Simultaneous Determination of Salsolinol Enantiomers and Dopamine

Anal. Chem. 2007, 79, 9166-9173

Simultaneous Determination of Salsolinol Enantiomers and Dopamine in Human Plasma and Cerebrospinal Fluid by Chemical Derivatization Coupled to Chiral Liquid Chromatography/ Electrospray Ionization-Tandem Mass Spectrometry Jeongrim Lee, Bill X. Huang, Zhixin Yuan, and Hee-Yong Kim*

Laboratory of Molecular Signaling, National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, Bethesda, Maryland 20892-9410

A sensitive, specific, and robust method to simultaneously determine enantiomeric salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, SAL), a potential biomarker implicated in alcohol-related neurotoxicity in a stereoselective manner, and its precursor dopamine (DA) has been developed using simple chemical derivatization and chiral separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS). SAL enantiomers and DA were converted to stable pentafluorobenzyl (PFB) derivatives directly from aqueous media. Bulky PFB groups introduced into the SAL structure enabled baseline separation of SAL stereoisomers on a chiral column without cumbersome chiral derivatization to unstable SAL diastereomers. Subsequent analysis by ESI-MS/MS with multiple reaction monitoring (MRM) in the presence of deuterium-labeled internal standards allowed specific detection of both derivatives with a wide dynamic range (SAL, 0.5-5000 pg; DA, 0.02-20 ng). The limit of quantitation assayed in the plasma matrix was below 10 pg for each SAL enantiomer and 100 pg for DA. Both coefficient of variance and error for inter- and intraday measurements in the blank plasma were less than 10% for SAL and DA in the concentration range of 10-4000 pg/mL and 0.1-8 ng/mL, respectively. This strategy enabled routine and specific determination of both SAL enantiomers and DA from 0.5 mL of human plasma and cerebrospinal fluid, which has not been possible using existing methodologies. The involvement of dopamine-derived tetrahydroisoquinoline alkaloids (TIQs), particularly salsolinol (SAL; 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), in the biochemical basis underlying alcoholism has been intensively studied since the discovery of SAL biosynthesis in vitro during ethanol metabolism.1 SAL is found in urine, cerebrospinal fluid (CSF), blood, and brain * Corresponding author. E-mail: [email protected]. Phone: 301-402-8746. Fax: 301-594-0035. (1) Yamanaka, Y.; Walsh, M. J.; Davis, V. E. Nature 1970, 227, 1143-1144.

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of both alcoholic and nonalcoholic subjects.2,3 Several research groups have reported that the SAL concentration in plasma and urine was significantly elevated in chronic alcoholics in comparison to control subjects.4-7 It has also been documented that SAL levels were significantly raised in the rat brain during ethanol intoxication and in the postmortem brains of alcoholic patients.8,9 Moreover, SAL infusion into rat brain ventricles promoted alcohol consumption, suggesting a role of SAL in mediating alcohol addiction.10,11 In addition, SAL as well as its metabolites has been indicated as a neurotoxin involved in Parkinson’s disease in a stereoselective manner.12-14 SAL has two stereoisomeric forms due to a chiral center at the C-1 position. The enantiomers of SAL can be formed through at least two different biosynthetic pathways. Racemic (R/S)-SAL can be formed in vivo by nonenzymatic Pictet-Spengler reaction between dopamine (DA) and acetaldehyde, an active metabolite of ethanol.15 In contrast, condensation of pyruvate and DA followed by enzymatic decarboxylation and reduction can lead to only the (2) Sjoquist, B.; Eriksson, A.; Winblad, B. Prog. Clin. Biol. Res. 1982, 90, 5767. (3) Haber, H.; Winkler, A.; Putscher, I.; Henklein, P.; Baeger, I.; Georgi, M.; Melzig, M. F. Alcohol Clin. Exp. Res. 1996, 20, 87-92. (4) Faraj, B. A.; Camp, V. M.; Davis, D. C.; Lenton, J. D.; Kutner, M. Alcohol Clin. Exp. Res. 1989, 13, 155-163. (5) Collins, M. A.; Nijm, W. P.; Borge, G. F.; Teas, G.; Goldfarb, C. Science 1979, 206, 1184-1186. (6) Rommelspacher, H.; Baum, S. S.; Dufeu, P.; Schmidt, L. G. Alcohol 1995, 12, 309-315. (7) Sjoquist, B.; Magnuson, E. J. Chromatogr. 1980, 183, 17-24. (8) Collins, M. A. Trends Pharmacol. Sci. 1982, 3, 373-375. (9) Matsubara, K.; Fukushima, S.; Fukui, Y. Brain Res. 1987, 413, 336-343. (10) Duncan, C.; Deitrich, R. A. Pharmacol. Biochem. Behav. 1980, 13, 265281. (11) Myers, R. D.; Melchior, C. Pharmacol. Biochem. Behav. 1977, 7, 19-35. (12) Wanpen, S.; Govitrapong, P.; Shavali, S.; Sangchot, P.; Ebadi, M. Brain Res. 2004, 1005, 67-76. (13) Maruyama, W.; Boulton, A. A.; Davis, B. A.; Dostert, P.; Naoi, M. J. Neural Transm. 2001, 108, 11-24. (14) Antkiewicz-Michaluk, L.; Krygowska-Wajs, A.; Szczudlik, A.; Roman´ska, I.; Ventulani, J. Biol. Psychiatry 1997, 42, 514-518. (15) Sandler, M.; Carter, S. B.; Hunter, G.; Stern, G. Nature 1973, 241, 439443. 10.1021/ac0715827 Not subject to U.S. Copyright. Publ. 2007 Am. Chem. Soc.

Published on Web 11/01/2007

(R)-isomer.16 Therefore, determination of endogenous SAL enantiomers and their precursor DA in biological samples is important to reveal the stereoselective biosynthetic origin of SAL and the possibility of SAL as a biomarker in alcoholism or neurodegenerative diseases. SAL enantiomers were first identified by GC and nitrogenphosphorus detection after derivatization with diazomethane and then with N-trifluoroacetyl-L-proyl chloride.17 However, routine analyses of SAL enantiomers from biological matrices have been difficult. A GC/MS method was developed by cumbersome twostep derivatization to diastereomers with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) and (R)-(-)-2-phenylbutyryl chloride as a chiral derivatizing agent.18,19 Although baseline separation of SAL enantiomers was demonstrated, this method used watersensitive derivatization procedures that required evaporation of aqueous solvent to complete dryness, often promoting oxidative degradation of SAL. In addition, the resulting chiral derivatives were unstable, hampering reliable analysis. Chiral HPLC using β-cyclodextrin stationary phases or sulfated β-cyclodextrins as chiral mobile phase additives has also been used in conjunction with electrochemical detection (ECD).20-22 Similarly, determination of SAL enantiomers by HPLC/ECD has been carried out as diastereoisomeric derivatives, after reaction with (S)-1-(1-naphthyl) ethyl isothiocyanate as a chiral derivatizing agent.23 However, poor resolution, specificity, and sensitivity as well as lack of positive identification were still severe limitations in HPLC/ECD methods. Recently, LC/MS methods using electrospray ionization (ESI) tandem MS24 or atmospheric pressure photoionization (APPI) MS25 have been used for SAL analysis without enantiomeric separation. In this study, we demonstrate a new robust analytical technique to determine SAL enantiomers and their precursor DA simultaneously based on chemical derivatization and chiral HPLC/ ESI-tandem mass spectrometry. We devised a direct single-step pentafluorobenzyl (PFB) derivatization scheme in an aqueous media, without extractive alkylation using phase transfer catalysts.26 This procedure allowed high derivatization yields with minimal sample degradation and low chemical background. As stable PFB derivatives, SAL enantiomers were baseline separated on a chiral phase HPLC column. Coupling with ESI-MS/MS analysis in the multiple reaction monitoring (MRM) mode allowed the detection of SAL enantiomers and DA with increased specificity and sensitivity. In the presence of deuterium-labeled internal standards, this approach allowed accurate and reliable quantitative (16) Dostert, P.; Strolin, B. M.; Bellotti, V.; Allievi, C.; Dordain, G. J. Neural Transm. 1990, 81, 215-223. (17) Dostert, P.; Benedetti, M.; Dordain, G. Pharmacol. Toxicol. 1987, 60 (Suppl. 1), 13. (18) Haber, H.; Henklein, P.; Georgi, M.; Melzig, M. F. J. Chromatogr., B 1995, 672, 179-187. (19) Musshoff, F.; Schmidt, P.; Dettmeyer, R.; Priemer, F.; Jachau, K.; Madea, B. Forensic Sci. Int. 2000, 113, 359-366. (20) Deng, Y. L.; Maruyama, W.; Kawai, M.; Dostert, P.; Yamamura, H.; Takahashi, T.; Naoi, M. J. Chromatogr., B 1997, 689, 313-320. (21) Baum, S. S.; Rommelspacher, H. J. Chromatogr., B 1994, 660, 235-241. (22) Stammel, W.; Woesle, B.; Thomas, H. Chirality 1995, 7, 10-19. (23) Pianezzola, E.; Bellotti, V.; Frontana, E.; Moro, E. J. Chromatogr. 1989, 495, 205-214. (24) Song, Y.; Xu, J.; Hamme, A.; Liu, Y.-M. J. Chromatogr., A 2006, 1103, 229234. (25) Starkey, J. A.; Mecherf, Y.; Muzikar, J.; McBride, W. J.; Novotny, M. V. Anal. Chem. 2006, 78, 3342-3347. (26) Davis, B. Anal. Chem. 1977, 49, 832-834.

analysis of enantiomeric (R/S)-SAL and DA in human plasma and CSF. EXPERIMENTAL SECTION Materials and Reagents. (()-Salsolinol hydrochloride (SAL‚ HCl), dopamine hydrochloride (DA‚HCl), N,N′-diisopropylethylamine (DIPEA), and acetaldehyde were purchased from SigmaAldrich (St. Louis, MO). Pentafluorobenzyl bromide (PFBBr) was purchased from Pierce Chemical Company (Rockford, IL). Deuterium-labeled (S)-SAL-d4‚HBr (1′, methyl-d4) and (R)-SAL-d4‚HBr (1′, methyl-d4) were prepared by Cambridge Isotope Laboratories (Andover, MA). Deuterium-labeled 1,1,2,2-d4-DA‚HCl was purchased from Cambridge Isotope Laboratories. Since (S)-SAL and (R)-SAL enantiomers are not separately available, standard solutions of SAL were prepared by dissolving the racemic mixture of (R/S)-SAL‚HCl in methanol. Standard stock solutions of (R/S)SAL‚HCl and DA‚HCl were prepared in methanol at 1 mg/mL in amber test tubes. Internal standard stock solutions were also made in methanol at 100 ng/mL for (S)-SAL-d4‚HBr and (R)-SAL-d4‚ HBr and 1 mg/mL for DA‚HCl. These solutions were stored in the dark at -20 °C until further use. The working internal standard solutions were prepared by further dilution of the stock solutions to 500 pg/mL of each SAL-d4 enantiomer and 500 pg/mL of DAd4. Bond Elute phenylboronic acid (PBA, 100 mg, 1 mL) cartridges were purchased from Varian, Inc. (Palo Alto, CA). The Chiralpak AD-H column was purchased from Chiral Technologies, Inc. (West Chester, PA). Organic solvents were HPLC grade from Burdick & Jackson (Muskegon, MI), and deionized water was obtained using a Milli-Q reagent water purification system (Millipore, Bedford, MA). Sample Preparation. Human plasma samples were collected from healthy volunteers and were either analyzed immediately or stored at -80 °C until the time of analysis. The samples were not subjected to more than three freeze and thaw cycles during which no appreciable degradation of SAL or DA was observed (90% in comparison to the neat derivatization in acetonitrile) with improved consistency. After SPE on a PBA cartridge, the pH of the aqueous solvent (0.1 M HCl/MeOH ) 1:1) eluting SAL and DA was adjusted to 8-8.2. Apparently, at this pH both phenol and amine groups of SAL were deprotonated for efficient alkylation, and yet degradation of SAL, DA, and PFBBr was minimal.27 Reaction for 2 h at 68 °C was found to be optimum for producing fully derivatized tri-PFB-SAL and tetra-PFB-DA. Chromatographic Separation and Mass Spectrometric Detection. The formation of tri-PFB-SAL was identified by GC/ NCI-MS, GC/PCI-MS and HPLC/ESI-MS. The spectrum obtained by GC/NCI-MS contained a peak at m/z 538, corresponding to the loss of PFB radical from the molecular ion [M]•(Figure 2A). Further loss of the PFB group or sequential losses of HF from [M - PFB]- led to the ions at m/z 358 or m/z 518 and 498, respectively. The positive ion spectrum obtained by GC/ PCI-MS contained [M + H]+ at m/z 720 (Figure 2B). The extracted ion chromatograms from GC/NCI-MS analysis indicated that the tri-PFB-SAL was the predominant product, whereas the mono- and di-derivatized forms were the minor components (Figure 2C). Reversed-phase HPLC/ESI-MS and HPLC/ESI-MS/ MS analyses confirmed the results obtained by GC/MS analyses (data not shown). Although the fully derivatized SAL could be detected by GC/ NCI-MS, chiral HPLC/ESI-MS/MS was chosen since separation and elution of tri-PFB-SAL enantiomers from a chiral GC column were problematic. In addition, under the HPLC condition, detection of tri-PFB-SAL by APCI in the negative ion electron capture mode was not as sensitive as the detection by ESI in the positive ion mode. To improve the specificity of the detection, MRM mode was employed. The ESI mass spectra of tri-PFB derivatives of SAL and SAL-d4 contained [M + H]+ ion as the base peaks at m/z 720 and m/z 724, respectively. The CID of [M + H]+ produced structurally characteristic product ions (Figure 3). The product ion spectra of [M + H]+ obtained from SAL derivatives contained major peaks at m/z 181, 210, and 358 for SAL and at m/z 181, 210, and 362 for SAL-d4, respectively. The most abundant product (27) Gyllenhaal, O. J. Chromatogr. 1978, 153, 517-520.

Figure 2. Full scan mass spectra and extracted ion chromatograms obtained by GC/MS. (A) Full scan mass spectrum of tri-PFB-SAL by GC/NCI-MS. The spectrum contained [M - PFB]- along with [M - PFB - HF]- and [M - PFB - 2HF]-. (B) Full scan mass spectrum of tri-PFB-SAL by GC/PCI-MS containing [M + H]+ ion at m/z 720 and [M + C2H5]+ adduct ion at m/z 748. (C) GC/NCI-MS ion chromatograms extracted for the most intense signals of tri- (m/z 518), di- (m/z 358), and mono-PFB-SAL (m/z 178).

Figure 3. ESI-MS/MS product ion mass spectra of [M + H]+ produced from tri-PFB derivatives of (A) SAL and (B) SAL-d4.

ion appeared at m/z 181 corresponding to the PFB fragment ion. The product ions at m/z 358 and 362 were derived from the loss of two PFB groups from the corresponding [M + H]+ ions of SAL and SAL-d4 derivatives, respectively. The product ion at m/z 210, derived from the ring cleavage with one PFB group on the

amine group, was common for SAL and SAL-d4. PFB derivatization allowed baseline separation of SAL enantiomers on a Chiralpak AD-H column using an isocratic IPA/MeOH (3:2) mobile phase. Typical MRM chromatograms of transitions m/z 720 f 181, 210, 358 for (R/S)-SAL and m/z 724 f 181, 210, 362 for (S)-SAL-d4 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 4. Representative ion chromatograms from the PFB derivatives of 50 pg each of (R/S)-SAL and (S)-SAL-d4 acquired in the multiple reaction monitoring (MRM) mode. The SAL derivatives were separated on a Chiralpak AD-H column prior to the mass spectrometric detection.

Figure 5. ESI-MS/MS product ion mass spectra of [M + H]+ produced from tetra-PFB derivatives of (A) DA and (B) DA-d4.

after separation on a chiral column are shown in Figure 4. The MRM chromatograms of derivatized (R/S)-SAL and (S)-SAL-d4 clearly indicated that the (S)-form (retention time, 8.2 min) eluted before the (R)-form (retention time, 12.6 min) and that interconversion between two enantiomers did not occur during analysis. The MRM chromatograms of (R/S)-SAL further indicated that each isomer was detected with approximately 1:1 relative peak area ratio. The SAL-d4 internal standards were stable, and no deuterium/hydrogen exchange was observed in an aqueous medium with a wide range of pH, according to MS and MS/MS analyses (data not shown). Although monitoring all three transitions in the MRM mode yielded higher intensity readout, the sensitivity of the assay was not improved due to the loss of specificity when plasma samples were analyzed. Therefore, the MRM corresponding to the ring cleavage, m/z 720 f 210 (SAL) and m/z 724 f 210 (SAL-d4), was selected to ensure the selectivity of detection. Postcolumn addition of 1% acetic acid in water (∼50 µL/min) to the main column flow substantially improved the sensitivity (by more than 10-fold) by enhancing ionization efficiency without adversely affecting the chiral column due to increased acidity. With the use of the same protocol, simultaneous determination of DA was also possible. The HPLC/ESI-MS/MS analysis revealed the presence of four derivatives of DA corresponding to three triPFB-DA isomers (m/z 694) and a fully derivatized tetra-PFBDA derivative (m/z 874). Since the tetra-PFB-DA is the major derivative under the conditions employed (data not shown), we chose this form for quantitative analysis of DA. The mass spectrum obtained by CID of tetra-PFB-DA produced major product ions at m/z 181, 316, and 497. The characteristic fragment ion at m/z 497 resulted from the loss of NH-(PFB)2 from [M + H]+ ions as depicted in Figure 5. The quantitation of DA was performed by MRM using the transitions of m/z 874 f 497 (DA) and m/z 878 f 501 (DA-d4). Under the HPLC conditions used for the enantiomeric separation of SAL, DA and (S)-SAL derivatives were

partially separated. However, the selectivity of MRM allowed simultaneous determination of partially resolved (S)-SAL and DA. Assay Evaluation in Plasma Matrix. Although semicarbazide was used as an aldehyde trapping reagent, SAL can be formed during the assay. To test the possibility, 1 mL of plasma was spiked with deuterated DA (1,1,2,2-d4-DA, 20 ng) with or without acetaldehyde addition, and the formation of 1,1,2,2-d4-SAL during the assay was monitored based on the MRM transition of m/z 724 f 212. As seen in Figure 6A, the plasma sample contained endogenous (S)- and (R)-SAL and DA but did not convert the spiked deuterated DA to 1,1,2,2-d4-SAL. In contrast, the plasma sample prepared in the presence of acetaldehyde showed the formation of the racemic mixture of (S)- and (R)-1,1,2,2-d4-SAL, further increase of (S)- and (R)-SAL, and decrease of DA and DA-d4 (Figure 6B), suggesting that nonenzymatic condensation of DA with acetaldehyde can readily occur during the sample preparation process. These data indicated that the current assay condition can successfully trap aldehydes and, thus, is suitable for reliable determination of endogenous SAL. The blank plasma prepared by alkaline treatment showed that the endogenous SAL and DA were successfully removed. The blank plasma spiked with SAL-d4 and DA-d4 showed no MRM peaks corresponding to endogenous SAL and DA (Figure 7A). The efficiency and reproducibility of the assay were evaluated by spiking known amounts of SAL and DA into 1 mL of blank plasma and processing as described in the Experimental Section. The recoveries of SAL spiked into blank plasma at 0.2, 1, and 4 ng were 82% ( 5%, 84% ( 5%, and 94% ( 2%, respectively (n ) 5). The recoveries for 1 and 5 ng DA were 89% ( 2% and 87% ( 4%, respectively (n ) 5). The instrumental response was linear in the range of 0.5 pg to 5 ng for each SAL enantiomer and 20 pg to 20 ng for DA with regression coefficient r2 > 0.999. The relative response factors of SAL/SAL-d4 determined by the slope of calibration curves were 1.06 and 1.01 for (S)- and (R)-SAL, respectively. Each isomer was detected with approximately 1:1 relative peak area ratio, indicating

9170 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Figure 6. MRM chromatograms demonstrating the formation of SAL during the assay in the presence of acetaldehyde. (A) MRM chromatograms of (S)-SAL, (R)-SAL, and DA from the human plasma sample spiked with 20 ng of 1,1,2,2-d4-DA. No formation of 1,1,2,2d4-SAL was detected. The endogenous SAL and DA levels in the sample were found to be 246 pg/mL, 375 pg/mL, and 1.95 ng/mL for (S)-SAL, (R)-SAL, and DA, respectively, when an aliquot was separately analyzed in the presence of internal standards (S)-SALd4, (R)-SAL-d4, and DA-d4. (B) MRM chromatograms of SAL, 1,1,2,2d4-SAL, DA, and 1,1,2,2-d4-DA from the same plasma sample spiked with 20 ng of 1,1,2,2-d4-DA and 5 µL of acetaldehyde. Significant formation of 1,1,2,2-d4-SAL is apparent.

Figure 7. MRM chromatograms of SAL and DA in the LOQ sample prepared with the blank plasma. (A) MRM ion chromatograms obtained from 1 mL of blank plasma spiked with 1 ng each of (S)SAL-d4 and (R)-SAL-d4 and 5 ng of DA-d4. No endogenous SAL or DA was detected. (B) MRM ion chromatograms obtained from 1 mL of blank plasma spiked with 20 pg of (R/S)-SAL and 100 pg of DA as well as 1 ng each of (S)-SAL-d4 and (R)-SAL-d4 and 5 ng of DA-d4. MRM ion chromatograms were acquired by monitoring the transition of m/z 720 f 210 (SAL), m/z 724 f 210 (SAL-d4), m/z 874 f 497 (DA), and m/z 878 f 501 (DA-d4).

that racemic (R/S)-SAL standard indeed consists of equal amount of each enantiomer. The minimum detectable amount of SAL injected on column was estimated to be approximately 8 fg at a signal-to-noise ratio (S/N) higher than 5/1 (data not shown). The variations of the MRM response ratios against the internal standard were less than 2% at 25 pg level over a period of 2 months (n ) 15). The accuracy and precision of the assay was evaluated by analyzing quality control (QC) samples consisting of 1 mL of blank plasma spiked with known amounts of standard SAL and DA at low (LQC, 160 pg/mL SAL, 0.6 ng/mL DA), middle low (MQC1, 1200 pg/mL SAL, 2 ng/mL DA), middle high (MQC2, 2500 pg/mL SAL, 4 ng/mL DA), and high (HQC, 4000 pg/mL SAL, 8 ng/mL DA) concentrations. The coefficients of variance (CV) and errors representing inter- and intraday analysis precision and accuracy for the QC samples are shown in Table 1. The % error determined for intra- (n ) 5) and interday (n ) 5) accuracy was found to be within (5% for both SAL and DA at all levels tested. The % CV determined for intra- and interday precision was less than 4%. The LOQ, defined as the lowest quantifiable amount

for which % CV and % error are within 10%, was found to be less than 10 pg for each SAL isomer and 0.1 ng for DA in 1 mL of blank plasma. Figure 7B shows MRM chromatograms for SAL and DA at a LOQ level, suggesting that even lower concentrations may be quantifiable. The excellent reproducibility and accuracy observed with this assay may be the combined outcome of using inherently selective MRM detection and deuterium-labeled internal standards as well as the improved chromatographic resolution due to stable PFB derivatization. In addition, increasing molecular weight by PFB derivatization improved mass spectrometric detection capability since analysis in the high-mass region avoided backgrounds of low molecular mass ions, generally yielding a higher S/N ratio. Analysis of SAL and DA in Biological Samples. The assay method described above allowed the routine analysis of SAL and DA in 0.5 mL of human plasma and CSF from healthy human subjects. This is a significant improvement in sensitivity of the enantiomeric assay of SAL which generally requires at least 5 mL Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Table 1. Precision and Accuracy of the Assay for SAL Enantiomers and DAa (a) Intraday (n ) 5) Precision and Accuracy (S)-SAL (R)-SAL

spiked amount

LOQb LQC MQC1 MQC2 HQC

DA (ng)

% CV

% error

% CV

% error

% CV

% error

10 160 1200 2500 4000

0.1 0.6 2.0 4.0 8.0

8.5 3.4 0.7 2.4 1.3

-4.0 2.5 -0.4 1.7 5.5

4.5 1.9 1.0 1.2 0.9

-2.0 1.1 -0.6 -1.1 1.4

5.9 1.7 1.6 1.0 0.4

4.1 5.3 0.4 3.2 3.8

(b) Interday (n ) 5) Precision and Accuracy (S)-SAL (R)-SAL

spiked amount

LQC MQC1 MQC2 HQC

DA

SAL (pg)

DA

SAL (pg)

DA (ng)

% CV

% error

% CV

% error

% CV

% error

160 1200 2500 4000

0.6 2.0 4.0 8.0

2.2 0.9 1.7 1.1

1.9 -1.6 -1.2 3.6

2.8 2.3 1.0 2.0

-0.5 -1.5 -2.2 1.0

3.4 1.9 0.6 1.0

3.3 0.8 3.5 4.4

a Indicated amounts of (R/S)-SAL and DA were spiked into 1 mL of blank plasma together with 1 ng each of (S)-SAL-d and (R)-SAL-d and 5 4 4 ng of DA-d4 and extracted using PBA cartridges followed by derivatization and analysis using HPLC/ESI-MS/MS. b LQC, MQC1, MQC2, and HQC represent low, middle low, middle high, and high QC concentrations.

Table 2. (S)-SAL, (R)-SAL, and DA Levels in Plasma and CSF from Healthy Human Subjects analytes

plasma (n ) 17)

CSF (n ) 10)

(S)-SAL (pg/mL)

136 ( (15-628)b

92 ( 109 (17-384)

(R)-SAL (pg/mL)

173 ( 220 (22-784)

115 ( 128 (17-409)

DA (ng/mL)d

4.73 ( 1.27c,d (2.63-6.62)

1.67 ( 0.78 (0.29-2.55)

167a

a Data are expressed as mean ( SD. b Figures in parenthesis represent the concentration range. c The plasma DA level is significantly different from the CSF level. d p < 0.001.

Figure 8. Representative MRM chromatograms of endogenous SAL and DA found in 0.5 mL of human plasma (A) and CSF (B) spiked with 1 ng each of (S)-SAL-d4 and (R)-SAL-d4 and 5 ng of DA-d4 as internal standards. (A) Plasma: the concentrations of (S)-SAL, (R)SAL, and DA were determined to be 122 pg/mL, 171 pg/mL, and 2.93 ng/mL, respectively. (B) CSF: the concentrations of (S)-SAL, (R)-SAL, and DA were determined to be 18 pg/mL, 25 pg/mL, and 0.60 ng/mL, respectively.

of biological fluids with the limit of quantitation claimed to be 100 pg/mL.18 A representative ESI-MS/MS ion chromatogram of a plasma sample is shown in Figure 8, indicating that the sample 9172 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

contained 122 pg/mL, 171 pg/mL, and 2.93 ng/mL of (S)-SAL, (R)-SAL, and DA, respectively. Table 2 summarizes the quantitation results for SAL enantiomers and DA obtained from 17 plasma and 10 CSF samples. The plasma concentrations of SAL in healthy subjects ranged from 15 to 628 pg/mL (mean ( SD, 136 ( 167 pg/mL) for (S)-SAL, and from 22 to 784 pg/mL (mean ( SD, 173 ( 220 pg/mL) for (R)-SAL. The DA concentrations in plasma were 2.63-6.62 ng/mL (mean ( SD, 4.73 ( 1.27 ng/mL). The CSF levels of (S)-SAL and (R)-SAL were in the range of 17-384 pg/ mL (mean ( SD, 92 ( 109 pg/mL) and 17-409 pg/mL (mean ( SD, 115 ( 128 pg/mL), which is similar to the plasma SAL level in healthy subjects. However, the DA level was in the range of 0.29-2.55 ng/mL (1.67 ( 0.78 ng/mL), which is significantly lower than the plasma DA level (p < 0.001). No statistical correlations (r2 < 0.2) were found between DA and (S)-SAL or (R)-SAL in both plasma and CSF samples. Our results for (S)and (R)-SAL and DA are in the similar range as some of the data previously reported for healthy individuals.21,28-30 Our present study showed that the level of (R)-SAL was higher in comparison (28) Naoi, M.; Maruyama, W.; Dosert, P.; Nagy, G. M. Neurotoxicology 2004, 25, 193-204. (29) Musshoff, F.; Daldrup, Th.; Bonte, W.; Leitner, A.; Lesch, O. M. J. Chromatogr., B 1996, 683, 163-176. (30) Mu ¨ ller, T.; Baum, S. S.; Ha¨ussermann, P.; Przuntek, H.; Rommelspacher, H.; Kuhn, W. J. Neurol. Sci. 1999, 164, 158-162.

to (S)-SAL in both plasma and CSF ((R)/(S) ≈ 1.2). The higher level of the (R)-enantiomer may reflect the fact that SAL is produced by the (R)-stereospecific enzymatic pathway in addition to the nonenzymatic pathway. The individual variations of SAL concentrations in plasma and CSF were high, possibly due to genetic predisposition or diet, although the cause for this variation is not clearly understood. CONCLUSIONS The chemical derivatization coupled with chiral phase HPLC/ MS/MS method described in this report allowed reliable determination of SAL stereoisomers and DA in human plasma and CSF with significantly improved sensitivity, selectivity, and reliability. Simple one-step PFB derivatization of SAL and DA in aqueous solution considerably simplified the sample preparation procedure. Introduction of bulky PFB groups into the SAL structure enabled the baseline separation of SAL on a chiral column using a mobile phase compatible with the analysis by ESI-MS. Detection of the PFB derivatives by tandem mass spectrometry using MRM further increased selectivity of the detection. This assay method was found to be robust, as evidenced by high reproducibility and accuracy (both % CV and % error within (10%). The PFB derivative of SAL was easily detected by GC/NCI-MS, suggesting that this assay

can be extended to GC/NCI-MS or GC/NCI-MS/MS analysis when a suitable chiral phase GC column becomes available. In addition, the same strategy may be easily applicable to the analysis of other catecholamines including metabolites of DA and SAL in various biological matrices. In conclusion, we have developed for the first time a method to reliably determine stereoisomers of SAL and DA from a reasonable amount of biological fluids readily amenable in clinical settings. The sensitive, reliable, and versatile nature of the method described in this study should prove extremely useful in clarifying a potential role of DA and SAL as potential biomarkers, neuromodulators, or neurotoxins involved in alcoholism or other neurological diseases. ACKNOWLEDGMENT The authors acknowledge Satjit Brar in Virginia Commonwealth University, for his excellent assistance for validating this assay. This research was supported by the Intramural Research Program of NIAAA, NIH.

Received for review July 26, 2007. Accepted September 14, 2007. AC0715827

Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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