Identification Strategy Using Combined Mass Spectrometric

Oct 25, 2010 - Identification Strategy Using Combined Mass Spectrometric Techniques for Elucidation of Phase I and Phase II in Vitro Metabolites of Li...
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Anal. Chem. 2010, 82, 9329–9335

Identification Strategy Using Combined Mass Spectrometric Techniques for Elucidation of Phase I and Phase II in Vitro Metabolites of Lipophilic Marine Biotoxins Katrin Kittler, Angelika Preiss-Weigert, and Anja These* National Reference Laboratory for Marine Biotoxins, Federal Institute for Risk Assessment, Thielallee 88-92, 14195 Berlin, Germany Combining mass spectrometric tools, a total of 47 in vitro metabolites of okadaic acid (OA), dinophysistoxins 1 and 2 (DTX1 and DTX2), yessotoxin (YTX), azaspiracid1 (AZA1), and pectenotoxin 2 (PTX2) could be detected and confirmed after an incubation with rat liver S9-mix. In a first step, liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) was used as a screening tool for the identification of in vitro metabolites of lipophilic marine biotoxins. Metabolic phase I and phase II reactions were screened for metabolites by calculating and subsequently monitoring theoretical MS transitions. In a second step, metabolites were confirmed by determination of accurate masses using high resolution MS provided by Orbitrap technology. Subsequently, product ion spectra, precursor ion spectra, and MS3 spectra were recorded for structure elucidation of metabolites. While all investigated toxins were found to form various oxygenated metabolites during the oxidative phase I metabolism, those metabolites varied in the number of added oxygen atoms and in the number of individual isomers. No hints were obtained concerning the formation of glutathione adducts, and a conjugation with glucuronic acid was detected for AZA1 only. Marine biotoxins are secondary metabolites of certain marine algae species and can accumulate in filter feeding mussels. After consumption of those mussels, serious intoxication will be caused in humans.1-5 Marine biotoxins can be classified either by structure or by symptoms of intoxication.6 The most frequently occurring toxins in Europe belong to the group of lipophilic toxins, which, subdivided into the following four groups, are subject to regulations regarding maximum levels: the azaspiracid group (AZA), the yessotoxin group (YTX), the pectenotoxin group (PTX), and the okadaic acid group (OA), which is also known as the * To whom correspondence should be addressed. E-mail: anja.these@ bfr.bund.de. (1) Yasumoto, T.; Oshima, Y.; Yamaguchi, M. Bull. Jpn. Soc. Sci. Fish. 1978, 44, 1249–1255. (2) Torgersen, T.; Aasen, J.; Aune, T. Toxicon 2005, 46, 572–578. (3) Kat, M. Antonie Van Leeuwenhoek 1983, 49, 417–427. (4) Vale, P.; Antonia, M.; Sampayo, M. Toxicon 1999, 37, 1109–1121. (5) McMahon, T.; Silke, J. Harmful Algae 1996, 14, 2. (6) Marine Biotoxins, FAO Food and Nutrition Paper 80; UN Food and Agriculture Organization (FAO): Rome, 2004. 10.1021/ac101864u  2010 American Chemical Society Published on Web 10/25/2010

diarrhetic shellfish poisoning (DSP) group. Each of these subgroups contains 2-4 individual toxins that mostly are structure or alkyl homologues.6 Incidents have been reported in many countries worldwide, but information on doses or toxin profiles is very limited as leftovers of meals are not always available and up to now postintoxication analysis has not been applied.7,8 This is mainly due to the fact that neither sensitive methods for the detection in body fluids have been developed nor metabolites of lipophilic marine biotoxins have been identified, and, beyond that, reference material is available for some of the toxins only. In view of this situation, which underlines the growing need for sensitive and specific analytical methods, we consider LC/MS to play an expanding role to guarantee food safety, on the one hand, and to ensure the identification of potential intoxication cases by measuring metabolites in body fluids, on the other hand.9,10 First results of OA metabolism have been published for in vitro studies with human recombinant cytochrome P 450s (CYP) that were confirmed by incubation with pooled human liver microsomes.11 CYP3A4 and CYP3A5 were shown to convert OA into four metabolites that subsequently were structurally characterized by mass spectrometry.11 No further metabolites of lipophilic marine biotoxins are known, and consequently no reference standards for sensitivity optimization of instruments are available. If metabolites are unknown and instrument parameters cannot be checked for their appropriateness, those analytes often remain undetected. Our strategy involved that target analytes were identified in a first step. Typical routes of metabolism of xenobiotics are well described in the literature, and those metabolic pathways should also be assumed for marine biotoxins.12-14 Furthermore, the structure, the molecular masses, and fragmentation patterns of lipophilic (7) EFSA, EFSA J. 2008, 589, 1–62. (8) EFSA, EFSA J. 2008, 723, 1–52. (9) Hess, P.; Grune, B.; Andersen, D. B.; Aune, T.; Botana, L. M.; Caricato, P.; van Egmond, H. P.; Halder, M.; Hall, S.; Lawrence, J. F.; Moffat, C.; Poletti, R.; Richmond, J.; Rossini, G. P.; Seamer, C.; Vilageliu, J. S. ATLA, Altern. Lab. Anim. 2006, 34, 193–224. (10) Pico, Y.; Barcelo, D. TrAC, Trends Anal. Chem. 2008, 27, 821–835. (11) Guo, F.; An, T.; Rein, K. S. Toxicon 2010, 55, 325–332. (12) Nelson, S. D.; Gordon, W. P. J. Nat. Prod. 1983, 46, 71–78. (13) Gao, H. Y.; Materne, O. L.; Howe, D. L.; Brummel, C. L. Rapid Commun. Mass Spectrom. 2007, 21, 3683–3693. (14) Zhu, M.; Ma, L.; Zhang, D.; Ray, K.; Zhao, W.; Humphreys, W. G.; Skiles, G.; Sanders, M.; Zhang, H. Drug Metab. Dispos. 2006, 34, 1722–1733.

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marine biotoxins are well-known.15-18 In a second step, theoretical MRM transitions for the investigation of the likely metabolic reactions were predicted and calculated. Depending on the fragmentation of the parent toxin, two theoretical options were considered: the transformation occurred either at the molecule part, which leaves the molecule as neutral, or at the fragment. In the latter case, the molecule mass (Q1 mass) and the fragment mass (Q3 mass) increase (decrease) by the mass change associated with the respective metabolic reaction. The detection by triple stage quadrupol MS in MRM mode increases specificity and sensitivity as compared to a screening in scan mode applied on a single MS. Third, elemental composition of metabolites was confirmed by well-directed analysis using high resolution mass spectrometry (A > 50.000, see Results and Discussion). In a fourth step, for structure investigation, all metabolites were analyzed by recording product ion spectra. To locate the position in the substrates where metabolic reaction took place, further steps, that is, recording precursor ion spectra and MS3, appeared to be necessary in some cases. The choice of the most reliable detection mode depends on the mass spectrometric behavior of respective analytes, their fragmentation pattern, as well as signal intensity plays a decisive role. We applied this strategy for the identification of in vitro metabolites of lipophilic toxins using the S9-fraction of rat liver and supplement of cofactors. The combination of different mass spectrometric detection modes has already been demonstrated to be successful for the elucidation of metabolites of xenobiotics.19-22 The S9-mix has originally been used for reflecting metabolism within bacterial reverse mutation tests and has been widely applied in the screening for potential mutagenic compounds.23 EXPERIMENTAL SECTION Solvents, Standards, and Reagents. Ammonium hydrogen carbonate, alamethicin (Trichoderma viride), d-saccharolactone, L-glutathione reduced (GSH), and uridine 5′-diphosphoglucuronic acid (UDPGA) were of analytical grade and obtained from SigmaAldrich (Steinheim, Germany). Reduced nicotinamide adenine dinucleotide phosphate (NADP+), glucose 6-phospate (G6P), magnesium chloride hexahydrate, potassium chloride, Na2HPO4, and NaH2PO4, all pro analysis grade, were purchased from Roth (Karlsruhe, Germany). Water was produced by a TKA water purification system (TKA, Niederelbert, Germany). Solutions of OA, YTX, PTX2, and AZA1 dissolved in MeOH were purchased from the National Research Council - Institute for Marine Biosciences (Halifax, Canada), DTX2 was from Labo(15) Brombacher, S.; Edmonds, S.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2002, 16, 2306–2316. (16) Gerssen, A.; Mulder, P.; van Rhijn, H.; de Boer, J. J. Mass Spectrom. 2008, 43, 1140–1147. (17) Torgensen, T.; Bremnes, N. B.; Rundberget, T.; Aune, T. Toxicon 2008, 51, 93–101. (18) Torgersen, T.; Wilkins, A. L.; Rundberget, T.; Miles, C. O. Rapid Commun. Mass Spectrom. 2008, 22, 1127–1136. (19) Jian, W. Y.; Yao, M.; Zhang, D. X.; Zhu, M. S. Chem. Res. Toxicol. 2009, 22, 1246–1255. (20) Wen, B.; Fitch, W. L. Expert Opin. Drug Metab. Toxicol. 2009, 5, 39–55. (21) Anari, M. R.; Sanchez, R. I.; Bakhtiar, R.; Franklin, R. B.; Baillie, T. A. Anal. Chem. 2004, 76, 823–832. (22) Kostiainen, R.; Kotiaho, T.; Kuuranne, T.; Auriola, S. J. Mass Spectrom. 2003, 38, 357–372. (23) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347–364.

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ratorio Cifga (Lugo, Spain), and DTX1 was from BlueBioTech GmbH (Husum, Germany). Incubation with S9-mix for Identification of Phase I Metabolites. The essential enzymes for the metabolization were provided by S9-mix. The S9-mix consisted of cofactors and a rat S9 liver homogenate (supernatant of 9000g centrifugation of rat liver homogenate),23 here with a protein content of 30.6 mg protein/mL. Male rats (Wistar) were previously treated with a combination of phenobarbital and β-naphthoflavone for inducing individual cytochroms. The phase I cofactor consisted of the NADPH regenerating system (NADP+ [4 mM] and G6P [5 mM]), KCl (33 mM), MgCl2 (8 mM), and 0.1 M sodium phosphate buffer (Na2HPO4 [0.2 M] + NaH2PO4 [0.2 M] to pH 7.4). Incubation was adopted from a protocol that had previously been established for the detection of mutagenesis. An experimental volume of 1.5 mL contained cofactor I (final concentration as described above), S9-homogenate (final concentration 3 mg/mL), and toxins dissolved in MeOH (final MeOH concentration below 1%). The following toxin concentrations were used as single solutions for metabolic reaction: OA, 310 nM; DTX1, 50 nM; DTX2, 50 nM; AZA1, 130 nM; YTX, 92 nM; PTX2, 100 nM. Solutions were incubated in a water bath at 37 °C for 3 h. The reaction was terminated by adding 1.5 mL of ice cold MeOH. Each sample was concentrated by a factor of 3 using solid-phase extraction according to an established protocol.24 To ensure that no analytes were lost during SPE procedure, one direct LC/MS/MS analysis was done per sample. For control purposes, the whole procedure was performed as described above, but S9-homogenate was inactivated at a temperature of 60 °C for 20 min before incubation. Incubation with S9-mix for Identification of Phase II Metabolites. Examination of phase II formation of glutathione adducts was performed as described above except that cofactor phase I solution contained 5 mM GSH additionally. For the investigation of glucuronidation, a volume of 1.5 mL was mixed as follows: 225 µL of S9-homogenate and 15 µL of alamethicin (2.5 mg/mL) were cooled on ice. After 15 min, 63 µL of d-saccharolactone (50 mg/mL), 37.5 µL of MgCl2 (0.4 M), 750 µL of 0.2 M sodium phosphate buffer (pH 7.4), 112.5 µL of UDPGA (50 mg/mL), and dissolved toxins were added and incubated. Final toxin concentrations, incubation, termination, and SPE procedure of phase II reaction were the same as for the phase I reaction. HPLC. Analyses (in combination with tandem mass spectrometry) were performed on a system of Agilent Technologies 1200 series, and analyses (in combination with high resolution mass spectrometry) were performed on a system of Thermo Fisher Accela LC Systems. Chromatography was carried out on a 150 × 2 mm, particle size 3 µm Gemini NX column with guard protection (Phenomenex, Aschaffenburg, Germany). Eluent A was prepared with 100% water containing 5 mM ammonium hydrogen carbonate, and eluent B was with 95% ACN and 5% water containing 5 mM ammonium hydrogen carbonate. A gradient elution was used starting with 20% eluent B, increasing B to 90% within 15 min, holding 90% B (24) These, A.; Scholz, J.; Preiss-Weigert, A. J. Chromatogr., A 2009, 1216, 4529– 4538.

Figure 1. Extracted ion chromatograms of lipophilic toxins incubated with S9-mix: OA (top, left), YTX (top, right), AZA1 (bottom, left), and PTX2 (bottom, right). The following metabolic reactions are represented: (a) nonmetabolized toxin, (b) +1×O-H2, (c) +1×O (oxygen located at the fragment), (d) +1×O (oxygen located at the neutral), (e) +2×O, (f) +3×O, (h) -1×H2, and (g) +C6H8O6. For each toxin, the respective chromatograms of control groups incubated with inactivated S9-mix are included.

for 5 min, decreasing B to 20% within 1 min, and holding for 20% B for 6 min. A flow rate of 200 µL/min was applied, and 5 µL was injected. Mass Spectrometry. Tandem Mass Spectrometry. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) data were acquired on a 4000 Q Trap system (Applied Biosystems, Ontario, Canada). The mass spectrometer was operated with the following source parameter: capillary voltage of +5000 V in positive and -4500 V negative ionization mode, desolvation temperature of 500 °C, and 35 psi ions source gas 1 and 2. Q1 and Q3 operated at unit resolution. As MS conditions and fragmentation patterns of nonmetabolized toxins were well-known, theoretical MRM transitions could be calculated and were monitored for detection of metabolites. This approach is exemplarily shown for OA. In OA routine analysis, the following transition using the settings in brackets is monitored: OA, 803.5 > 255.1 (DP, -170 V; CE, -62 eV). Possible oxidation reactions, for instance, the addition of an oxygen, were investigated once by assuming that the inserted oxygen was located at that part of the molecule ion where it leaves with the neutral fragment, OA+O, 819.5 > 255.1 (DP, -170 V; CE, -62 eV), or remaining at the fragment ion, OA+O, 819.5 > 271.1 (DP, -170 V; CE, -62 eV). The same procedure was applied for DTX1, 817.5 > 255.1 (DP, -170 V; CE, -62 eV) and DTX2, 803.5 > 255.1 (DP, -170 V; CE, -62 eV). AZA1, PTX2 metabolites were investigated by calculating MRM transition reflecting the loss of water: PTX2, 876.5 > 823.5 (DP, 150 V; CE, 27 eV), and AZA1, 842.5 > 824.5 (DP, 100 V; CE, 40 eV), or for YTX the loss of SO3, YTX, 1141.6 > 1061.6 (DP, -150 V; CE, -42 eV).

Accordingly, the following metabolic products were monitored: toxin+O, toxin+2×O, toxin+3×O, toxin+H2O, toxin+H2O2, toxin+O-H2, toxin+H2, toxin-H2, toxin+CH2, toxin-CH2, toxin+2×CH2, toxin+C6H8O6 (glucuronidation), toxin+2×C6H8O6, toxin+C6O6H8O, and toxin+C10H15N3O6S (glutathione).13,14,22 Enhanced product ion spectra of m/z 803 and 819 were acquired for a mass range of m/z 150-825 using a CE of -60 eV and applying a CE ramp of 20 eV. Enhanced product ion spectra of m/z 876 and 892 were acquired for a mass range of m/z 80-900 using a CE of 27 eV, and, for generating product ion spectra of m/z 842 and 858, a mass range of m/z 100-863 was scanned using a CE of 50 eV and applying a CE ramp of 20 eV. Precursor ion spectra of m/z 672 were acquired for a mass range of m/z 700-1025 using a CE of 53 eV. MS3 spectra of m/z 378 and m/z 362 were scanned for m/z 80-385 by applying a CE of 40 eV and using an AF2 60 eV for an excitation time of 150 ms. High Resolution Mass Spectrometry. Electrospray ionization high resolution mass spectrometry (ESI-hRMS) data were acquired on an Orbitrap Exactive system (Thermo Fisher, Bremen, Germany). The mass spectrometer was equipped with an H-ESI II source and operated at +3700 V in positive and -3700 V negative ionization mode. Tube lens voltage was set to 150 V, and capillary voltage was set to 32 V in negative and 10 V in positive mode. A capillary temperature of 350 °C was used. Positive and negative ion spectra were generated in parallel by permanent polarity switching applying a time of 25 ms per switch. For both polarities, the mass range between 100 and 2000 Da was recorded. Operating at “ultrahigh mode”, a mass resolution of 100.000 was achieved. Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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RESULTS AND DISCUSSION In Figure 1, the extracted ion chromatograms of MRM transitions of metabolites of lipophilic toxins are shown, which were detected with a signal-to-noise (S/N) ratio higher than 3/1. None of these signals was attended at the respective negative control. In terms of phase II reactions, only the conjugation of glutathione and the glucuronidation were investigated. No formation of glutathione adducts for any toxin could be observed, and only for AZA1 could a glucuronidation be identified. All metabolites were confirmed by determination of accurate masses using high resolution mass spectrometry. The mass error between detected ion and the suggested sum formula is given in the respective tables. As enzyme activity was exhaustible and detectable amounts of nonmetabolized educts remained in solution, their accurate mass determination could be used for quality control of analysis and assessment of reliableness of metabolite confirmation. The confirmation by high mass resolution was an effective tool to avoid misinterpretation or identification of false positive results as demonstrated in the following case. All investigated toxins contain between 44 and 55 C atoms. The probability of the carbon composition for molecules with sum formulas of 50 C atoms is 57% pure 12C, 32% one 13C, and the probability of containing two 13C atoms is estimated to be 8.7%.25 Consequently, for a toxin consisting of 50 C atoms, the relative signal intensity of the molecule containing two 13C atoms is 15% as compared to the pure 12C signal, which in most cases is solely monitored in MRM mode. In MRM mode, molecules containing one or two 13 C atoms will not be detected, but, nevertheless, analysts have to be aware that during metabolism, besides, for instance, the addition of oxygen toward a molecule consisting of pure 12C, also a significant amount of mono-oxygenated toxins containing two 13C atoms will be formed. For both the toxin containing none or two 13C atoms, the exact mass increases by 15.99492 amu [oxygen], but due to the mass difference between 12C and 13 C (2 × 1.00335 amu) in MRM mode where the pure 12C signal is monitored, this will be detected as a mass increase of 18 amu and could be interpreted as the addition of H2O to a pure 12 C molecule (2 × 1.00783 [hydrogen] + 15.99492 [oxygen] ) 18.01057). The mass difference between the addition of water to a pure 12C molecule and the addition of oxygen to a molecule containing two 13C atoms is only of 0.00894 amu and can only be distinguished by mass spectrometers providing a mass resolution of 50.000 or higher. Identification of Phase I and Phase II Metabolites. DSP Toxins. For OA, two metabolic reactions, a single addition of oxygen and a single addition of oxygen with a parallel loss of H2, could be detected (Table 1). The addition of oxygen can be assigned to a formation of either an epoxide or a hydroxylation. In most cases, epoxides are unstable and tend to form diols after addition of water.26 The formation of diols could not be observed for OA metabolites, and OA does not contain structures as aromatic ring systems suitable to stabilize epoxides either. Thus, it is very likely that the addition of oxygen can be assigned to a hydroxylation. Because of chromatographic separation prior to MS detection, eight isomers (25) von Ardenne, M.; Steinfelder, K.; Tuemmler, R. Naturwissenschaften 1960, 47, 492–493. (26) Choi, W. J.; Lee, E. Y.; Yoon, S. J.; Yang, S. T.; Choi, C. Y. J. Biosci. Bioeng. 1999, 88, 339–341.

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Table 1. Phase I in Vitro Metabolites of OA, DTX1, and DTX2 Identified by Combining Triple Stage Quadrupole and Exact Mass Spectrometry toxin and metabolic reaction [OA-H]+1×O +1×O

+O-H2 [DTX1-H]+1×O +1×O +O-H2 [DTX2-H]+1×O +1×O

rel. Rt

transition

sum formula of proposed ion

error (pmm)

1.00 0.92 0.89 0.91 0.87 0.86 0.84 0.81 0.79 0.96 1.00 0.91 0.91 0.90 0.85 0.97 1.00 0.98 0.97 0.93 0.91 0.89 0.88 0.86 0.84 0.83 0.81 0.77 0.76

803.5f255.1 819.5f271.1 819.5f271.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1 817.5f269.1 817.5f255.1 833.5f271.1 833.5f255.1 833.5f255.1 833.5f255.1 831.5f269.1 803.5f255.1 803.5f255.1 803.5f255.1 819.5f271.1 819.5f271.1 819.5f271.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1 819.5f255.1

C44H67O13 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H65O14 C45H69O13 C45H69O14 C45H69O14 C45H69O14 C45H69O14 C45H67O14 C44H67O13 C44H67O13 C44H67O13 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14 C44H67O14

2.15 2.09 2.23 2.09 2.23 2.17 a 2.22 a 2.33 0.67 1.48 1.48 1.06 0.41 1.39 0.45 0.55 0.51 0.88 0.50 a 0.78 0.88 0.56 0.98 0.74 a 0.71

a Too low sensitivity for determination of exact mass in high resolution scan mode.

of monohydroxylated OA metabolites could be determined. As these isomers were chromatographically separated by C18 material, these isomers can be assumed to be positional or stereo isomers rather than enantiomers. Fragmentation of OA has been studied intensively16,18 so that the recorded product ion spectra of OA allowed one to subdivide the structure of OA into fragments I, II, and III (Figure 2c). The monohydroxylated isomers were investigated by generating enhanced product ion spectra. The respective chromatogram of m/z 819 (OA+O) in Figure 2a revealed several peaks for which two different types of fragmentation patterns could be observed. For analytes eluting between Rt ) 8.98 and 9.73 min (Figure 2b), the same product ions as for OA were detected, indicating that the oxygen atom had to be added at fragment III, which had left the molecule as neutral. In such a case, no clear statement can be made, and, consequently, when it comes to determining the position in the molecule where the metabolic reaction took place, no conclusions could be drawn. The two analytes eluting between Rt ) 10.20 and 10.49 min (Figure 2b) exhibited product ions with a mass increase of 16 amu as compared to OA fragments (Figure 2b, Rt ) 11.21 min). As for both metabolites, the product ion of m/z 271 instead of m/z 255 was detected, and the oxygen atom had to be added at fragment I. The intensity of the both MS3 spectra of m/z 255 and 271 was not high enough to allow further interpretation and successful structure elucidation. Enhanced product ion spectra were also recorded for the two isomers for which an oxygen addition with a parallel loss of hydrogen was

Table 2. Phase I in Vitro Metabolites of YTX Identified by Combining Triple Stage Quadrupole and Exact Mass Spectrometry toxin and metabolic reaction [YTX-H]+1×O

rel. Rt

transition

1.00 1141.5f1061.5 0.90 1157.5f1077.5

sum formula of error proposed ion (pmm) C55H81O21S2 C55H81O22S2

-0.26 -0.29

Table 3. Phase I and Phase II in Vitro Metabolites of AZA1 Identified by Combining Triple Stage Quadrupole and Exact Mass Spectrometry toxin and metabolic reaction

rel. Rt

transition

sum formula of proposed ion

error (pmm)

[AZA1+H]+

1.00 1.03 1.06 0.92 0.95 0.94 0.86 0.85 0.83 0.81 0.78 0.75 0.73 0.74 0.71 0.65 0.56 0.96 0.71

842.5f824.5 842.5f824.5 842.5f824.5 1018.5f848.5 1018.5f848.5 858.5f824.5 858.5f824.5 858.5f824.5 858.5f824.5 858.5f824.5 858.5f824.5 874.5f856.5 874.5f856.5 890.5f872.5 890.5f872.5 890.5f872.5 890.5f872.5 840.5f822.5 840.5f822.5

C47H72O12N C47H72O12N C47H72O12N C53H80O18N C53H80O18N C47H72O13N C47H72O13N C47H72O13N C47H72O13N C47H72O13N C47H72O13N C47H72O14N C47H72O14N C47H72O15N C47H72O15N C47H72O15N C47H72O15N C47H70O12N C47H70O12N

1.02 1.07 0.83 0.98 0.24 1.20 0.13 -0.60 1.14 1.70 1.15 0.31 1.25 1.07 1.38 0.99 1.38 1.14 1.95

+(C6H8O6) +1×O

+2×O +3×O

-H2

Figure 2. (a) Enhanced product ion chromatograms of m/z 803.5 (OA) and m/z 819.5 (OA+1×O). Respective spectra are shown under (b). Whereas metabolites eluting between Rt ) 8.98 and 9.73 exhibited comparable fragmentation patterns, which are exemplarily shown in the middle (red), spectra for analytes between Rt ) 10.20 and 10.49 are shown in the back (blue). (c) Structure of DSP toxins and main fragments under ESI negative conditions are included to localize metabolic reaction.

observed (refer to Table 1). For both of these metabolites, the transformation could be assigned to fragment I. This metabolic reaction can be related either to the oxidation of an alkyl moiety to a carbonyl group or to the oxidation of an alcohol to a carboxyl group.14 Guo et al.11 detected four metabolites after incubation of OA with nine human recombinant CYPs as well as pooled human liver microsomes and identified three of them as monohydroxylated OA and one as ketone. By chemical interconversion and by applying tandem mass spectrometry, the authors proposed that two of the monohydroxylated metabolites as well as the ketone are located at fragment I, precisely at the C 11 position of OA, and one monohydroxylated metabolite has to be located at fragment III. Although we used a lower toxin concentration for the S9-mix experiments as compared to Guo et al., a higher number of isomers could be detected. This could be explained by a different chromatographic separation or an improved sensitivity in MRM mode (after SPE enrichment) as compared to the

scan mode used there. Besides, it may be assumed that the rat S9-mix contained a larger number of CYP isoforms, which could have been involved in OA metabolism, as compared to those investigated by Guo et al. Yessotoxin. For YTX, only one metabolite, a single addition of oxygen, could be detected (Table 2). Concerning the reactive part of the molecule, no restrictions could be made, and no conclusion could be drawn as during the fragmentation of YTX only the loss of SO3 could be monitored. Referring to the structure of YTX, especially due to the partially unsaturated side chain, more than one metabolite should be expected. However, with the response of YTX in the mass spectrometer being generally weak, the absence of further metabolites might be explained by poor sensitivity for metabolites formed in low concentrations. AZA1. For AZA1, different metabolic reactions like a single, a double, or a triplicate addition of oxygen could be detected (Table 3). Enhanced product ion spectra were recorded for AZA1 and the mono-oxygenated isomers (Figure 3). As compared to the AZA1 product ion spectrum, all product ions detected for AZA1+O are increased by 16 amu, except for the product ion at m/z 168. This fragmentation pattern revealed the oxygen to be located at that part of the molecule between fragment ion of m/z 362 and m/z 168 (Figure 3b,c). For a more precise localization, a MS3 spectrum of m/z 378 was recorded for AZA1+O (Figure 3c1) and compared to the MS3 spectrum of m/z 362 of AZA1 (Figure 3b1). The MS3 spectrum of AZA1+O exhibited m/z 278, which is increased by 16 amu as Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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Table 4. Phase I in Vitro Metabolites of PTX2 Identified by Combining Triple Stage Quadrupole and Exact Mass Spectrometry toxin and metabolic reaction

rel. Rt

transition

sum formula of proposed ion

error (pmm)

[PTX2+NH4]+ +1×O +2×O

1.00 0.90 0.88 0.80 0.65 0.65

876.5f823.5 892.5f839.5 908.5f855.5 908.5f855.5 908.5f855.5 924.5f871.5

C47H74O14N C47H74O15N C47H74O16N C47H74O16N C47H74O16N C47H74O17N

1.35 -3.62 1.66 2.56 2.51 1.78

+3×O

fragmentation known from AZA1. In those cases, no conclusions from product ion spectra could be drawn; however, the recording of precursor ion spectra proved to be helpful. Therefore, the precursor ion spectrum of m/z 672 was recorded and revealed m/z 672 to be a product ion of m/z 1018 (AZA1Gluc) instead of m/z 842 (AZA1), which confirmed the glucuronic acid to be bound at C1 via an ester linkage. PTX2. For PTX2, a mono, double, and triplicate addition of oxygen could be identified (Table 4). For the mono-oxygenation, a single isomer with high intensity could be detected. Fragmentation of PTX2 was already intensively investigated by Gerssen et al.16 We recorded enhanced product ion spectra of PTX2 and the mono-oxygenated metabolite (shown in Figure 4b). Labeled product ions can be assigned to certain parts of the PTX2 structure (Figure 4a). For PTX2, product ions of m/z 551 and m/z 439 were detected, whereas for the mono-oxygenated metabolite, both product ions with a 16 amu mass increase could be detected instead. In the further fragmentation process, m/z 551 is fragmented to m/z 213 and m/z 439 to m/z 275. Both fragments were Figure 3. Structure of AZA1 and main fragments under ESI positive conditions (a). Enhanced product ion spectra of m/z 842 (AZA1) are shown in black (b), while the enhanced product ion spectra of m/z 858 (AZA1+1×O) are shown in red (c). MS3 spectra of m/z 362 (b1) and m/z 378 (c1) are included. Pale blue marks denote the molecule part to which the oxygen is added during oxidative phase I reaction.

compared to m/z 262 of AZA1. Thus, the mono-oxygenation can be restricted to that part of AZA1 that is marked in pale blue (Figure 3a). Furthermore, a loss of hydrogen could be detected for two isomers. This reaction can be explained either by the oxidation of an alcohol to a carbonyl group or by desaturation or by a ring closure.27 A conjugation of glucuronic acid by UDP-glucuronyl transferase could be observed for two isomers of AZA1. As glucuronic acid has to be linked to AZA1 via a carboxylic or an alcoholic group, this linkage can a priori be limited to C1, C20, or C21. For these metabolites, product ion spectra of m/z 1018 were recorded, which exhibited a loss of glucuronic acid in a first step and subsequently the same fragmentation pattern with the same fragments as observed for AZA1. As both the ester and the ether glucuronides are very fragile and easily prone to fragmentation,28 it could not be excluded that the glucuronic acid is fragmented at first and followed by a (27) Guo, J.; Nikolic, D.; Chadwick, L. R.; Pauli, G. F.; van Breemen, R. B. Drug Metab. Dispos. 2006, 34, 1152–1159.

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Figure 4. Structure of PTX2 and main fragments under ESI positive conditions (a). In the front (b), the enhanced product ion spectra of m/z 876 (ammonia adduct of PTX2), and in red (c), enhanced product ion spectra of m/z 892 (ammonia adduct of PTX2+1×O) are shown. Pale blue marks denote the molecule part to which the oxygen is added during oxidative phase I reaction.

also detected as product ions of PTX2+O. Thus, the monooxygenation can be localized in the part of the molecule marked in pale blue (refer to Figure 4a). CONCLUSIONS The combination of MS strategies proved to be successful for the identification of phase I and phase II metabolites of lipophilic marine biotoxins. A total number of 47 metabolites from six toxins could be identified by in vitro studies using the S9-fraction of rat liver and supplement of cofactors for which the elemental composition was confirmed by high resolution mass spectrometry. The combination of mass spectrometric features such as spectra of product and precursor ions as well as MS3 spectra allowed for a specific designation of the carbon atom to which the functional group is attached or at least for a restriction to a certain part of within the molecule structure. For a full structure elucidation, the isolation of metabolites, followed by the application of NMR spectroscopy, would be necessary. All investigated toxins were found to be converted into oxygenated phase I metabolites, which varied in the number of individual isomers. A second and a third addition of oxygen could (28) Yan, Z. Y.; Caldwell, G. W.; Jones, W. J.; Masucci, J. A. Rapid Commun. Mass Spectrom. 2003, 17, 1433–1442. (29) Martignoni, M.; Groothuis, G. M. M.; de Kanter, R. Expert Opin. Drug Metab. Toxicol. 2006, 2, 875–894. (30) Silva, J. M.; Morin, P. E.; Day, S. H.; Kennedy, B. P.; Payette, P.; Rushmore, T.; Yergey, J. A.; Nicoll-Griffith, D. A. Drug Metab. Dispos. 1998, 26, 490– 496.

be observed for PTX2 and AZA1, whereas for AZA1, also a loss of hydrogen was identified. In terms of phase II reactions, only the conjugation of glutathione and the glucuronidation were investigated. No formation of glutathione adducts for any toxin could be detected. A glucuronidation could only be observed for AZA1 but for no other investigated toxin. To which extent these results can be transferred to humans cannot be clarified. Main reactions and metabolites are comparable between species, but differences within species can lead to different susceptibility to xenobiotics.29,30 The use of rat S9-mix revealed the potential of lipophilic marine toxins to be metabolized and encourages further research. Experiments with pooled human liver microsomes have only been published with respect to OA.11 With regard to sum formulas and MS fragmentation patterns, we observed the same metabolic reactions like the addition of oxygen and the addition of oxygen with a parallel loss of hydrogen for OA incubated with liver preparations from rats treated with phenobarbital and β-naphthoflavone. The identification of in vitro metabolites provides a first step toward a more substantiated knowledge regarding intake, transformation, and excretion of this naturally occurring toxin group, which has serious effects on human health.

Received for review July 13, 2010. Accepted October 4, 2010. AC101864U

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