Anal. Chem. 2006, 78, 3342-3347
Determination of Salsolinol and Related Catecholamines through On-Line Preconcentration and Liquid Chromatography/Atmospheric Pressure Photoionization Mass Spectrometry Jason A. Starkey,† Yehia Mechref,† Jan Muzikar,† William J. McBride,‡ and Milos V. Novotny*,†
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, and Department of Psychiatry, Indiana University, Indianapolis, Indiana 46202
A new analytical approach has been developed for simultaneous measurements of endogenous salsolinol and major catecholamines in brain tissue of experimental animals. This procedure involves a combination of online phenyl boronate affinity preconcentration and microcolumn liquid chromatography, followed by mass spectrometry equipped with an atmospheric pressure photoionization (APPI) source. Flow conditions of the APPI source were optimized for detection sensitivity while different dopants were evaluated. The on-line preconcentration was found essential for the sensitivity requirements of salsolinol measurements in the brain tissue from alcohol-preferring rats subjected to different levels of alcohol exposure. Various brain functions are involved with the metabolic pathways of catecholamines. Whereas some catechol derivatives (e.g., dopamine) serve important roles as neurotransmitters involved in neuronal pathways,1 their metabolically related compounds may also be mediators of other important biochemical processes and are, thus, widely considered as biomarkers of certain disease states.2-4 One example has been salsolinol, presumably a key factor in alcoholism induction, which is thought to be derived through a nonenzymatic condensation of acetaldehyde and dopamine via the Pictet-Spengler mechanism.5
Several attempts have been made to confirm the association of salsolinol and alcoholism through its quantification in specific * Corresponding author. Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405. Tel: 812-855-4532. Fax: 812-855-8300. Email:
[email protected]. † Indiana University, Bloomington. ‡ Indiana University, Indianapolis. (1) Melis, M.; Spiga, S.; Diana, M. In International Review of Neurobiology; Bradley, R. J., Harris, R. A., Jenner, P., Eds.; Elsevier Academic Press: San Diego, 2005; Vol. 63, pp 101-154.
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regions of the brain that are known to be involved in the induction of addictive behaviors. Many of these attempts focused on the nucleus accumbens, a brain structure involved in motivational and emotional behaviors as well as memory processes.6-8 Current interest in this anatomically heterogeneous structure originates from the fact that it has been described as a functional interface between the limbic and motor systems and is believed to be primarily responsible for the induction of alcoholism in the rat where the reinforcement of salsolinol becomes possible.9 Recently, McBride and co-workers have examined the endogenous formation of salsolinol in the rat lines selectively bred for disparate alcohol consumption.9 Salsolinol has been formed in both alcoholic and nonalcoholic subjects, thus suggesting the existence of normal endogenous salsolinol sources. They have discovered, however, that the endogenous levels of salsolinol in the nucleus accumbens of alcohol-preferring rats are lower, leading support to the hypothesis that lower levels of salsolinol may be associated with innate high preference for alcohol. Accordingly, additional studies to measure the levels of salsolinol following different alcohol exposures are deemed necessary to determine the possible reinforcing effect of salsolinol in the nucleus accumbens, among the other regions of brain. However, such measurements are analytically challenging due to the very low levels of salsolinol in brain tissue and interferences originating from a very complex biological matrix. Salsolinol analytical methodologies can benefit from a very extensive set of bioanalytical techniques and tools developed for detection of catecholamines in biological fluids and tissues. Traditionally, liquid chromatography (LC) with electrochemical detection has been used for this type of analysis due to its excellent detection limits.10-12 However, the inability to positively identify the detected compounds, or verify a peak identity, in a (2) Kågedal, B.; Goldstein, D. S. J. Chromatogr. 1988, 429, 177-233. (3) Roden, M. Wien. Klin. Wochenschr. 2002, 114, 246-251. (4) Yamada, S.; Urayama, A.; Kimura, R.; Watanabe, H.; Ohashi, K. Life Sci. 2000, 67, 3051-3059. (5) Sandler, M.; Glover, V.; Armando, I.; Clow, A. Prog. Clin. Biol. Res. 1982, 90, 215-226. (6) Schacter, G. B.; Yang, C. R.; Innis, N. K.; Mogenson, G. J. Brain Res. 1989, 494, 339-349. (7) Le Moal, M.; Simon, H. Physiol. Rev. 1991, 71, 155-234. (8) Seamans, J. K.; Phillips, A. G. Behav. Neurosci. 1994, 108, 456-468. (9) McBride, W. J.; Murphy, J. M.; Ikemoto, S. Behav. Brain Res. 1999, 101, 129-152. 10.1021/ac051863j CCC: $33.50
© 2006 American Chemical Society Published on Web 04/08/2006
complex biological extract is a serious limitation. To circumvent these problems, various approaches have been developed to convert polar catechols to their volatile derivatives for gas chromatographic/mass spectrometric (GC/MS) analysis.13,14 Although the GC/MS methods principally offer the sensitivity and selectivity for positive identification together with low-level quantification, they introduce new challenges of their own: a need for multiple derivatization steps with multifunctional solutes, variation in sample injection reproducibility, and analyte instability at the elution temperatures.15 Therefore, it seemed most logical to develop a method in which the selectivity of mass spectrometry could be utilized without the need for prior sample derivatization. In this paper, we present a novel approach to the measurement of salsolinol, together with a profile of related metabolites, which substantially minimizes sample handling and the losses that are commonly associated with sample preparation/derivatization. Its sensitivity and selectivity are secured through the use of an iontrap mass spectrometer provided with an atmospheric pressure photoionizaton (APPI) source. This measurement system is coupled to a low-flow (microcolumn) LC, which is, in turn, preceded by a concentration precolumn. A brain tissue extract is introduced first onto this precolumn, followed by a chromatographic separation of catecholamines and their MS detection and measurement. When compared to other ionization alternatives (e.g., electrospray ionization, ESI), APPI is well-suited for the structural features of catechol derivatives, that is, their aromaticity, provided a suitable eluant dopant is being utilized. To optimize the detection conditions, certain fundamental parameters of APPI (flow rate and mobile-phase composition) were briefly investigated together with analytical reproducibility and limits of detection (with and without preconcentration). Finally, the analytical approach has been validated through the determinations of salsolinol levels in brain tissues. EXPERIMENTAL METHODS Chemicals. Acetone, anisole, toluene, formic acid, dopamine, salsolinol, epinephrine, norepinephrine, 3,4-dihydroxybenzylamine, and ammonium bicarbonate were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Norsalsolinol was obtained from Acros Organics (Morris Plains, NJ). Tetrahydrofuran and 0.1 N hydrochloric acid were products of Fisher Scientific (Fairlawn, NJ). HPLC-grade acetonitrile was acquired from EMD Chemicals, Inc. (Gibbstown, NJ). A Milli-Q water purifying system (Millipore Corp, Bedford, MA) was utilized to generate 18.2-MΩ deionized water. Liquid nitrogen was purchased from Airgas, Inc. (Radnor, PA). Absolute ethanol was received from AAPER Alcohol and Chemical Co. (Shelbyville, KY). Preparation of Standard Stocks Solutions and Sample Extraction. Standard stock solutions were prepared in a 0.1% formic acid solution to assist dissolution and minimize oxidation (10) He, H.; Stein, C. M.; Christman, B.; Wood, A. J. J. J. Chromatogr., B 1997, 701, 115-119. (11) Raggi, M. A.; Sabbioni, C.; Nicoletta, G.; Mandrioli, R.; Gerra, G. J. Sep. Sci. 2003, 26, 1141-1146. (12) Sabbioni, C.; Saracino, M. A.; Mandrioli, R.; Pinzauti, S.; Furlanetto, S.; Gerra, G.; Raggi, M. A. J. Chromatogr., A 2004, 1032, 65-71. (13) Haber, H.; Haber, H. M.; Melzig, M. F. Anal. Biochem. 1995, 224, 256262. (14) Haber, H.; Stender, N.; Mangholz, A.; Ehrenreich, H.; Melzig, M. F. J. Chromatogr., B 1999, 735, 299-303. (15) Raffaelli, A.; Saba, A. Mass Spectrom. Rev. 2003, 22, 318-331.
Figure 1. Flowchart summarizing the steps involved in sample preparation.
of samples. These solutions were stored at -70 °C until use (for a maximum of 1 week). The standards utilized for construction of calibration curves were diluted to the proper concentration with water prior to injection. Young adult male rats from the selectively bred alcoholpreferring (P) line were used in this study. This selectively bred rat line was generated and is maintained at the School of Medicine Animal Facility, Indiana University, Indianapolis, IN. Rats were housed individually in a climate-controlled vivarium and maintained on a 12:12 reverse light cycle (lights off at 0900 h) with food and water provided ad libitum. Rats were killed by decapitation, and the brain tissue was rapidly removed and chilled; the different regions of the brain were hand-dissected, weighed, and stored at -20 °C. Extraction of salsolinol and catecholamines was performed using multiple extraction steps, as summarized in Figure 1. First, the weighed tissue was suspended in 0.5 mL of 0.1% formic acid, and the internal standard was then added prior to homogenization using sonic dismembrator model 550 (Fisher Scientific, Fairlawn, NJ). The internal standard, 3,4-dihydroxybenzylamine, was chosen because of its structural similarity to the catecholamines of interest and because it would be trapped efficiently on the phenylboronic acid (PBA) trapping column. Next, the sample was sonicated on ice for 30 s prior to the addition of 0.5 mL of cold (-20 °C) ethanol to “crash” proteins. The sample was then incubated in the freezer for 20 min prior to centrifugation for 15 min at 4 °C, at the end of which the supernatant containing salsolinol and catecholamines was collected and dried. Next, the dried sample was resuspended in 0.5 mL of 5% acetonitrile and then applied to a conditioned Waters C18 SPE (Milford, MA) column. The aqueous fraction was passed through a 0.5-µm syringe filter and dried again. The final extract was resuspended in 30 µL of water prior to analysis. On-Line Trapping and Chromatographic Conditions. All analyses were performed on an Agilent 1100 series LC system (Agilent Technologies, Waldbronn, Germany) consisting of a binary pump, temperature-controlled autosampler, isocratic pump, and a 10-port nanovalve. The configuration of the 10-port nanovalve in the load and injection positions is depicted in Figures 2a and b, respectively. For the experiments without trapping (preconcentration), a 0.35-µL aliquot of the sample was loaded directly Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
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Figure 2. On-line trapping flow diagram used to analyze samples containing catecholamines and salsolinol. (A) Sample loading and trapping connections; (B) sample injection and separation and MS analysis connections.
onto the column. For the trapping experiments, an 8-µL aliquot of the sample was preconcentrated on a 20.0 × 0.1 mm homemade trapping cartridge packed with PBA medium acquired from the SPE cartridge, purchased from Varian (Palo Alto, CA). The isocratic pump was utilized at 20 µL/min flow rate to load the sample on the trapping cartridge. The loading process was performed for 5 min. The chromatographic separation was performed on a porous activated graphite column (3 µm, 100 × 0.5 mm) received from Thermo Electron (Bellefonte, PA). A gradient separation was employed at 15 µL/min flow rate using solvents A and B consisting of 3% acetonitrile in 96% water and 1% formic acid versus 3% water in 96% acetonitrile and 1% formic acid, respectively. Samples were separated using a multistep gradient. First, B% was increased from 0 to 2% over 5 min, followed by a 2-12% increase over another 5-min period. A third step followed in which B% was increased from 12 to 17 over 5 min. Finally, the column was washed with 80% B for 5 min and reconditioned with the initial mobile phase for another 5 min. Mass Spectrometric Conditions. The LC system was coupled to an Agilent XCT Plus ion trap mass spectrometer (Agilent Technologies, Waldbronn, Germany). An APPI source was equipped with a krypton discharge lamp, emitting 10.0-eV photons perpendicularly to the vaporized capillary effluent. Nitrogen was used as the nebulizing and drying gas. Initially, the vaporizer temperature was varied to determine the optimal condition (determined to be 335 °C). The drying gas temperature was set to 300 °C at a flow rate of 4 L/min, and the nebulizing gas was set at a pressure of 30 psi. The instrument was operated in the positive ion mode, and the scan range was set to 120-300 m/z with an average count of 5. Ion charge control was enabled with a smart target of 200 000 counts or 200 ms, whichever was attained first. Other parameters were as follows: heated capillary voltage, -1500 V; capillary exit voltage, 120 V; skimmer voltage, 40 V; rolling averaging, value of 1. The LC eluant was mixed with an appropriate dopant using a Harvard Apparatus syringe pump (Holliston, MA) prior to introduction to the APPI source. Animals and Alcohol Drinking Procedure. The procedure for repeated episodes of excessive alcohol drinking was conducted as described previously.16 After the acclimation period, rats received continuous, concurrent free-choice access in the home cage to 10, 20, and 30% (v/v) EtOH and water for at least 6 weeks. The positions of the three EtOH solutions were randomly changed each day, and fluid intake was recorded to the nearest 0.1 g by 3344
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weighing the water and EtOH containers. EtOH intake was calculated on the basis of grams of EtOH per kilogram of body weight per day or grams of EtOHper kilogram per 2 h period (on first reexposure day). At the end of the initial 6-week period, EtOH was removed for 2 weeks. At the end of this 2-week abstinence period, the three EtOH solutions were returned at the onset of the dark cycle (0900 h). ETOH intakes were measured after the first 2 h of reexposure, or after 4 h of reexposure on the first day, for 2 weeks. RESULTS AND DISCUSSION Optimization of APPI Parameters. APPI can be viewed as a technique that is complementary to ESI and atmospheric pressure chemical ionization (APCI) due to its unique ionization mechanism and performance, allowing the possibility of ionization of molecules that are difficult to deal with effectively in the other two sources.15 Although the compounds which were analyzed in this study (see Figure 3 for their structures) are readily ionized in ESI,17,18 APPI is utilized in this study to allow higher sensitivity and lower detection limits as a result of the low background signal typically associated with this ionization source. Moreover, the ionization efficiency in APPI could be substantially enhanced through the use of an appropriate dopant. These factors happen to be critical to analyzing salsolinol in small tissue samples. An obvious advantage of APPI over APCI and ESI is the lack of dependence of the flow rate on the ionization efficiency. APPI is viewed as a mass-flow-sensitive source, as opposed to the other two sources, which are concentration-sensitive. This beneficial feature of APPI is readily seen at flow rates lower than 100 µL/ min, where APCI is not very stable. Recently, interfacing microcolumns (even capillary electrophoresis) to APPI were reported19-21 using a sheath liquid at a flow rate of 25 µL/min. Although there is little or no dependence of the ionization efficiency on flow rate in APPI, there may be significant instrumental factors affecting (16) Rodd-Henricks, Z. A.; Bell, R. L.; Kuc, K. A.; Murphy, J. M.; McBride, W. J.; Lumeng, L.; Li, T.-K. Alcohol.: Clin. Exp. Res. 2001, 25, 1140-1150. (17) Kushnir, M. M.; Urry, F. M.; Frank, E. L.; Roberts, W. L.; Shushan, B. Clin. Chem. 2002, 48, 323-331. (18) Chan, E. C. Y.; Ho, P. C. Rapid Commun. Mass Spectrom. 2000, 14, 19591964. (19) Nilsson, S. L.; Andersson, C.; Sjo ¨berg, P. J. R.; Bylund, D.; Petersson, P.; Jo ¨rnte´n-Karlsson, M.; Markides, K. E. Rapid Commun. Mass Spectrom. 2003, 17, 2267-2272. (20) Mol, R.; de Jong, G. J.; Somsen, G. W. Electrophoresis 2005, 26, 146-154. (21) Mol, R.; de Jong, G. J.; Somsen, G. W. Anal. Chem. 2005, 77, 5277-5282.
Figure 4. Ionization efficiency as a function of eluant flow rate. The curves reflect a fixed infusion of 33 ng/µL of each solute at a rate of 1 µL/min, whereas the solvent flow rate was varied. (1) Norsalsolinol, (2) salsolinol, and (3) 3,4-dihydroxybenzylamine.
of the lamp used in the APPI source. After ionization of the dopant (1a), an intermediate is formed that could react with the analytes via a proton transfer (1b) or charge exchange (1c) mechanism.15 Figure 3. Chemical structures of the compounds analyzed in this study.
D + hν f D+ +
the vaporization process. In this work, the influence of flow rate on signal-to-noise ratios was investigated by varying the flow rate at a constant sample flux. This was achieved through using two pumps, one responsible for delivering a fixed sample amount, and the other pump being varied to reflect the change in total flow. As illustrated in Figure 4, we see some effect on the signal intensity as the flow rate is varied; accordingly, optimal flow rate of ∼12.5-µL/min was utilized for the remainder of this study. Next, the influence of the nebulizer temperature on the signal intensity was investigated. Three compounds were chosen to cover the mass range of interest in the analysis of endogenous catecholamines; namely, the internal standard 3,4-dihydroxybenzylamine (m/z 123), salsolinol (m/z 180), and tetrahydropapaveroline (m/z 288). It is well-known that the vaporization temperature affects the desolvation process, since higher temperatures generally lead to a more efficient evaporation. Additionally, an increase in optimal ionization temperature is dependent on the polarity of analytes. Accordingly, higher temperatures are needed to desolvate highly polar compounds. Since salsolinol is the main target compound, a temperature of 335 °C was determined experimentally to provide optimum results (data not shown). The use of an appropriate dopant is another factor that may substantially influence the analyte detection. All solvents that are sprayed orthogonally to the lamp in APPI are subject to ionization, including additives to the mobile phases used in chromatographic separations. Certain additives are commonly doped into the postcolumn effluents to enhance signal intensities for analytes.15,22-24 Dopants are added to the column effluent mixture in relatively large amounts to increase the number of ions. Selection of a dopant is based on its ionization potential targeted to fall below that
+
D + M f MH + D[- H] +
+
D +MfM +D
(1a) (1b) (1c)
The krypton lamp emits radiation at 10 eV; the ionization potentials of the dopants investigated here are below this energy. Several commonly used dopants were investigated to determine the most appropriate one for catecholamines and salsolinol. These included acetone, toluene, anisole, and tetrahydrofuran. The percentages of each as a component of the eluant were varied, and the optimal composition is deduced from Figure 5. It was clear that acetone at 7.5% provided the greatest sensitivity relative to the other dopants as well as the situation when no dopant was employed. Toluene and tetrahydrofuran increased the sensitivity as compared to no dopant used, whereas anisole adversely influenced the results. Although 7.5% acetone addition increased substantially the detection sensitivity of our analytes, there was a need to determine its optimum percentage. The optimal percentage of acetone was determined by varying this solvent relative to the mobile-phase flow rate. The variation in the signal intensity as a function of acetone percentage is shown in Figure 6. It appears that 7.5% is the optimal percentage of dopant. Values higher than 7.5% decreased the signal intensity and increased the background noise. Reproducibility of PBA On-Line Trapping of Catecholamines. Theoretically, a desirable injection volume for the small (22) Hsieh, Y.; Merkle, K.; Wang, G.; Brisson, J.-M.; Korfmacher, W. A. Anal. Chem. 2003, 75, 3122-3127. (23) Kauppila, T. J.; Kostiainen, R.; Bruins, A. P. Rapid Commun. Mass Spectrom. 2004, 18, 808-815. (24) Wang, G.; Hsieh, Y.; Korfmacher, W. A. Anal. Chem. 2005, 77, 541-548.
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Table 1. Linearity, Limits of Detection, and Precision for Standard Solutions of Catecholamines with and without On-Line Trapping through Packed PBA Cartridges compound
Figure 5. Relative photoionization efficiency of analytes for different dopants. A 3.3 ng/µL aliquot of analyte solution infused at a flow rate of 12.5 µL/min while using different dopants at their optimal percentage.
R2 a
slope
LOD (ng)b
epinephrine norepinephrine dopamine norsalsolinol salsolinol
Without PBA Trapping 8.27 1.00 6.29 0.98 9.36 1.00 10.34 0.99 13.25 0.99
1.00 0.50 1.00 0.50 0.25
epinephrine norepinephrine dopamine norsalsolinol salsolinol
With PBA Trapping 9.09 0.96 6.83 0.99 10.13 0.98 10.06 0.97 12.14 0.98
0.05 0.05 0.10 0.05 0.03
a Linear fit was based on a 1/Y weighted curve. b Detection limit is defined as S/N ) 3.
Table 2. Run-to-Run and Cartridge-to-Cartridge Reproducibility for On-Line Trapping Using PBAa run-to-runb
column-to-columnc
analyte
amount (ng)
%RSD
amount (ng)
%RSD
salsolinol epinephrine norepinephrine dopamine
10.01 10.03 9.98 9.72
1.1 2.5 2.4 5.9
9.94 9.99 10.01 9.90
1.9 6.3 2.1 3.7
a A 10-ng aliquot of each analyte was loaded using on-line trapping and analyzed through LC/APPI-MS. b N ) 6. c N ) 3.
Figure 6. Effect of acetone concentration on the relative photoionization efficiency. A 3.3-ng/µL aliquot of analyte solution infused at a flow rate of 12.5 µL/min and different percentages of acetone. Analytes: (1) norsalsolinol, (2) salsolinol, (3) 3,4-dihydroxybenzylamine, and (4) tetrahydropapaveroline.
chromatographic column used in this study has been determined to be 0.35 µL. Such a volume would minimize band-broadening associated with this injection and originate from overloading; however, it is not experimentally easy to inject such a small volume reproducibly for quantification purposes. This problem, however, is easily rectified through on-line trapping and sample enrichment. This approach improves detection through loading the sample onto a selective adsorbent material that isolates the compounds of interest while washing away salts and contaminants. Using a sample enrichment cartridge, we can elute the sample as a very narrow concentration impulse. Moreover, the use of appropriate trapping media minimizes sample preparation through selectively binding the catecholamines. Accordingly, we could increase throughput and minimize sample losses due to excessive sample handling. Due to the high polarity of catecholamines and salsolinol, standard C18 trapping cartridges were not suitable for accomplishing this task. Phenyl boronate-based affinity (PBA) 3346 Analytical Chemistry, Vol. 78, No. 10, May 15, 2006
media packed in trapping cartridges were far more effective in trapping the analytes, permitting the development of a robust methodology for determination of salsolinol and catecholamines at subnanogram levels as a profile. Interaction between phenyl boronate and catecholamines is a pH-mediated complexation in which the vicinal diols of the analytes bind to the boronic acid functional group under slightly alkaline conditions. On the other hand, this binding is reversible at acidic conditions, such as the acidic composition of the mobile phase used in the separation utilized in this study. Effective preconcentration of salsolinol and catecholamines prior to LC/APPI-MS analysis was achieved utilizing PBA trapping cartridges in an on-line format. The use of a ten-port valve (see Figure 2) permitted the direct loading of protein and peptidedepleted biological samples to the PBA trapping column, thus substantially reducing the sample handling and cleanup procedures. Subsequently, preconcentrated salsolinol and catecholamines were then eluted and loaded onto an activated graphite column for a chromatographic separation. Several figures of merit for the trapping and separation process are seen in Table 1, illustrating the improvements offered by trapping the sample. The detection limits for all solutes were significantly improved as a result of on-line trapping. The analytical approach is also robust and highly reproducible. Although the cartridge was repacked after every 60 runs, no noticeable loss in selectivity or trapping efficiency was observed over that span. The %RSD for trapping and injection was determined to be
