this study, sulfates and glucuronides of HVA, DOPAC, 5-HT, and 5-HIAA (Figure 1) were chemically and enzymatically synthesized to provide reference compounds for a direct LC-MS/MS method. Unlike with indirect methods employing hydrolysis of the conjugates, the direct analysis method used in this study permits the detection of different regioisomers of intact glucuronides and sulfates. Synthesis and Characterization of Glucuronides. The glucuronides of 5-HT, 5-HIAA, HVA, and DOPAC were synthesized enzymatically using rat liver microsomes as a biocatalyst, and the products were characterized by LC-MS. The synthesis and characterization of DA-G are described elsewhere.2 LC-MS ion chromatograms of the deprotonated molecules [M - H]corresponding to monoglucuronidated compounds in the negative ion mode showed one peak for 5-HT-G, two peaks for 5-HIAA-Gs, and four peaks for DOPAC-Gs and HVA-Gs (Figure 2). The respective MS/MS spectra of [M - H]- showed product ions at m/z 175 [M - H - aglycone]-, m/z 157 [M - H aglycone - H2O]-, and m/z 113 [M - H - aglycone - H2O CO2]- derived from the glucuronic acid moiety confirming the presence of the glucuronic acid moiety in the molecule (Table 1). A product ion at m/z 193 (glucuronic acid) was observed in the MS/MS spectrum of three HVA-Gs (rt 6.65, 7.20, and 7.35 (26) Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates, 2nd ed.; Academic Press: San Diego, CA, 1986.

Figure 2. LC-ESI-MS extracted ion chromatograms of the glucuronides (API 3000) and sulfates of 5-HT, 5-HIAA, DOPAC, and HVA (Agilent 6410) analyzed in the negative ion mode. The ions extracted (m/z) corresponding to the [M - H]- ion appears in the figure. Compounds were synthesized enzymatically, except 5-HT-S and 5-HIAA-S were synthesized chemically. Linear gradients were used: glucuronides, 5-35% ACN/ MeOH (2:1) for 0-10 min; sulfates, 5% ACN/MeOH (1:1) for 0-1 min, and 5-20% ACN/MeOH (1:1) for 1-11 min.

min), two DOPAC-Gs (rt 5.18 and 5.76 min), and one 5-HIAA-G (rt 7.31 min). It has been reported earlier that in the MS/MS spectra of aliphatic hydroxyl glucuronides, a product ion m/z 193 is observed, whereas it is not observed in the MS/MS spectra of phenol-linked glucuronides.27-29 It is therefore reasonable to assume that those HVA-Gs, DOPAC-Gs, and 5-HIAA-G showing a product ion at m/z 193 in the MS/MS spectra were glucuronidated to the carboxylic group, thus producing acyl glucuronides. The DOPAC-Gs with retention times of 6.23 and 6.47 min, HVA-G with a retention time of 6.79 min, and 5-HIAA-G with a retention time of 6.56 min were most probably glucuronidated at phenolic groups since m/z 193 was not observed in the MS/MS spectra. The acyl glucuronides are prone to acyl migration30 (i.e., (27) Wen, Z.; Tallman, M. N.; Ali, S. Y.; Smith, P. C. Drug Metab. Dispos. 2007, 35, 371–380. (28) Niemeijer, N. R.; Gerding, T. K.; De Zeeuw, R. A. Drug Metab. Dispos. 1991, 19, 20–23. (29) Fenselau, C.; Johnson, L. P. Drug Metab. Dispos. 1980, 8, 274–283. (30) Johnson, C. H.; Wilson, I. D.; Harding, J. R.; Stachulski, A. V.; Iddon, L.; Nicholson, J. K.; Lindon, J. C. Anal. Chem. 2007, 79, 8720–8727.

the aglycone is moving from one hydroxyl group of glucuronic acid to another through molecular internal reactions forming a new regioisomer). This explains why the number of glucuronides formed for HVA and DOPAC (four peaks) exceeded the expected number of products (two and three, respectively) based on the possible glucuronidation sites (Figure 1). It is known that the acyl migration is reduced at low temperatures and under acidic conditions.30 However, basic conditions (MeOH containing 2% ammonia) were needed in the purification step of 5-HIAA-G standard when it was eluted from the mixed-mode SPE cartridge, otherwise acidic conditions were used throughout the study. In the analysis of brain microdialysates, the acyl migration was controlled by freezing the brain microdialysates immediately after sampling and thawing the samples just before LC-MS/MS analysis. The brain microdialysates were injected as such without any sample pretreatment which also minimized the possible acyl migration. The glucuronidation site of 5-HT-G was determined by NMR (Table 2). The HMBC experiment showed 3J coupling of an acetal CH proton (H1′) to the quaternary aromatic carbon Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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Table 1. m/z Values of Main Ions in the LC-MS/MS Spectra of 5-HT-, 5-HIAA-, HVA-, and DOPAC-Sulfates and Glucuronides Analyzed in the Negative Ion Mode compound Glucuronides 5-HT-G 5-HIAA-G DOPAC-G

HVA-G

Sulfates 5-HT-S 5-HIAA-S DOPAC-S HVA-S a

rta (min)

precursorion [M - H]-

6.82 6.56 7.31 5.18 5.76 6.23 6.47 6.65 6.79 7.20 7.35

351 366 366 343 343 343 343 357 357 357 357

113 113 113 113 113 113 113 113 113 113 113

(33), 157 (10), 175 (100) (41), 146 (54), 157 (26), 175 (94), 190 (100) (95), 131 (19), 133 (19), 146 (29), 157 (24), 175 (52), 190 (86), 193 (100) (100), 123 (100), 157 (20), 167 (11), 175 (27), 193 (25) (77), 123 (100), 131 (18), 157 (18), 167 (50), 175 (45), 193 (77) (100), 123 (92), 157 (24), 175 (22) (100), 123 (98), 157 (20), 167 (16), 175 (27) (100), 133 (10), 157 (10), 175 (22), 193 (10) (100), 137 (23), 157 (13), 175 (25), 181 (25) (100), 131 (16), 133 (15), 157 (13), 175 (19), 193 (12) (55), 131 (23), 137 (21), 157 (18), 175 (23), 181 (22), 193 (100)

9.40 8.37 9.64 6.64 7.04 7.97

255 270 270 247 247 261

175 226 226 203 203 181

(100), 131 (42), 80(26) (100), 147 (14), 146 (14), 80 (62) (58), 146 (100), 131 (26), 80 (19) (18), 167 (11), 123 (78) (38), 167 (10), 123 (75) (51), 137 (100), 122 (44), 80 (8)

m/z in the product ion spectrum of [M - H]- (abundance %)

rt ) retention time.

Table 2. 1H and 5-HT-G

13

C NMR Chemical Shifts of 5-HT and

1

5-HT

5-HT-G

H1 H2R H2β H4 H6 H7 H1′ H5′ H2′,3′, 4′

7.25 3.08 3.28 7.07 6.86 7.39

7.24 3.05 3.24 7.31 7.03 7.41 5.04 3.83 3.60 - 3.61

H

13

C

5-HT

5-HT-G

C1 C2 C2R C2β C3 C4 C5 C6 C7 C8 C1′ C2′ C3′ C4′ C5′ C6′

125.3 108.4 22.6 39.6 127.1 102.4 148.7 111.8 112.8 131.6

125.5 109.1 22.5 39.6 126.6 105.5 150.5 113.6 112.7 133.0 101.9 72.9 75.4 71.7 76.2 175.3

ArCO (C5), indicating that the glucuronide group is attached to phenolic oxygen. An earlier study has shown that the glucuronidation of 5-HT with mouse liver microsomes occurred at the phenol group.31 The yield of 5-HT-G synthesized with rat liver microsomes was good, being 20.6 mg (59%). The yields for HVA-G, DOPAC-G, and 5-HIAA-G were clearly worse than for 5-HT-G, being 0.5-1.5 mg (1.4-4.4%) for phenol-glucuronides and 0.04-0.1 mg (0.1-0.4%) for acyl glucuronides, and therefore their structure was not determined by NMR. (31) Krishnaswamy, S.; Duan, S. X.; Von Moltke, L. L.; Greenblatt, D. J.; Sudmeier, J. L.; Bachovchin, W. W.; Court, M. H. Xenobiotica 2003, 33, 169–180.

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Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

Synthesis and Characterization of Sulfates. 5-HT-S and 5-HIAA-S were synthesized chemically and DOPAC-S and HVA-S enzymatically using the rat liver S9 fraction. The chemical synthesis method was also applied for DOPAC and HVA, but according to the LC-MS/MS measurements, the method failed to produce those sulfate isomers of DOPAC and HVA found in brain microdialysates (see results below). LC-MS ion chromatograms of the synthesis products showed one peak for 5-HT-S and HVA-S and two peaks for HIAA-Ss and DOPAC-Ss (Figure 2). All the negative ion mass spectra showed abundant [M - H]-, thus corresponding to monosulfated compounds. The product ion spectra of the [M - H]- of 5-HIAA-Ss, DOPAC-Ss, and HVA-S showed abundant product ions at m/z 226, m/z 203, and m/z 217, respectively, formed by the direct loss of the carbon dioxide (44 Da) from the [M - H]- ion, thus indicating that no sulfate is attached to the carboxylic acid group of the substrate (Table 1). It is known that a hydroxyl group is a common acceptor group of sulfate in the reaction catalyzed by SULT,32 and that the most obvious sulfatation site is therefore a phenolic hydroxyl group. The sulfation sites of 5-HT-S and 5-HIAA-S (rt ) 9.64 min) were verified by NMR. The peak in the extracted ion chromatogram of 5-HIAA-S with the retention time of 8.37 min (Figure 2) is a side product formed in the chemical synthesis of sulfates. It was not observed in the brain microdialysates and therefore its structure was not studied. The chemical shifts of the H4 and H6 protons of 5-HT-S and 5-HIAA-S (rt ) 9.64 min) clearly moved downfield compared to those of the free substrates, thus indicating that sulfation occurred at the phenolic group. The yields of HVA-S and DOPAC-Ss produced by the enzymatic synthesis method were too low to study the sulfation site by NMR. The yields for 5-HT-S and 5-HIAA-S (rt ) 9.6 min) were 2.6 (9%) and 2.4 mg (7%), respectively. Analysis of Rat Brain Microdialysates by LC-MS/MS. The glucuronides and sulfates of 5-HT, 5-HIAA, DA, HVA, and (32) Blanchard, R. L.; Freimuth, R. R.; Buck, J.; Weinshilboum, R. M.; Coughtrie, M. W. H. Pharmacogenetics 2004, 14, 199–211.

Table 3. Results of the Validation Tests of the LC-MS/MS Method for the Analysis of Monoamines and Their Metabolites in Rat Brain Microdialysatesa

compound 5-HT 177 f 5-HT-G 353 f 5-HIAA 190 f 5-HIAA-S 270 f DA 154 f DA-G 330 f DOPAC 167 f DOPAC-S (rt ) 6.6 min) 247 f HVA 181 f HVA-S 261 f a

SRM pairs, m/z 160 160 146 226 137 137 123 123 137 181

(132, 117, 115) (336, 177) (144, 116) (146, 131, 80) (91) (154, 91) (108, 95, 93) (203, 167, 80) (122) (217, 80)

LOD (nM), LOQ (nM), S/N ) 3-5 S/N > 10 0.1 0.1 1 4 0.3 0.1 10 1 20 0.5

0.5 0.5 5 15 0.8 0.5 30 5 60 2

linearity range (nM) 0.5-10 0.5-10 10-260 15-160 0.8-20 0.5-10 180-1000 50-500 70-1000 50-500

r2 0.997 0.999 0.998 0.996 0.998 0.998 0.999 0.999 0.999 0.999

repeatability 100 nM, % RSD 10 nM, accuracy % n)9 n)6 92-123 99-100 91-116 92-107 95-129 94-125 97-102 96-111 95-105 97-109

3.7 2.3 2.4 3.1 2.9 3.6 3.5 2.4b 3.9 2.5b

nm 2.9 nm