Journal of Analytical Toxicology,Vol. 27, October 2003
A Liquid Chromatographic-TandemMass Spectrometric Method for the Analysisof Serotoninand Related Indoles in Human Whole Blood Jonathan P. Danaceau 1, George M. Anderson2, William M. McMahon 3, and Dennis J. Crouch1,* 1Centerfor Human Toxicology, Departmentof Pharmacologyand Toxicology, Universityof Utah, 20 South 2030 East, Room 490, Salt Lake City, Utah 84112; 2Departmentsof LaboratoryMedicine and Child Psychiatry, Yale UniversitySchool of Medicine, New Haven, Connecticut 06510; and 3Departmentof Psychiatry, Universityof Utah, School of Medicine, 30 North 1900 East, Room5Rl10, Salt Lake City, Utah 84112
Abstract I This paper describes a high.performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) method for the determination of serotonin (5-HT) and the related indoles tryptophan (TRP), 5.hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid (IAA), and indole-3-proprionic acid (IPA) in whole blood. Ionization was achieved by atmospheric pressure chemical ionization (APCI). The method uses a solvent gradient to achieve baseline separalion of all analytes. Linearity was established for all components from 10 to 500 ng/mL, with the exception of TRP,which was calibrated from 0.4 to 20 pg/mL. Limits of quantilation were established that were well below the normal endogenous concentrations of 5-HT and its related indoles. Recoveries of 5-HT, TRP, 5-HIAA, IAA, and IPA were 95%, 91%, 98%, 79%, and 65%, respectively, from whole blood matrix and varied less than 2%. Serotonin, TRP,and IAA results were verified by cross-validation with an external laboratory using a previously validated HPLC method with fluoromelric detection. A regression analysis of the two data sets resulted in correlation coefficients of 0.981, 0.964, and 0.987 for 5-HT, TRP,and IAA, respectively.
logical samples by high-performance liquid chromatography (HPLC) coupled to either an electrochemical detector (EC) or fluorometric detector (F). A number of methods have been reported for determining the compounds in whole blood, plasma, urine, cerebrospinal fluid (CSF) and brain (7-17). In preparation for studies examining whole blood concentrations of 5-HT in individuals with autism, we aimed to extend the available methods for determination of indoles in whole blood. Although Anderson et al. (7) have reported a validated, simple, and specific HPLC-F method for the determination of 5-HT and TRP in whole blood, we believedthat an HPLCtandem mass spectrometric (MS-MS) method offered advantages in terms of specificity and linear range, while permitting simultaneous determination of additional indolic species. This paper describes an HPLC-MS-MS method for the determination of 5-HT, TRP,5-HIAA,and the indole acids indole-3-acetic acid (IAA)and indole-3-propionic acid (IPA) in whole blood samples.
Materials and Methods Introduction
Standardsand materials
Serotonin (5-hydroxytryptamine, 5-HT) serves as a central and peripheral neurotransmitter/neuromodulator and has growth factor-like action in the developing nervous system (1,2). Serotonin mediates a range of critical behaviors (3), and it has been linked to a number of neuropsychiatric disorders including anxiety, depression, obsessive-compulsive disorder (OCD), and autism (4-6). Serotonin and related compounds, including its precursor amino acid tryptophan (TRP) and major metabolite 5-hydroxyindoleacetic acid (5-HIAA),are typically measured in physio9Author to whom correspondenceshould be addressed. E-mail:
[email protected].
440
Standard materials were obtained from the following suppliers: N-methylserotonin (NMS) oxalate salt and perchloric acid were from Aldrich Chemicals (Milwaukee,WI); serotonin creatinine sulfate, l-tryptophan (TRP), indole-3-propionicacid (IPA), 5-hydroxy-indole-3-aceticacid (5-HIAA),ascorbic acid, and sodium metabisulfite were from Sigma Chemicals (St. Louis, MO); and indole-3-acetic acid (IAA)was from Acros Chemicals (Pittsburgh, PA). Stock solutions of all standards were prepared by weighing the appropriate amount of the standard material to obtain 10.0 mg of the free base (or acid). The free base material was dissolved in 100 mL of 0.2% ascorbic acid, resulting in stock solutions of 0.1 mg/mL. The stock solutions were further diluted in 0.2% ascorbic acid to the
Reproduction(photocopying)of editorialcontentof thisjournal is prohibitedwithoutpublisher'spermission.
Journal of Analytical Toxicology, Vol. 27, October 2003
appropriate concentrations for the working standards. All standards were stored at --80~
Equipmenl An Agilent(HP) 1100 series HPLCsystem (PaloAlto, CA)was coupled to a Thermo-Finnigan Triple Stage Quadrupole (TSQ7000) MS employingatmospheric pressure chemical ionization (APCI) (San Jose, CA). Analyte separation was achieved on a Waters| (Milford, MA) Polarity column (3-pro packing; 2.1mm i.d. x 100-mm length). The data system used Excalibur software (Thermo-Finnigan, San Jose, CA).
Collection of whole blood Whole blood samples were collected in 5-mL vacutainers containing K3EDTAfrom non-fasting subjects. Aliquots of 250 mL of each WB sample were transferred into 2-mL plastic Eppendorf tubes. Samples were frozen at -80~ until extraction. The use ofhuman subjects in this study was approvedby the Institutional ReviewBoard of the Universityof Utah.
Extractionof whole blood samples Whole blood contains endogenous 5-HT and other indoles, making it unsuitable as a matrix for calibration standards. Therefore,calibration standards were prepared in 0.2% ascorbic acid at concentrations of 10, 20, 50, 100, 200, 400, and 500 ng/mL from the working standards. 1Yyptophan concentrations were 0.4, 0.8, 2, 4, 8, 16, and 20 mg/mL to better reflect whole blood concentrations of this amino acid. Two method blanks consistingof 250 mL of 0.2% ascorbicacid were also prepared. Quality control samples were prepared by adding 5 mL of a 10 ng/l~Lworking standard containing 5-HT, 5-HIAA,IAA, and IPA and 10 mL of a standard containing 100 pg/mL TRP to whole blood. This resulted in samples fortified with an additional 200 ng/mL of 5-HT, 5-HIAA,IAA,and IPA and an additional 4 pg/mL TRP. All blanks, unknown samples, quality control samples, and standards were then extracted according to the followingprocedure that is a modification of the proceTable I. MS Instrumentation Parameters Vaporizertemp Capillary temp Corona current Collision gas pressure EM voltage Sheathgas pressure Collision chamber voltage
Table II. Analyte and Fragmentation Masses MW
M+H § (Parent)
D1
5-HT 5-HIAA NMS TRP IAA IPA
176 191 190 204 175 189
177 192 191 205 176
160 146 160 188 130 130
190
Probabletoss -I 7 .-46 -31 -I 7 -46 .-60
Analysis by HPLC-MS--MS The analytes were separated using a solvent gradient. The aqueous phase consisted of 0.1% formic acid and the organic phase was 100% acetonitrile (ACN). The initial condition of 100% aqueous was held for 5 rain. From 5 to 10 rain, the solvent mixture was ramped to 35% ACNand held there for 5 rain. The gradient was then returned to 100% aqueous in 1 rain and maintained at 100% aqueous for 7 rain. Twenty microliters of each sample was injected on column. As stated, samples were analyzed by HPLC-MS-MS using APCI.Target compounds were analyzedusing multiple reaction monitoring (MRM) in which the first quadrupole was used to select the protonated molecular ion (M+H+) of each analyte. Fragmentation was achieved in the collision cell using argon and a collision energy of-16 V. This produced secondary (product) ions that were selected in the third quadrupole. The MS parameters used in the analysis are shown in Table I. The collision conditions were adjusted such that the major product ion and the precursor ion were in a ratio of approximately 10:1 in the product ion spectrum. A collisioncell voltage of-16 V proved optimum to produce this ratio for each analyte. Table II shows the analyte molecular weights, mass-to-charge ratio of precursor ions, primary product ions, mass lost, and the probable fragment lost for each analyte. Analyte quantitation was performed using the Excalibur software (Thermo-Finnigan). The peak-area ratio (analyte area/IS area) was used to quantitate each analyte. A 1/x weighting of a linear regression was used in order to optimize the assay accuracy at lower concentrations.
Analysis by HPLC-fluorometry
350~ 200~ 4 IJA 3 mTorr -2500 V 40 PSl -I 6 V
Analyte
dure described by Anderson et al. (7): ice-cold solutions of 400 mL of 25% ascorbic acid, 10 mL of 10 ng/mL NMS (internal standard), 400 mL of 5% sodium metabisulfite, and 300 mL of 3.4M perchloric acid were added in order to the still frozen sampies. Each sample was vortex mixed for 10 s and placed on ice for 10 rain. The samples were then centrifuged twice at 20,000 • g for 10 min. Sample supernatants were transferred to new 2-mL Eppendorf tubes and frozen at-80~
(-NH3) (-COOH2) (-CH3NH2) (-NH3) (-COOH2) (-CH2COOH2)
Samples were prepared as described, and 10 laLwas injected on a 4- • 250-mm Altex Uitrasphere| 5-m Cls reversed-phase column eluted with 1.0 mL/min of a mobile phase composed of 80% 0.1M phosphate buffer with 50 mg/L of disodium EDTA and 150 mg/L of sodium octylsulfate/20% methanol. Compounds were detected using a Shimadzu RF-10AXL fluorometric detector and quantitated by comparison of peaks heights to that of the internal standard, NMS. Retention times for TRP, 5-HT, NMS,and IAAwere 4.6, 8.8, 10.0, and 11.8 rain, respectively. Recovery and variance data have been previously reported for 5-HT, TRP, and NMS (7,18) and a similar HPLC-F method cross-validated with fluorometric and HPLC-EC methods for 5-HT (19).
Results Figure I shows the HPLC--MS-MSchromatogram of 5-HT, 441
Journal of Analytical Toxicology,Vol. 27, October 2003
TRP, NMS, 5-HIAA, IAA, and IPA from a whole blood sample using the column and mobile phase conditions described. The chromatogram demonstrates that excellent peak shape and baseline separation were achieved for the analytes. Retention times were extremely stable, varying less than 6 s across analytical runs. Standard curves were linear with correlation coefficients of 0.99 or greater for all analytes. In agreement with previous reports (4,7,20,21), whole blood concentrations of 5-HT were generally in the 100-300 ng/mL range and averaged 133.8 • 83.6 ng/mL (mean • S.D.; N = 33). However, 5-HT concentrations as low as 5-10 ng/mL were seen in subjects currently taking drugs that inhibit platelet 5HT reuptake as previously reported by Epperson et al. (18). 1Yyptophan concentrations, as expected, were in the range of 4.98--8.78 Iag/mL with a mean • S.D. of 6.6 • 1.30 mg/mL. Average concentrations for 5-HIAA, IAA,and IPA were 14.3 • 2.4, 100 • 40, and 91 • 48 ng/mL, respectively (mean • S.D.; N = 11). The observed ranges for the remaining analytes were 7.7-19.6 ng/mL for 5-HIAA, 44-174 ng/mL for IAA,and 55-207 ng/mL for IPA and are consistent with previous reports of these compounds in blood and plasma (9,20-22). Limits of detection (LOD) and quantitation (LOQ) studies Studies to determine the LOQs and LODs were performed
with 0.2% ascorbic acid fortified with low concentrations of all the analytes. Five replicate samples at 2, 5, and 10 ng/mL were extracted as described in the materials and methods. Although all compounds were easily detected at 2 ng/mL, the LOQs were established where the relative percent deviations (%RSDs) and absolute % deviations were 15% or less (Table III). The mean signal-to-noise (S/N) ratio for each analyte was greater than 5. Using those criteria, the LOQs were 2 ng/mL for 5-HIAA, 5 ng/rnL for 5-HT, IAA,and IPA, and 10 ng/mL for TRP. Standard addition recovery study Whole blood samples were fortified with additional analytes as described in the materials and methods section. These fortified samples were extracted in triplicate and analyzed along with the calibration curve and duplicate unfortified samples. Samples previously analyzed by HPLC-MS--MS that had been determined to contain very low 5-HT concentrations were used for these experiments. The recoveries of 5-HT, TRP,and 5-HIAA were 95.4%, 90.9%, and 97.6%, respectively. All were within 10% of the actual fortified analyte concentration. Recoveries for IAAand IPA were considerably lower (with recoveries of 78.8% and 64.8%, respectively). Values reported for IAAand IPA were not corrected for % recovery.
Intra-assay repeatability Replicate injections of individual samples, or standards, as well as replicate extractions of the same samples or standards were analyzed to assess the repeatability of analysis and extractions. Triplicate extractions of fortified whole blood samples had % RSDs of less than 1.25%. In addition, triplicate injections of standards and samples consistently had % RSDs of less than 4% (data not shown).
30000 20000 10000 0 40000
30000 20000 10000 0
A
1000000 t 500000
--
~TRP;205->188;] 10.3min. I
o C
Interassay repeatability/stability of frozen samples To assess the repeatability of the method and the stability of frozen extracted samples, 12 duplicate samples were analyzed for 5-HT before and after storage at --80~ for eight months. The observed mean (• S.D.) pre- and post-storage 5-HT con-
i
1500000
9
L ~NMS; 191-->160;J 8,0 rain
2000 1500 1000 500 0
i
i
~5-H~A;192-->146;] 11.3min.
i
Table III. Limit of Quantitation Data for 5-HT and Related Indoles
l
5-HT
l
5-HIAA
IAA
IPA
Concentration(ng/mL)
4000
3000 2000 1000 0 1000
I ~IAA; 176->130;13,5rain. I
600
I
~IPA; 190-->130;14.5min.
40O 2OO
01 ~176 4
Replicate
5
10
2
5
5
1 2 3 4 5
4.65 5.26 4.62 5.06 4.00 4.72 0.48
11.13 10.59 10.99 12.27 10.71 11.14 0.67
1.83 1.88 2.06 2.02 2.20 2.00 0.15
5.24 5.45 6.10 5.10 4.02 5.18 0.76
5.38 5.80 6.77 5.13 5.69 5.75 0.63
Mean 6
8
10
Time (min)
12
14
16
Figure1. HPLC-MS-MS determination of 5-HT, NMS (the internal standard), TRP,5-HIAA, IAA, and IPA from a prepared whole blood sample.
Anal~ename,precursorto productiontransition,and retentiontimeare listed alongside each chromatogram.
442
TRP
S.D.
%RSD
%Deviation* -5.64%
10.26%
6.00% 7.31% 14.58% 10.89% 11.38% -0.02% 3.61% 15.08%
MeanS/N Ratio 5.8
38.0
5.2
15.0
9% deviationof observedmeanfrom knownpreparedconcentration.
7.8
Journal of Analytical Toxicology, Vol. 27, October 2003
centrations were 172 • 74 and 170 • 77 ng/mL (N --- 12), respectively and the values were highly correlated (r = 0.909, slope = 0.997). Stability of 5-HT in whole blood To test the stability of 5-HT in a whole blood samples stored at room temperature, blood was drawn and portions removed for analysis at 5 min and at hourly intervals from I to 11 h and after 23 h. Samples were analyzed by HPLC-F and no tendency to decrease or increase was seen over the entire period. The mean (• SD) observed for the sample studied was 190 • 8.3 nglmL (%RSD = 4.4%., N = 9). Similar stability was observed for TRP concentrations in the same whole blood kept at room temperature for 23 h (8.14 • 0.36 pglmL, %RSD = 4.5%,N = 9). Accuracy: cross-validationof HPLC-MS-MS resultswith fluorescencedata As an additional assessment of the accuracy of the method, a cross-laboratory validation study was conducted for 5-HT, TRP, and IAA.This cross-validation provided the advantages of comparing the HPLC-MS--MSmethod under development with an established, HPLC-fluorescence method that was validated. ~elve whole blood samples were used for the cross-validation. The data are shown in Figure 2. A correlation coefficient of 0.981 was calculated from comparing the HPLC-F concentrations of 5-HT with those obtained by HPLC-MS--MSand the mean absolute percent deviation [(HPLC-MS--MSconc.- HPLC conc.)/mean conc.] was 6.11%, indicating excellent agreement between the two methods. A paired t-test indicated no significant difference between the methods (bias = 5.97 • 13.25; p = 0.147; N = 12). The HPLC-MS--MS and the HPLC-F concentrations obtained for TRP and IAAwere also highly correlated with r valuesof 0.964and 0.987 and mean absolute % deviations of 5.00 and 6.56%, respectively. Paired t-tests revealed no significant difference between the results for TRP and IAA obtained by the two methods. The biases were -0.19 • 0.36 pg/mL (p = 0.113) and -3.55 • 6.78 ng/mL (p = 0.943) for TRP and IAA,
300
~
y = 0.8699x + 18.954 r 2 = 0.9626 r = 0.9811
250
c'~ 200
,-,-i 100. 5O 50
100
150 200 HPLC-F (ng/mL)
250
300
Figure 2. Comparison 5-HT results in whole blood samples by HPLC-F and HPLC-MS-MS. Resultsof a cross-laboratory and cross-method validation of 5-HT determination in whole blood. The r value of 0.981 and the slope of 0.87 demonstrate excellent agreement between the two methods. Cross-comparison results obtained for IAA and TRP were also highly correlated (seetext).
respectively. 5-HIAA and IPA were not determined using the HPLC-F method.
Discussion This paper describes a specific and sensitive HPLC-MS--MS procedure for the analysis of 5-HT and related indoles (TRP,5HIAA, IAA,and IPA) in whole blood. All analytes eluted within 15 rain and baseline separation was achieved between chromatographic peaks. In addition, the LOQs for each analyte were well below endogenous concentrations seen in whole blood. The specificity of the HPLC-MS--MSmethod arises from the multi-stage selectivity inherent in the system and resulted in ion chromatograms that were virtually devoid of interference. Although the cross-method comparison experiment indicated that the HPLC-F method was not affected by interfering compounds, a number of extraneous peaks were observed in the chrornatograms and the possibility for interference was clearly greater with the HPLC-F method. The analyte fragmentation patterns listed in Table II are based on expected losses of functional groups from the molecule. The losses of 17 and 31 were most likely a result of loss of NH3 and CH3NH2from the side chain of 5-HT and NMS, respectively. In both cases, the remaining fragment was the 5-hydroxylated indole ring, protonated at the indole N with a -CH=CH2group at the 3 position. McCleanet al. (23) found identical fragmentation patterns for these two molecules using an electrospray ionization ion trap setup for MSn analysis of indole alkaloids from frog skin. The method had LOQs well below the normal endogenous concentrations of 5-HT and its related indoles. The LOQ concentrations chosen all had %RSDs and % deviations of less that 15%. Even TRP, which was calibrated at a much higher concentration than the other analytes (0.4-20 lag/mL)had a % deviation of only 11.4% from the actual concentration of 10 ng/mL. Mean signal-to-noise ratios at the LOQ were greater than 5 for all of the analytes. In addition, the calibration curves had regression coefficients of 0.99 or greater. These data show that the HPLC-MS--MSmethod had ample sensitivity and it had the accuracy to quantitate these analytes at even the lowest endogenous concentrations in whole blood. One advantage of the HPLC-MS--MSmethod was the ability to simultaneously detect all of the analytes in a single assay.This advantage was due to the selectivity and the dynamic range of tandem MS. For example, current methods for quantitating 5HIAA in plasma or blood are unable to analyze 5-HT and TRP concurrently, and determining 5-HT and TRP in the same run can be problematic because of a 100- to 1000-fold difference in their whole blood concentrations. The method described in this paper is designed to measure 5-HT and related indoles in whole blood. However, there is substantial and continuing interest in measuring these compounds in a wide range of biological samples, including brain tissue, cerebrospinal fluid, plasma, urine, media, and foodstuffs. With slight modification, the method described in this paper should prove effectivefor analyzing 5-HT and related indoles in 443
Journal of Analytical Toxicology, Vol. 27, October 2003
any of these matrices because it was rigorous enough to analyze a complex matrix such as whole blood, which requires cell lysis, deproteinization and antioxidant treatment. Limitations One limitation of the method was the relatively low recovery of ~ and IPA from whole blood. Table Ill shows that only 78.8% of [AA and 64.8% of ]PA were recovered from a supplemented whole blood sample. The likely explanation for this low recovery was their relatively non-polar nature compared to the other indoles analyzed by the method. Under the acidic extraction conditions, the carboxylic acid groups of IAA and IPA would be protonated and uncharged. In addition, they lack the polar hydroxyl substitution that 5-HIAA has on the indole ring. For these reasons, [AA and IPA could potentially adsorb to any lesspolar components of whole blood. As the indole acids become less and less polar, their recovery decreases from 97.6% for 5-
HIAAto 78.8% for IAAto 64.8% for IPA.
Acknowledgments This work was supported in part by the Utah Autism Foundation, HD4293 and HD42438 to WMM and DA13367-02 to DJC.
References 1. N.M. Barnes and T. Sharp. A review of central 5-HT receptors and their function. Neuropharmacology 38:1083-1152 (1999). 2. P.M. Whitaker-Azmitia. Serotonin and brain development: role in human and developmental diseases. Brain Res. Bull. 56:479-485 (2001). 3. I. Lucki. The spectrum of behaviors influenced by serotonin. Biol. Psychiatry. 44:151-162 (1998). 4. P.A. McBride, G.M. Anderson, M.E. Hertzig, M.E. Snow, S.M. Thompson, V.D. Khait, T. Shapiro, and D.J. Cohen. Fffects of diagnosis, race, and puberty on platelet serotonin levels in autism and mental retardation. J. Am. Acad. Child Adolesc. Psychiatry37: 767-779 (1998). 5. A. Sjoerdsma and M.G. Palfreyman. History of serotonin and serotonin disorders. Ann. N.Y Acad. Sci. 60:1-8 (1990). 6. J. Veenstra-VanderWeele, G.M. Anderson, and E.H. Cook, Jr. Pharmacogenetics and the serotonin system: initial studies and future directions. Eur. J. PharmacoL 410:165-181 (2000). 7. G.M. Anderson, F.C. Feibel, and D.J. Cohen. Determination of serotonin in whole blood, platelet-rich plasma, platelet-poor plasma and plasma ultrafiltrate. Life 5ci. 40:1063-1070 (1987). 8. J.C Alvarez, D. Bothua, I. Collignon, C. Advenier, and O. SpreuxVaroquaux. Simultaneous measurement of dopamine, serotonin, their metabolites and tryptophan in mouse brain homogenates by
444
high-performance liquid chromatography with dual coulometric detection. Biomed. Chromatogr. 13:293-298 (1999). 9. I.P. Kema, W.G. Meijer, G. Meiborg, B. Ooms, RH. Willemse, and E.G. de Vries. Profiling oftryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection. Clin. Chem. 47" 1811-1820 (2001). 10. D.D. Koch and RT. Kissinger. Determination of tryptophan and several of its metabolites in physiological samples by reversed-phase liquid chromatography with electrochemical detection. J. Chromatogr. 164:441-455 (1979). 11. I. Morita, T. Masujima, H. Yoshida, and H. Imai. Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma. Anal. Biochem. 118:142-146 (1981). 12. I. Morita, T. Masujima, H. Yoshida, and H. Imai. Direct plasma injection method for the analysis of tryptophan metabolites by highperformance liquid chromatography coupled with precolumn deproteinization. Anal. Biochem. 151:358-364 (1985). 13. M. Picard, D. Olichon, and J. Gombert. Determination of serotonin in plasma by liquid chromatography with electrochemical detection. J. Chromatogr. 341:445-451 (1985). 14. D.D. Koch and P.T.Kissinger. Determination of serotonin in serum and plasma by liquid chromatography with precolumn sample enrichment and electrochemical detection. Anal Chem. 52:27-29 (1980). 15. D.D. Koch and P.T. Kissinger. Current concepts: I. Liquid chromatography with pre-column sample enrichment and electrochemical detection. Regional determination of serotonin and 5-hydroxyindoleacetic acid in brain tissue.Life Sci. 26:1099-1017 (1980). 16. P.C.Tagari, D.J. Boullin, and C.L. Davies. Simplified determination of serotonin in plasma by liquid chromatography with electrochemical detection. Clin. Chem. 30:131-135 (I 984). 17. W.H. Lyness, N.M. Friedle, and K.E. Moore. Current concepts: II. Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in discrete brain nuclei using reverse phase liquid chromatography with electrochemical detection. Life 5ci. 26: 1109-1114 (1980). 18. N. Epperson, K.A. Czarkowski, D. Ward-O'Brien, E. Weiss, R. Gueorguieva, P. Jatlow, and G.M. Anderson. Maternal sertraline treatment and serotonin transport in breast-feeding mother-infant pairs. Am. J. Psychiatry 158:1631 -I 637 (2001). 19. N. 8adcock, J.G. Spence and L.M. Stern. Blood serotonin levels in adults, autistic and non-autistic children--with a comparison of different methodologies. Ann. Clin. Biochem. 24:625-634 (1987). 20. J. Ortiz, F. Artigas, and E. Gelpi. Serotonergic status in human blood. Life 5ci. 43:983-990 (1988). 21. M.J. Sarrias, P. Cabre, E. Martinez, and F. Artigas. Relationship between serotoninergic measuresin blood and cerebrospinal fluid simultaneously obtained in humans. J. Neurochem. 54:783-786 (I 990). 22. S. Laer, J. Scholz, C. Ritterbach, C. Laer, E. Rahme, and J. Schulte Am Fsch. Association between increased 5-HIAA plasma concentrations and postoperative nausea and vomiting in patients undergoing general anaesthesia for surgery. Eur. J. Anaesthesiol. 18: 833-835 (2001). 23. S. McClean, R.C. Robinson, C. Shaw, and W.F. Smyth. Characterisation and determination of indole alkaloids in frog-skin secretions by electrospray ionisation ion trap mass spectrometry. Rapid Commun. Mass Spectrom. 16:346-354 (2002).
