Quantitative Determination of Acetylcholine and Choline in

Anal. Chem. 2010, 82, 922–929

Quantitative Determination of Acetylcholine and Choline in Microdialysis Samples by MALDI-TOF MS Markus Persike,*,† Martina Zimmermann,‡ Jochen Klein,‡ and Michael Karas† Cluster of Excellence “Macromolecular Complexes”, Institute of Pharmaceutical Chemistry, and Institute of Pharmacology, Goethe University, Frankfurt, Germany A simple, sensitive, and fast matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) method was developed for the determination of acetylcholine (ACh) and choline (Ch) in microdialysis samples. An optimized dried droplet preparation with the common matrix r-cyano-4-hydroxycinnamic acid (CHCA) was used. Limits of detection [signal-to-noise ratio (S/N) ) 3] of 0.3 fmol/µL for ACh and 20 fmol/µL for Ch were found using standards diluted in artificial cerebrospinal fluid (aCSF). The limit of quantification (LOQ) for ACh was 1 fmol/µL, and excellent linearity (R2 ) 0.9996) was maintained over the range of 1-1000 fmol/µL. Choline was quantified over the range of 0.1-50 pmol/ µL, also with excellent linearity (R2 ) 0.9995). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of ACh and Ch in mouse brain microdialysis samples. The samples were quantified by a calibration curve and also by the method of standard addition. Despite the high salt content of the perfusion fluid (>150 mM), a direct measurement was possible. To the authors’ knowledge, this is the first published method to determine acetylcholine and choline in microdialysis samples by MALDI-TOF MS. The method presented significantly reduces the needed analysis time, as only approximately 10 s/sample is required, and it is also possible to improve the temporal resolution, because only ∼1 µL of sample is needed. The essential neurotransmitter acetylcholine (ACh) is involved in signaling cascades in the peripheral and central nervous system (CNS). In the CNS, for example, ACh plays a major role in functions of memory and learning, attention, and sleep regulation.1 It has been shown that pathological conditions such as schizophrenia; Tourette’s syndrome; and Huntington’s, Parkinson’s, and Alzheimer’s diseases are at least partially caused by cholinergic dysfunction.2,3 For instance, levels of ACh have been shown to * To whom correspondence should be addressed. Address: Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt, Germany. E-mail: [email protected]. † Institute of Pharmaceutical Chemistry. ‡ Institute of Pharmacology. (1) Wessler, I.; Kirkpatrick, C. J.; Racke, K. Pharmacol. Ther. 1998, 77, 59– 79.

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be decreased in the brain of trangenic mice presenting in vivo models of Alzheimer’s disease.4 Microdialysis is a commonly used method for studying extracellular levels of neurotransmitters in in vivo systems.5-7 A probe containing a semipermeable membrane is implanted into the relevant brain region of a living animal such as a mouse or rat. Artificial cerebrospinal fluid (aCSF) is slowly pumped through the probe to extract the low-molecular-weight compounds from the extracellular space. Assessing the release of ACh by means of microdialysis has greatly contributed to the understanding of the physiology, pharmacology, and behavioral roles of cholinergic systems.8 Because of the fast enzymatic hydrolysis of ACh to choline (Ch) by acetylcholinesterase (AChE) in the synaptic cleft, the concentration of ACh is typically in the low nanomolar region, depending on the dialyzed brain area and the surgical technique applied, whereas the concentration of Ch in extracellular fluid is high (low micromolar range).9-12 Therefore, a common method to raise the ACh level in the microdialysis sample is to use an AChE inhibitor, such as neostigmine or physostigmine, in the perfusion fluid.8,13,14 Nevertheless, highly sensitive and selective analytical methods are required to detect and quantify the cholinergic parameters of interest. Because of the high salt concentration (>150 mM) of the aCSF employed for dialysis and the similarity of cationic ACh to salts, a sophisticated chromatographic setup is needed. The analytes ACh and Ch in dialysate are traditionally detected by high-performance liquid chromatography (LC) in conjunction (2) Felician, O.; Sandson, T. A. J. Neuropsychiatry Clin. Neurosci. 1999, 11, 19–31. (3) White, K. E.; Cummings, J. L. Compr. Psychiatry 1996, 37, 188–195. (4) Ikarashi, Y.; Harigaya, Y.; Tomidokoro, Y.; Kanai, M.; Ikeda, M.; Matsubara, E.; Kawarabayashi, T.; Kuribara, H.; Younkin, S. G.; Maruyama, Y.; Shoji, M. Neurobiol. Aging 2004, 25, 483–490. (5) Zielke, H. R.; Zielke, C. L.; Baab, P. J. J. Neurochem. 2009, 109 (1), 24– 29. (6) van der Zeyden, M.; Oldenziel, W. H.; Rea, K.; Cremers, T. I.; Westerink, B. H. Pharmacol. Biochem. Behav. 2008, 90, 135–147. (7) Westerink, B. H. J. Chromatogr. B: Biomed. Sci. Appl. 2000, 747, 21–32. (8) Day, J. C.; Kornecook, T. J.; Quirion, R. Methods 2001, 23, 21–39. (9) Taylor, P.; Radic, Z. Annu. Rev. Pharmacol. Toxicol. 1994, 34, 281–320. (10) Zackheim, J. A.; Abercrombie, E. D. Methods Mol. Med. 2003, 79, 433– 441. (11) Huang, T.; Yang, L.; Gitzen, J.; Kissinger, P. T.; Vreeke, M.; Heller, A. J. Chromatogr. B: Biomed. Appl. 1995, 670, 323–327. (12) Massoulie, J.; Pezzementi, L.; Bon, S.; Krejci, E.; Vallette, F. M. Prog. Neurobiol. 1993, 41, 31–91. (13) Erb, C.; Troost, J.; Kopf, S.; Schmitt, U.; Loffelholz, K.; Soreq, H.; Klein, J. J. Neurochem. 2001, 77, 638–646. (14) Henn, C.; Loffelholz, K.; Klein, J. Naunyn Schmiedebergs Arch. Pharmacol. 1998, 357, 640–647. 10.1021/ac902130h  2010 American Chemical Society Published on Web 01/08/2010

with an enzyme reactor with electrochemical (EC) detection.11 The postcolumn immobilized enzyme reactor, containing AChE and choline oxidase, is needed to generate electrochemically detectable hydrogen peroxide.11 The limit of detection (LOD) for ACh by these traditionally employed LC/EC systems varies between 2 and 50 fmol on column.11,15-17 Another frequently used method to analyze microdialysate is LC coupled to mass spectrometry (MS) that, in turn, does not require any postcolumn modification. The ionization is realized by different techniques such as fast atom bombardment (FAB)18 in the earlier years and later electrospray ionization (ESI)19 or atmospheric-pressure chemical ionization (APCI).20 Compared to the classical LC/EC system, LC/ESI and LC/APCI setups achieve higher sensitivity as well as improved selectivity. Because of this obvious advantage, recently published methods for the analysis of ACh are generally MS-based.19-23 Various modes of chromatographic separation such as C18, SCX, HILIC, or some combination of these materials are used. The LOD varies between 0.2 and 1.4 fmol on column.19-23 However, a problem with LC-based analytics is the relatively poor temporal resolution due to sampling times of up to 20 min. Furthermore, because of the rather lengthy time spans required for the LC analysis of a single sample, the throughput of these systems is restricted to a limited number of samples.19 Recently, some improvements were developed to reduce these problems. Keski-Rahkonen et al. reduced the run time to 3 min with a reverse-phase column and an APCI source to raise the sample throughput.20 However, a 15-µL injection volume was needed, thus still resulting in a relatively poor temporal resolution. In contrast, Shackman et al. used online capillary LC/MS that allowed for a temporal resolution of 2.4 min.24 This experimental setup for collecting microdialysis samples, however, requires that the living animal be very close to the noisy LC/MS instrument. Despite the higher salt and buffer tolerance of matrix-assisted laser desorption ionization (MALDI) compared to ESI or APCI, only the latter two techniques have found widespread acceptance for the analysis of small organic molecules.25 This is due to some obvious limitations of the MALDI technique. First, the use of a large excess of organic matrixes generates intense matrix background in the relevant mass range up to 800 Da. Second, singlelaser-shot MALDI mass spectra show a high fluctuation of ion signals, typically aggravated by the inhomogeneous matrix crystal(15) Ichikawa, J.; Meltzer, H. Y. Brain Res. 2000, 858, 252–263. (16) de Boer, P.; Westerink, B. H.; Horn, A. S. Neurosci. Lett. 1990, 116, 357– 360. (17) Tsai, T. R.; Cham, T. M.; Chen, K. C.; Chen, C. F.; Tsai, T. H. J. Chromatogr. B: Biomed. Appl. 1996, 678, 151–155. (18) Ikarashi, Y.; Itoh, K.; Maruyama, Y. Biol. Mass. Spectrom. 1991, 20, 21– 25. (19) Zhu, Y.; Wong, P. S.; Cregor, M.; Gitzen, J. F.; Coury, L. A.; Kissinger, P. T. Rapid Commun. Mass Spectrom. 2000, 14, 1695–1700. (20) Keski-Rahkonen, P.; Lehtonen, M.; Ihalainen, J.; Sarajarvi, T.; Auriola, S. Rapid Commun. Mass Spectrom. 2007, 21, 2933–2943. (21) Hows, M. E.; Organ, A. J.; Murray, S.; Dawson, L. A.; Foxton, R.; Heidbreder, C.; Hughes, Z. A.; Lacroix, L.; Shah, A. J. J. Neurosci. Methods 2002, 121, 33–39. (22) Uutela, P.; Reinila, R.; Piepponen, P.; Ketola, R. A.; Kostiainen, R. Rapid Commun. Mass Spectrom. 2005, 19, 2950–2956. (23) Lacroix, L. P.; Ceolin, L.; Zocchi, A.; Varnier, G.; Garzotti, M.; Curcuruto, O.; Heidbreder, C. A. J. Neurosci. Methods 2006, 157, 25–31. (24) Shackman, H. M.; Shou, M.; Cellar, N. A.; Watson, C. J.; Kennedy, R. T. J. Neurosci. Methods 2007, 159, 86–92. (25) Cohen, L. H.; Gusev, A. I. Anal. Bioanal. Chem. 2002, 373, 571–586.

lization and cocrystallization of the analyte with the matrix.25 Numerous efforts have been made to solve these problems. One possible approach to reduce the interfering background is to couple MALDI with a triple quadrupole (QqQ) mass analyzer and to take advantage of single reaction monitoring (SRM).26,27 Other possibilities are the use of inorganic matrix species such as metal oxide or graphite powder28,29 or matrix-free approaches involving laser desorption ionization (LDI) from silicon substrates30 or nanostructures.31 However, none of these approaches are widely used, and their analytical performance and assumed advantages compared to conventional MALDI analysis remain to be established.32 The success of a MALDI experiment is highly dependent on the sample preparation. Different dedicated sample preparation protocols, such as vacuum crystallization,33 fast evaporation,34 spray techniques,35 multicomponent matrixes,36 and thin-layer preparations37 have been applied to reduce the problem of inhomogeneous crystallization and cocrystallization of the matrix and the analyte. To compensate for the remaining variations, an internal standard (IS) is a necessity for quantitative analysis.25 On the other hand, MALDI has some intrinsic advantages when compared to ESI, such as the temporal disconnection of chromatography and mass spectrometry. The offline coupling offers a high-throughput potential for MALDI, because the analysis time is not determined by the sample LC elution profile. Another MALDI advantage is the higher salt and buffer tolerance. This allows for the running of alternative LC methods that are ESIincompatible because of the salt and buffer content or even to omit the LC separation.38 The aim of this study was to develop a simple, sensitive, and fast MALDI-TOF MS method that does not require a specific sample purification or derivation for the determination of ACh and Ch in microdialysis samples. Specifically, we also want to show that MALDI MS can generally be used for the determination of small molecules. Moreover, we found that, despite the high salt concentration (>150 mM) of the sample, a cleaning or separation step was not necessary. In fact, MALDI MS is able to directly quantify ACh and Ch from the microdialysis sample. (26) Gobey, J.; Cole, M.; Janiszewski, J.; Covey, T.; Chau, T.; Kovarik, P.; Corr, J. Anal. Chem. 2005, 77, 5643–5654. (27) Hatsis, P.; Brombacher, S.; Corr, J.; Kovarik, P.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2003, 17, 2303–2309. (28) Sunner, J.; Dratz, E.; Chen, Y. C. Anal. Chem. 1995, 67, 4335–4342. (29) Wen, X.; Dagan, S.; Wysocki, V. H. Anal. Chem. 2007, 79, 434–444. (30) Wei, J.; Buriak, J. M.; Siuzdak, G. Nature 1999, 399, 243–246. (31) Woo, H. K.; Northen, T. R.; Yanes, O.; Siuzdak, G. Nat. Protoc. 2008, 3, 1341–1349. (32) Kovarik, P.; Grivet, C.; Bourgogne, E.; Hopfgartner, G. Rapid Commun. Mass Spectrom. 2007, 21, 911–919. (33) Ling, Y. C.; Lin, L.; Chen, Y. T. Rapid Commun. Mass Spectrom. 1998, 12, 317–327. (34) Nicola, A. J.; Gusev, A. I.; Proctor, A.; Jackson, E. K.; Hercules, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 1164–1171. (35) Axelsson, J.; Hoberg, A. M.; Waterson, C.; Myatt, P.; Shield, G. L.; Varney, J.; Haddleton, D. M.; Derrick, P. J. Rapid Commun. Mass Spectrom. 1997, 11, 209–213. (36) Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Anal. Chem. 1995, 67, 1034–1041. (37) Miliotis, T.; Kjellstrom, S.; Nilsson, J.; Laurell, T.; Edholm, L. E.; MarkoVarga, G. Rapid Commun. Mass Spectrom. 2002, 16, 117–126. (38) LeRiche, T.; Osterodt, J.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2001, 15, 608–614.


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EXPERIMENTAL SECTION Materials. The following chemicals were purchased from Sigma-Aldrich Laborchemikalien GmbH, Seelze, Germany: acetylcholine chloride (purity > 99%), choline chloride (>99%), acetylβ-methylcholine chloride (>98%), and choline-d9 chloride (N,N,Ntrimethyl-d9, 98% D enrichment). The artificial cerebrospinal fluid (aCSF) contains 147 mM NaCl, 2.7 mM KCl, 1.2 mM MgCl2, and 1.2 mM CaCl2 (all Fluka, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Neostigmine bromide and scopolamine hydrobromide were purchased from ACROS Organics (Geel, Belgium) and TCI (Tokyo, Japan), respectively. The used MALDI matrix, R-cyano-4hydroxy-cinnamic acid (CHCA), was purchased from Bruker Daltonik GmbH, Bremen, Germany. LC/MS-grade acetonitrile (ACN), trifluoroacetic acid (TFA) (both Carl Roth GmbH & Co KG, Karlsruhe, Germany), and Milli-Q organic-free water (Millipore, Bedford, MA) were used as solvents for the matrix solution. Only solvents of at least LC-grade purity were used for all experiments described in this study. Sample Preparation and MALDI Spotting. All stock solutions, standard solutions, and dilution series were prepared in aCSF. The stock solutions for acetylcholine, choline, acetyl-βmethylcholine, and choline-d9 were prepared at a concentration of 10-2 M. Acetyl-β-methylcholine and choline-d9 were used as internal standards (ISs). The stock solutions were used to prepare the dilution series. ACh was analyzed in the range from 2.5 pmol/µL to 0.5 fmol/µL. To mirror actual in vivo conditions, the Ch concentration of standard samples was prepared as 100 times the ACh concentration. The internal standards were used at constant final concentrations of 100 fmol/µL for acetyl-β-methylcholine and 2.5 pmol/µL for choline-d9. The calibration standard solutions for the microdialysis samples were also prepared with the stock solutions at concentrations of 0, 0.5, 1, 5, 10, 25, 50, 100, 250, 500, and 1000 fmol/µL for ACh and the relevant 100-fold-higher concentrations for Ch. The internal standards were used at the constant final concentrations of 50 fmol/µL for acetyl-β-methylcholine and 5 pmol/µL for choline-d9. The MALDI matrix, CHCA, was prepared at a concentration of 1.5 mg/mL in 80% ACN (v/v) with 0.01% TFA. Each sample was spotted using an optimized dried droplet preparation technique (1 µL of sample, 1 µL of matrix) on a 384-well insert OptiTOF-stainless steel MALDI plate (Applied Biosystems, MDS SCIEX, Foster City, CA). The optimized dried droplet preparation was realized to achieve a homogeneous layer. First, 1 µL of the sample was spotted on the target plate and left to dry completely. In addition, 1 µL of IS (50 fmol/µL acetyl-β-methylcholine and 5 pmol/µL choline-d9) was spotted on the in vivo microdialysis sample and left to dry completely. Shortly thereafter, 1 µL of matrix solution was pipetted onto the dried sample. After the crystals had dissolved completely, the spot was dried under a continuous air stream. The microdialysis samples were additionally analyzed by the method of standard addition. To that end, every sample was spotted four times on the target plate (1 µL each) and left to dry completely. Afterward, all samples were mixed with 1 µL of IS at final concentrations of 50 fmol/µL for acetyl-β-methylcholine and 924


5 pmol/µL for choline-d9. After the spots had dried, standard solutions with final concentrations of 50, 100, and 150 fmol/ µL ACh and the corresponding 100-fold-higher concentrations for Ch were added to one spot each, to accomplish the method of standard addition. Finally, 1 µL of matrix solution was pipetted onto the dried sample spots. After the crystals had dissolved completely, the spot was dried under a continuous air stream. Microdialysis Samples. For the present study, we assessed neurotransmitter release in the striatum of four CD1 mice. CD1 mice (Charles River, Sulzfeld, Germany) were anesthetized with isoflurane (4% induction, 1.5% maintenance) in synthetic air and placed in a stereotaxic frame. I-shaped concentric dialysis probes with an exchange length of 2 mm were constructed as previously described13,39 and implanted into the right striatum using the following coordinates (from bregma): AP, -0.5 mm; L, -2.2 mm; and DV, -3.8 mm. The animals were allowed to recover overnight. Experiments were carried out in the freely moving animal on the following day: Microdialysis probes were perfused with aCSF containing the AChE inhibitor neostigmine (1 µM) at a perfusion rate of 1 µL/min. Dialysate was sampled at intervals of 30 min (animal A1), 15 min (animals A2 and B), and 1 min (animal C) in order to obtain baseline values for a 90-min time span. Subsequently, the dialysis fluid was switched to aCSF containing not only neostigmine, but also scopolamine (100 nM), and the collection was continued for another 90 min, before the fluid was switched back to aCSF containing neostigmine only. Samples were collected for another 3 h. Samples from mice A and B were stored on ice immediately after collection with a fraction collector (Biorad, Munich, Germany) and frozen to -20 °C until analysis was carried out. The samples from mouse C were spotted automatically in intervals of 1 min via MALDI-Spotter (SunCollect, SunChrom, Friedrichsdorf, Germany) onto a 384-well insert Opti-TOF-stainless steel MALDI plate. On day 3, the animals were sacrificed, and their brains were extracted. Following the preparation of 1-mm coronal sections, the correct microdialysis probe location was assessed. The experimental procedures described here were carried out in accordance with the guidelines as set and approved by the responsible government agency (Regierungspra¨sidium Darmstadt, Darmstadt, Germany). Determination of in Vitro Recovery of Microdialysis Probes. Probes were immersed in slowly stirred aCSF containing 1 µmol/L each ACh and Ch and perfused with aCSF at 1 µL/min perfusion rate and room temperature. Samples were taken in either 15- or 1.5-min intervals for a total of 2 h after equilibrium was reached. Recovery was calculated as the average dialysate concentration of ACh and Ch over all samples collected, expressed as a percentage of the initial 1 µmol/L concentration in the stirred aCSF. MALDI MS Experiments. MALDI experiments were performed on a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems, MDS SCIEX, Foster City, CA) equipped with a 200-Hz Nd: YAG laser (355 nm). Mass spectra were acquired automatically in the positive reflector mode between m/z 10 and 600 with fixed laser intensity. Two thousand laser shots per spot were ac(39) Kopf, S. R.; Buchholzer, M. L.; Hilgert, M.; Loffelholz, K.; Klein, J. Neuroscience 2001, 103, 365–371.

cumulated at 50 different spot positions. To avoid the accumulation of blank spectra, all spectra with signal-to-noise (S/N) values below 200 (for each signal) were discarded automatically by the instrument. The operation parameters were optimized for the low-mass region with a delay time of 20 ns. Because of the high salt content of the samples, it was necessary to increase the laser intensity. However, to avoid detector saturation and to improve the spectra, the laser intensity was adjusted manually. The internal calibration that was obtained using matrix signals yielded a mass accuracy below 20 ppm. The MS/MS fragmentation was performed at a collision energy of 2 kV using air as the collision gas at a pressure of 1 × 10-6 Torr. Two thousand laser shots per precursor mass were accumulated at 50 different spot positions. The 4000 Series Explorer V3.5.3 software and the DataExplorer Software V4.8 (both Applied Biosystems/MDS SCIEX, Foster City, CA) were used for operating the mass spectrometer, as well as for data acquisition and processing. Method Validation. All data presented in this work were obtained by averaging three replicates unless otherwise noted. The linear range was assessed by plotting the intensity of the analyte relative to the intensity of the internal standard multiplied by the concentration of IS on the y axis. The concentration of the analyte in solution was plotted on the x axis. Linear regression (R2), relative standard deviations (RSDs), and accuracy were calculated with Microsoft Excel 2007 SP1. Accuracy was calculated by comparing the experimentally determined concentrations of analyzed standard solutions to their nominal values. The mean RSD and the mean accuracy in all measurements were averaged for nonzero concentrations, as well as concentrations that fulfilled the U.S. Food and Drug Administration (FDA) ±15/20 criteria in the quantification range.40 RESULTS AND DISCUSSION The analytics of molecules with low molecular masses (below 800 Da) by MALDI MS is generally not as established as the determination of larger biological molecules such as peptides or proteins. One major challenge is the intense background of matrixion signals, which span the range from matrix-molecule fragment ions to cluster ions up to at least m/z 800. More specifically, significant interference is caused by the CHCA matrix ions (M) [M + H - CO2]+ at m/z 146, [M + H - H2O]+ at m/z 172, and [M + H]+ at 190, as well as the salt adducts [M + Na]+, [M + K]+, and [M + 2Na - H]+ at m/z 212, 228, and 234, respectively. Other distinct matrix signals are the dimeric CHCA ions [2M + H - CO2]+ and [2M + H]+ at m/z 335 and 379, respectively. On one hand, these signals and their strong intensities clearly pose a problem. On the other hand, they can be used for the internal calibration of the instrument, yielding a high mass accuracy below 20 ppm. As a result of this accurate mass measurement and high resolution, the analytes were clearly separated from the interfering matrix background. Even the matrix signal [M + H - CO2]+ at m/z 146.06 was clearly separated from the signal of ACh at m/z 146.12. Figure 1A displays a spectrum obtained for 25 fmol/µL ACh in aCSF. The inset shows that the matrix and analyte signals are clearly (40) Guidance for Industry. Bioanalytical Method Validation. U.S. Department of Health and Human Services; FDA, CDER, CVM, 2001. http://www. fda.gov (accessed Dec 2009).

separated. This reflects the high resolution and mass accuracy of a modern TOF instrument and resulted in a high significance. In fact, as can be gathered from this spectrum, the mass accuracy for ACh was 150 mM), the matrix signals were predominantly cationized; however, the analyte signals showed no cation adducts. Importantly, obstacles to a reliable quantification by means of MALDI were, first, strong fluctuations in signal intensities from spot-to-spot and shot-to-shot quantification, and, second, potential inhomogeneous cocrystallization of the analyte with the matrix. However, those problems were successfully addressed. Specifically, fluctuations in signal intensities were overcome by averaging over a high number of laser shots and working with a large part of the sample spot. This example demonstrates that the relatively high-repetition-rate lasers used in modern MALDI-TOF/ TOF instruments considerably improve the reproducibility of MALDI-TOF mass spectra. To reduce the inhomogeneous cocrystallization of the analyte with the matrix, a fast drying protocol is usually employed that yields a more homogeneous crystal pattern. This optimized dried droplet preparation was already used for the quantification of different small drugs by means of MALDI-TOF MS.41 However, given that samples had to be prepared in 100% aqueous solution so that their composition matched that of dialysates obtained from in vivo setups, fast drying was not possible. To overcome this issue, the following amendments to the protocol were made: First, the sample was spotted and dried completely. Shortly thereafter, matrix solution (80% ACN v/v with 0.01% TFA) was pipetted onto the dried sample. After dissolution was complete, the spot was dried under a continuous air stream. This fast evaporation resulted in more homogeneous matrix/analyte cocrystallization with a smaller crystal size. Taken all together, the increased homogeneity of the matrix layer in combination with an increased number of accumulated laser shots per mass spectrum resulted in markedly minimized variations in signal intensity. Method Validation. Calibration curves for ACh were analyzed in the concentration range of 0.5-2500 fmol/µL using acetyl-βmethylcholine as the IS at a concentration of 100 fmol/µL. Ch was analyzed simultaneously at a 100-times-higher concentration to anticipate conditions expected for in vivo dialysates. Because of this significant difference between the two analyte concentrations, a separate IS for Ch was necessary. Therefore, Ch-d9 was used as the IS at a constant concentration of 2.5 pmol/µL. Generally, isotopically labeled standards present the best option as IS, but they are expensive and frequently unavailable. In the absence of labeled standards, it is common to use structurally analogous compounds. In this study, an isotopically labeled compound and a structurally analogous compound were used. No significant differences in RSD and accuracy between the two IS were noted. Table 1 summarizes the RSDs and accuracies for ACh and Ch in the analyzed concentration range. As expected, the RSD of the concentrations measured increased with decreasing sample concentration. In fact, for a concentration of 10 fmol/µL, the RSD was found to be 7%, and the measurement of 1 fmol/µL produced a deviation of 18.3%. The LOD was calculated in two different ways: (41) Persike, M.; Karas, M. Rapid Commun. Mass Spectrom. 2009, 23, 3555– 3562.


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Figure 1. (A) Mass spectrum of a sample with concentrations of 25 fmol/µL ACh, 100 fmol/µL acetyl-β-methylcholine (IS ACh), 2.5 pmol/µL Ch, and 2.5 pmol/µL choline-d9 in aCSF. The inset displays the clearly separated matrix (M - CO2 + H)+ and ACh peaks. MALDI-matrix signals are labeled with an asterisk (*). (B) Mass spectrum of an in vivo microdialysate sample collected from the right striatum of a CD1 mouse, containing both internal standards (acetyl-β-methylcholine, 50 fmol/µL; Ch-d9, 5 pmol/µL) and the AChE inhibitor neostigmine (1 pmol/µL). MALDImatrix signals are labeled with an asterisk (*). The insets display the MS/MS data for Ch and ACh.

First, the straightforward and often-used method that makes use of the ratio S/N ) 3. Second, the LOD was calculated with the signal of a blank sample plus 3 times the standard deviation of this blank signal. Both methods result in an LOD of 0.3 fmol/µL. The LOQ was determined experimentally and was found to be 1 fmol/µL. Notably, ACh shows an excellent linearity with a correlation coefficient (R2) of 0.9996 in the quantified region between 1 and 1000 fmol/µL. Even in the lower concentration range between 1 and 50 fmol/µL, the R2 value was still 0.9977. Choline also shows a good precision with low RSD values and an excellent linearity (R2 ) 0.9995) in the quantified region between 0.1 and 50 pmol/µL. We verified these results by measuring a dilution series with variable concentration of IS. In this experiment, the IS was present at the same concentration as the analyte. At a concentration of 1 fmol/µL, ACh was still visible, but the IS was not detectable. Hence, it was not possible to quantify this concentration. The results for precision, accuracy, and linearity were also excellent (see Table 1). More specifically, the mean RSDs for ACh and Ch were 3.3 and 2.0, respectively, and the R2 values were 0.999 and 1.000, respectively. The traditionally used method to detect ACh and Ch by LC/ EC has an LOD for ACh ranging from 2 to 50 fmol on column.11,15-17 In contrast, the recently published and more 926


sensitive methods that combine LC and MS have LODs ranging from 0.2 to 1.4 fmol on column.19-23 Importantly, the MALDI MS method has a lower LOD than the LC/EC application but, at the same time, an LOD comparable to that of LC/MS methods without using any chromatographic separation or desalting. In addition, the method as described here enables a high sample throughput because of a very short measuring time of approximately 10 s per sample. Moreover, given that sample amounts of only 1 µL are necessary for analysis, the temporal resolution significantly improves as compared to that of LC applications. Taken all together, these results illustrate the excellent linearity, precision, and accuracy of this method, as well as the general ability of MALDI-TOF systems to serve in the analysis and quantification of small molecules. To rule out the possibility that the results obtained were instrument-specific, the dilution series was also measured using another MALDI-TOF instrument (Voyager-DE STR, Applied Biosystems, Darmstadt, Germany). The results obtained when employing this alternative system confirmed the validity and reproducibility of the method. Although the data collected with the Voyager STR system also showed good precision, accuracy, and linearity, the LOQ with this old instrument was 25 fmol/µL (data not shown). At this point, we wanted to challenge the high

Table 1. Precision, Accuracy, and Linearity as Obtained for the Measurement of ACh and Ch Samples Prepared in aCSF When Using Either Constant or Variable Concentrations of ISa choline conc (pmol/µL)

RSD (%)

acetylcholine accuracy (% error)

conc (fmol/µL)

RSD (%)

accuracy (% error)

250 100 50 25 10 5 2.5 1 0.5 0.1 0.05 linearity (R2)

constant concentration of IS 9.82 -4.26 2500 5.18 -33.83 1000 10.16 -18.02 500 2.25 -18.41 250 1.95 -12.99 100 0.74 0.65 50 0.72 1.89 25 3.93 -12.27 10 1.01 -4.4 5 8.11 15.62 1 8.49 68.91 0.5 0.9995 linearity (R2)

1.39 9.55 1.53 4.78 8.99 6.13 5.01 7.02 15.67 18.32 50.97

66.62 -4.05 2.55 6.65 -4.86 -4.86 14.5 6.06 -1.27 10.75 41.15 0.9996

250 100 50 25 10 5 2.5 1 0.5 0.1 linearity (R2)

variable concentration of IS 0.31 4.14 2500 1.14 1.9 1000 0.64 8.7 500 3.45 9.08 250 3.72 5 100 0.71 -2.24 50 2.1 4.75 25 1.01 0.88 10 3.59 0.51 5 3.01 3 1 1.0000 linearity (R2)

1.76 1.55 7.79 7.85 3.2 1.5 1.27 0.41 4.86 ndb

-1.64 0.98 -9.56 -5.85 -2.27 -8.19 -7.16 -11.83 -8.26 ndb 0.9990

a Choline-d9, 2.5 pmol/µL; acetyl-β-methylcholine, 100 fmol/µL. b Not detectable.

sensitivity, reproducibility, and reliability, as well as the suitability of our newly developed MALDI-TOF method, by assessing in vivo microdialysis samples of an expected low concentration of ACh as is found in the extracellular space of murine striatum. Microdialysate Samples. To this end, we quantified levels of ACh and Ch in microdialysis samples of four CD1 mice, measuring amounts in two different ways. The recoveries of ACh and Ch in the microdialysis probes were 32.6% and 34.6%, respectively. To verifiy that ACh and Ch were analyzed, a microdialysis sample was measured in addition by means of MALDI MS/MS. The assumed ACh signal showed the following reactions in the MS/MS analysis: m/z 146 f 87 (100%) and m/z 146 f 60 (20%). The following mass transitions were monitored for the assumed Ch signal in the MS/MS experiment: m/z 104 f 60 (100%), m/z 104 f 45 (35%), m/z 104 f 58 (30%), and m/z 104 f 104 (20%). These results conform with the data obtained for standard ACh and Ch solutions and are also in agreement with the data from ESI or APCI methods in the literature. The combination of accurate masses and the results of the MS/MS experiment clearly demonstrate that ACh and Ch were analyzed. To provoke physiological changes of ACh and Ch levels that could be detected by means of this newly established method, a standard paradigm was used to enhance ACh release from the striatal system. The muscarinic antagonist scopolamine was employed to boost the release of ACh over a 90-min time span.42 Figure 1B displays a mass spectrum of an in vivo microdialysate sample collected from the right striatum of a CD1 mouse (42) Hartmann, J.; Kiewert, C.; Duysen, E. G.; Lockridge, O.; Klein, J. Neurochem. Int. 2008, 52, 972–978.

containing both internal standards (acetyl-β-methylcholine, 50 fmol/µL; Ch-d9, 5 pmol/µL) and the AChE inhibitor neostigmine. The insets show the MS/MS data for the Ch and ACh signals. Because of the high salt content, it was not possible to detect scopolamine. However, neostigmine with a concentration of 1 pmol/µL generated an intense signal. The spectrum also shows that, in the in vivo sample, no interfering signals appeared. Samples from mice A1 and A2 were measured three times each and quantified by means of a calibration curve (with constant concentrations for the ISs) that was used in the range between 5 and 500 fmol/µL, showing excellent linearity (R2 ) 0.9995) and precision (mean RSD ) 7%). Figure 2 displays the ACh (red line) and Ch (blue line) levels that were measured for the dialysates of both mice A1 and A2 across the microdialysis sampling time quantified using the calibration curve. The upper chart shows the data from mouse A1 with a temporal resolution of 30 min. The first four data points show baseline ACh levels as dialyzed from the right striatum of the CD1 mouse. More specifically, addition of the AChE inhibitor neostigmine to the perfusion fluid yielded an ACh baseline concentration of approximately 30 fmol/µL in the samples. Notably, it was possible to detect ACh in murine striatum without additional neostigmine being used, but the amounts ranged below 1 fmol/µL, so reliable and reproducible quantification was not possible (data not shown). After the addition of the muscarinic antagonist scopolamine to the perfusion fluid, the ACh levels increased by a factor of 4-5. This standard paradigmsadding the competitive muscarinic antagonist scopolamine to the perfusion liquidsis frequently used to provoke a large and transient increase of ACh levels.42 After the original perfusion fluid (not containing any scopolamine) had been restored, the ACh levels slowly but continuously reversed to the original levels. When measuring these samples by means of the method described in the present study, we obtained RSD values all ranging below 15% with a mean of 8.8%. As can be gathered from the graph in Figure 2, the Ch levels oscillated around baseline values throughout the experimental period. Compared to the good precision of the standard solutions, the Ch data exhibited larger error bars. However, the precision is acceptable in that a mean RSD of 9.9% was observed. The ACh and Ch levels as actually measured in the microdialysate samples of mouse A1 are listed in Table 2. The lower chart in Figure 2 shows the data from mouse A2 with a temporal resolution of 15 min. A curve progression similar to that obtained from the assessment of dialysates of mouse A1 was obtained, in that an increase in the ACh levels by a factor of 4 following scopolamine perfusion was observed. However, the absolute values were higher, which is due to interindividual variations between animals as documented in numerous microdialysis studies.43 More specifically, baseline ACh levels ranged between 60 and 90 fmol/µL, and the addition of scopolamine to the perfusion liquid caused an increase of the ACh level to approximately 370 fmol/µL. After the dialysis fluid had been switched back to the original perfusion liquid, a return to baseline levels was observed. Also, the Ch levels oscillated around the baseline throughout the experimental period. However, the (43) Hartmann, J.; Erb, C.; Ebert, U.; Baumann, K. H.; Popp, A.; Konig, G.; Klein, J. Neuroscience 2004, 125, 1009–1017.


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Figure 2. Graphs showing changes in microdialysate levels of ACh (red line) and Ch (blue dashed line) during the sampling time in the right striatum of two CD1 mice. Scopolamine was added to the neostigmine containing aCSF after 90 min for 90 min to boost the release of ACh. Table 2. Concentration and Precision as Obtained for the Measurement of ACh and Ch in in Vivo Microdialysis Samples Collected from the Right Striatum of a CD1 Mouse (Animal A1)a choline

acetylcholine

time (min)

conc (pmol/µL)

RSD (%)

conc (fmol/µL)

RSD (%)

-120 -90 -60 -30 0 30 60 90 120 150 180 210

1.60 1.82 1.70 1.42 1.41 1.37 1.49 1.27 1.55 1.60 1.64 1.45

14.31 8.70 6.62 16.05 3.87 13.95 9.10 0.32 11.26 7.08 10.67 16.56

31.45 30.68 32.09 28.85 37.74 117.13 139.20 101.32 73.99 62.66 58.79 39.84

8.20 6.62 6.48 6.28 10.77 12.75 4.83 13.25 13.70 14.68 1.85 6.78

a

Scopolamine was added after 90 min for 90 min to boost the release of ACh.

precision in this experiment was good in that mean RSDs of 4.1% for Ch and 5.8% for ACh were observed. To validate the results of the actual quantification method (by means of a calibration curve that employs constant concentrations for the IS) as presented so far and, in a second instance, to test whether disturbing compounds were extracted from the brain, dialysate samples were also analyzed by the method of standard addition. A separate animal was dialyzed to obtain samples to be quantified by standard addition and calibration curve. Only a limited amount of sample was available, because, first, the perfusion rate was limited 928


Figure 3. Curve progression representing the ACh and Ch levels determined by the method of standard addition (ACh, dashed blue line; Ch, dashed violet line) and by a calibration curve (ACh, red line; Ch, green line) during the sampling time in the right striatum of a CD1 mouse. Scopolamine was added to the neostigmine containing aCSF after 90 min for 90 min to boost the release of ACh.

to 1 µL/min and, second, the temporal resolution would further decrease if sampling intervals were increased. Because of the “relatively high” sample consumption of both the standard addition method and the simultaneous analysis by calibration curve method, only a single assay was measured using each approach. Thus, no statement regarding the precision can be given. However, the former results of the standard solution and the dialysate samples demonstrate that analyzing ACh and Ch with MALDI-TOF is able to achieve good precision. Figure 3 displays changes in the ACh and Ch levels in mouse B that are comparable to those observed for mice A. The dashed

Figure 4. Graphs showing changes in microdialysate levels of ACh (red line) and Ch (blue line) during the sampling time in the right striatum of a CD1 mouse spotted with a time resolution of 1 min via a MALDI spotter. Scopolamine was added to the neostigmine containing aCSF for 90 min to boost the release of ACh.

lines represent the data obtained from the method of standard addition, whereas the continuous lines represent the data achieved with the calibration curve method. A curve progression similar to that obtained from the assessment of dialysates of mouse A was obtained, in that an increase of ACh levels by a factor of 4 following scopolamine perfusion was observed. However, the absolute values were lower, which is due to interindividual variation between animals as documented in numerous microdialysis studies.43 More specifically, the baseline ACh levels ranged between 10 and 20 fmol/µL, and the addition of scopolamine to the perfusion liquid caused an increase of ACh levels to approximately 75 fmol/µL. After the original perfusion liquid was restored, a slow return to baseline levels was observed. The red continuous curve progression in Figure 3 represents the ACh levels as determined by the calibration curve method. The fact that the results obtained using the method of standard addition are similar to those collected with the calibration curve method demonstrates that no interfering matrix background in the dialysate samples interfered with the measurements. The green and dashed violet lines in Figure 3 display the Ch levels of mouse B. Again, quantitation by means of the calibration curve resulted in nearly the same values as obtained using the method of standard addition. Taken all together, the similarity of the results obtained by the two quantification methods demonstrates that the two methods are equally suitable for the direct quantification of ACh and Ch in microdialysate by MALDI-TOF MS. Furthermore, we demonstrate that no ion-suppressing or -enhancing compounds, which would interfere with the analysis, were extracted from the in vivo dialysates. In fact, the absence of interfering compounds promotes the quantitation with a calibration curve, because it is faster and less sample volume is needed. To improve the temporal resolution, a MALDI spotter was used to spot the dialysates of mouse C onto the target plate at a time resolution of 1 min. Figure 4 displays the ACh (red line) and Ch (blue line) levels that were measured for the dialysates of mouse C across the microdialysis sampling time using the calibration curve for quantification. A curve progression similar to those obtained from the assessment of dialysates of the other mice was

obtained in that an increase of ACh levels by a factor of 5 following scopolamine perfusion was observed. More specifically, the first 75 data points show baseline ACh levels as dialyzed from the right striatum of the CD1 mouse. The addition of the AChE inhibitor neostigmine to the perfusion fluid yielded an ACh baseline concentration of approximately 35 fmol/µL in the samples. The addition of scopolamine to the perfusion liquid caused an increase in the ACh level to approximately 200 fmol/µL. After the fluid had been switched back to the original perfusion liquid, a return to baseline levels was observed. Also, the Ch levels oscillated around the baseline values throughout the experimental period. Additionally, the delivery of neostigmine of mouse C was analyzed. Using acetyl-β-methylcholine as the IS showed that the delivery of neostigmine was constant. A fluctuation of ∼10% in the delivery of neostigmine during the experiment was observed (data not shown). In summary, the curve progression showed the improved temporal resolution of the in vivo dialysate samples. Moreover, we consider the reliability of the data obtained as high, as the reflection of the experimental paradigm in the curve progression is excellent from one data point to the next. CONCLUSIONS The performed studies demonstrate that the quantification of ACh and Ch in microdialysis samples can be achieved using a conventional MALDI-TOF mass spectrometer following a optimized dried droplet preparation protocol and common MALDI matrix. Despite the high salt content in the perfusion fluid (>150 mM), the direct measurement of microdialysate samples was possible. Therefore, MALDI MS presents a highly suitable alternative to LC/EC or LC/MS combinations for the purpose of analyzing ACh and Ch levels in microdialysis samples. The established quantitation strategy yielded excellent linearity, precision, and accuracy. In fact, the LOD and LOQ were 0.3 and 1 fmol/µL, respectively, for ACh and 20 and 50 fmol/µL, respectively, for Ch, where these limits are comparable to or even better than those reported when other methods were used.11,15-17,19-23 Finally, it was shown that no other compounds that are naturally dialyzed from the brain in conjunction with ACh and Ch interfere with the analysis following the method described herein. Taken all together, the method presented here displays the following advantages: First, employing the method as described significantly reduces the analysis time because only approximately 10 s are required per sample. Second, the temporal resolution of physiological alterations following pharmacological or environmental stimuli is improved, as only about 1 µL of sample is needed per data point. Furthermore, given that no LC preparation of the sample is required, the overall handling of the dialysate samples is simplified. To the authors’ knowledge, this is the first published method to determine ACh and Ch in microdialysis samples by MALDITOF MS.

Received for review September 22, 2009. Accepted December 17, 2009. AC902130H


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Quantitative Determination of Acetylcholine and Choline in

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