Structural Characterization of Contaminants Found in Commercial

It has been reported that melatonin consumption has resulted in eosinophilia in some humans taking high doses of this supplement. Although there has n...
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Chem. Res. Toxicol. 1998, 11, 234-240

Structural Characterization of Contaminants Found in Commercial Preparations of Melatonin: Similarities to Case-Related Compounds from L-Tryptophan Associated with Eosinophilia-Myalgia Syndrome Brian L. Williamson,† Andy J. Tomlinson,† Prasanna K. Mishra,§ Gerald J. Gleich,‡ and Stephen Naylor*,†,| Biomedical Mass Spectrometry Facility and Department of Biochemistry and Molecular Biology, Analytical NMR Facility, Department of Immunology and Medicine, and Department of Pharmacology and Clinical Pharmacology Unit, Mayo Clinic/Foundation, Rochester, Minnesota 55905 Received November 5, 1997

On-line HPLC/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) in conjunction with NMR has been successfully employed to identify and structurally characterize seven contaminants found in three different commercial preparations of melatonin. Six of these contaminants were identified as analogues of impurities found in contaminated L-tryptophan (an over-the-counter dietary supplement) associated with the eosinophilia-myalgia syndrome (EMS) epidemic that occurred in the United States during 1989. In particular, our studies identified two compounds with MH+ ) 249 to be hydroxymelatonin isomers. Four other compounds with MH+ ) 477 were identified as melatonin-formaldehyde condensation products. These compounds are structural analogues of L-tryptophan contaminants, namely, ‘peak C’ and ‘peak E’ that were previously implicated as etiological agents causing EMS. It has been reported that melatonin consumption has resulted in eosinophilia in some humans taking high doses of this supplement. Although there has not been a major outbreak of EMSlike symptoms from consumption of melatonin, this study clearly suggests that tighter control and regulation of nutritional supplements sold and used as drugs is necessary.

Introduction Melatonin is a hormone produced in the brain by the pineal gland and has received considerable interest since its endocrine action was first reported (1). It is secreted only in the dark (2) to regulate sleep. Melatonin and light provide complementary endocrine signals, the latter providing a daytime signal to the body’s master clock and the former, one for nighttime. Secretion is greatest during childhood and declines with increasing age. Recently, synthetic melatonin has found widespread use in the treatment of sleep disorders (3-5), as well as the alleviation of jetlag symptoms (sleep disturbances and fatigue due to rapid time zone change) (6-8). It was reported that administration of 5 mg of melatonin for no more than 7 days prevented or reduced the symptoms of jet lag. In addition melatonin has shown potential in the fight against Alzheimers disease (9), through activity thought to arise from its potential antioxidant properties (10). However the effects of melatonin are still not wellunderstood, and a number of side effects have been reported (11, 12). For example, use of melatonin by

individuals being treated for tumors caused increased eosinophilia in these patients (13, 14). Melatonin has been found in some foods and therefore can be sold ‘over-the-counter’ as a dietary supplement in the United States under the Dietary Supplement Health and Education Act. Safety and efficacy evaluations of such supplements are currently not required by the Food and Drug Administration (FDA).1 It is estimated that ‘over 20 million new consumers’ in the United States used melatonin in 1995 (11). This has produced cause for concern given the lack of comprehensive knowledge about the effects of melatonin (11-15). The purity of this overthe-counter hormone has also raised concerns. It was recently noted that four of six melatonin products obtained from health food stores contained unknown impurities that could not be identified (16). The results of the analysis of commercially available melatonin (17) are reminiscent of those obtained from similar studies of L-tryptophan (Trp). This latter product is another naturally occurring indole-based compound that is sold over the counter as a dietary supplement (18). Contaminant-

* Corresponding author, at the Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, C009B Guggenheim Bldg., Rochester, MN 55905. Phone: (507) 284-5220. Fax: (507) 284-8433. E-mail: [email protected]. † Biomedical Mass Spectrometry Facility and Department of Biochemistry and Molecular Biology. § Analytical NMR Facility. ‡ Department of Immunology and Medicine. | Department of Pharmacology and Clinical Pharmacology Unit.

1 Abbreviations: FDA, Federal Drug Administration; Trp, L-tryptophan; EMS, eosinophilia-myalgia syndrome; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; LC/ ESI-MS/MS, on-line high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry; NMR, nuclear magnetic resonance; CID, collision-induced dissociation; CD3OD, deuterated methanol; DMSO-d6, deuterated dimethyl sulfoxide; DFCOSY, doublequantum-filtered correlated spectroscopy; NOESY, nuclear Overhauser effect spectroscopy.

S0893-228x(97)00202-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/14/1998

Contaminants in Commercial Melatonin

(s) in Trp were reported to be responsible for the 1989 outbreak of eosinophilia-myalgia syndrome (EMS) (19), which affected ∼1500 people and resulted in ∼30 deaths in the United States (20). Epidemiological studies revealed that at least six contaminants present in Trp from one company (Showa Denko K.K. of Japan) were caserelated (21, 22). Cases of EMS had been reported for several years before this large epidemic (23), possibly indicating that Trp contamination has been present at much lower levels for some time. The specific component that caused EMS has still not been determined. In fact in a recent study we have characterized many of the contaminants in the implicated samples of Trp, and we are currently evaluating the possible significance of these compounds (24).

The EMS epidemic highlighted the need for nutritional supplements sold and used as drugs to require monitoring and oversight as drugs (25). Furthermore, in regard to dietary supplements, it has been suggested that ‘another accident [epidemic] is waiting to happen’ (26). Taking into account that melatonin has been reported to induce eosinophilia (13, 14) and given the structural similarities of Trp and melatonin, a comparison of the contaminants in the two food supplements was needed. We therefore investigated the nature of the contaminants in commercial preparations of melatonin by the use of tandem mass spectrometry (MS/MS) coupled with on-line HPLC (LC/ESI-MS/MS) together with off-line NMR analysis. We report the detection and structural characterization of seven of these impurities.

Materials and Methods Materials. Three different preparations of commercial melatonin were obtained from a local pharmacy. Methanol and acetonitrile (HPLC grades) were obtained from EM Science (Gibbstown, NJ), and water (HPLC grade) was from Burdick and Jackson (Muskegon, MI). Acetic acid and high-purity synthetic melatonin were obtained from Sigma (St. Louis, MO). Formaldehyde was purchased from Aldrich (Milwaukee, WI). Synthesis of Melatonin-Formaldehyde Condensation Isomers. Melatonin-formaldehyde condensation isomers were prepared from a modified method for the synthesis of 1,1′ethylidenebis(L-tryptophan) (27). Specifically, 6 mM melatonin and 6 mM HCl were added to 5 mL of H2O to form a white slurry. This solution was continuously stirred and cooled to 0 °C in an ice bath. Then 3 mM formaldehyde was added, and a yellow precipitate formed after approximately 5 min. The reaction mixture was then neutralized with NH4OH to pH ) 7, and the precipitate was washed with 50 mL of H2O. HPLC/ESI-MS/MS Analyses. Melatonin tablets were crushed into a powder, and 10 mg was dissolved in 1 mL of 80: 20 H2O-MeOH. The resulting solution was placed in an ultrasonic bath for 30 min, and any undissolved matter was removed by centrifuging the sample. HPLC analysis of the melatonin solution was performed on a Hewlett-Packard (Palo Alto, CA) model 1090 liquid chromatograph. A C-18 guard column [Primesphere HC, 5-µm particle size, 30 × 3.20 mm (Phenomenex, Inc., Torrance, CA)] was attached to the front of a C-18 column [Primesphere HC, 5-µm particle size, 110-Å pore

Chem. Res. Toxicol., Vol. 11, No. 3, 1998 235 size, 150 × 3.20 mm (Phenomenex, Inc.)] at a flow rate of 0.4 mL/min with gradient elution (0-4 min, 4-5% B; 4-11 min, 5% B; 11-15 min, 5-20% B; 15-30 min, 20-25% B; 30-45 min, 25-40% B; 45-48 min, 40-80% B; 48-53 min, 80% B; 53-55 min, 80-100% B; 55-65 min, 100% B) and buffers (A, 97% water/2% acetonitrile/1% acetic acid; B, 89% acetonitrile/10% methanol/1% acetic acid). UV spectral data were obtained online from the HP 1090 chromatograph equipped with a diodearray detector (DAD) at a wavelength of 254 nm. MS and MS/ MS analyses were performed on-line with a Finnigan MAT 95Q (Bremen, Germany) mass spectrometer of BEQ1Q2 configuration (where B is the magnetic sector, E is the electrostatic sector, Q1 is the octapole collision cell, and Q2 is the mass filter quadrupole). A Finnigan MAT electrospray ionization (ESI) source was used in positive mode throughout with a capillary temperature of 250 °C and a 3-kV potential on the spray needle. A scan range from m/z 100 to 750 at a rate of 1 s/decade was used. Protonated molecular weight ([M + H]+) ions were subjected to collision-induced dissociation (CID) in the octapole collision cell, using argon (pressure ) 1 × 10 -4 mbar) as collision gas, with typical collision energies of 25 eV. The product ions were analyzed in Q2 with 5-10 scans acquired and averaged to generate a product ion spectrum. HPLC/ESI-MS3 Analysis. MS3 analyses was performed by inducing primary fragmentation in the skimmer region of the electrospray source. This was achieved by increasing the voltages on the heated capillary and tube lens in the electrospray source by 30 V. The product ion of interest was then isolated and subjected to CID in the normal way. NMR Analysis. Proton NMR analyses were performed on either a Bruker AMX300 spectrometer operating at 300 MHz or a Bruker AMX500 spectrometer operating at 500 MHz. All samples were run at 25 °C. Samples were dissolved in deuterated methanol (CD3OD) or deuterated dimethyl sulfoxide (DMSOd6). Synthetic melatonin was dissolved in deuterated methanol, and proton NMR spectra were obtained at 500 MHz. The ring amide proton of melatonin exchanged easily with the hydroxyl of methanol and therefore could not be seen in the proton NMR spectra. The observed proton NMR resonances were assigned based on their peak splittings due to homonuclear proton coupling. To observe the ring amide protons, subsequent proton NMR analysis was attempted at 300 MHz in DMSO-d6. With the solvent methanol methyl resonances referenced to 3.30 ppm and the dimethyl sulfoxide peak referenced to 2.49 ppm, the observed melatonin peaks are assigned as shown in Table 1.

Results Concomitant HPLC/UV (at 254 nm) and LC/ESI-MS analysis of three commercial preparations of melatonin revealed the presence of seven contaminants. On the basis of both their UV and MS responses, the contaminants were determined to be present at the 0.1-0.5% level of parent melatonin in all three samples. This is illustrated in Figure 1 by the extracted ion current (representing the sum of the following ion signals: m/z 249, 265, 233, and 477) resulting from LC/ESI-MS analysis of a H2O-MeOH extract of commercial melatonin. The contaminants are labeled [1]-[8], with peak [4] being that of melatonin. The partial single-ion chromatogram of MH+ ) 477 ion is inset into Figure 1 to clearly indicate the presence of four responses characterized by a molecular weight of 476. They are labeled peaks [5]-[8]. The occurrence of two responses at m/z 249 and four responses at m/z 477 suggested that stereoisomers and/or geometric isomers were present in these melatonin samples. The contaminants detected in the commercial melatonin samples were characterized by LC/ESI-MS/MS. The

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Figure 1. Extracted ion currents at m/z 249, 265, 233, and 477 from a typical HPLC/ESI-MS analysis of an extract from commercially available melatonin. A section of the single-ion chromatogram for the MH+ ) 477 ion is inset into the figure to clearly indicate the presence of four peaks. The extract was obtained from a 10 mg/mL solution of crushed melatonin tablets dissolved in 1 mL of 80:20 H2O-MeOH. The solution was ultrasonicated for 30 min, and any undissolved matter was removed by centrifugation; 10 µL of the supernatant was injected. LC/MS conditions are described in Materials and Methods.

Figure 2. Product ion MS/MS spectra of MH+ ) 233 ion (peak [4], Figure 1) from the HPLC/ESI-MS/MS analysis of an extract from commercially available melatonin. HPLC conditions are as for Figure 1. Interpretation of the spectra is inset into the figure. -H and -2H indicate the additional loss of one and two hydrogens, respectively.

product ion spectrum from the MH+ ion of melatonin (MH+ ) 233, 31:15 min, Figure 1) produced prominent product ions at m/z 174 [the loss of H2NC(O)CH3] and 159 resulting from fragmentation of the side chain connected to the indole ring (as shown in Figure 2). The product ion spectra of peaks [1] and [2] (MH+ ) 249, 22: 00 and 23:15 min, Figure 1) are shown in Figure 3a,b, respectively. Both spectra are almost identical suggesting structural similarities and the possible presence of positional isomers. The two main fragmentation products result from the loss of H2NC(O)CH3 (m/z 190) and CH3CH2NHC(O)CH3 (m/z 162) and suggest these contaminants are hydroxy isomers of melatonin (as illustrated in the insert of Figure 3a). Since hydroxylation imparts hydrophilic character, their elution several

Williamson et al.

Figure 3. Product ion MS/MS spectra of MH+ ) 249 ions (peaks [1] and [2], Figure 1) from the HPLC/ESI-MS/MS analysis of an extract from commercially available melatonin. HPLC conditions are as for Figure 1. Interpretation of the spectra is inset into the figure. -H indicates the additional loss of one hydrogen.

Figure 4. Product ion MS/MS spectra of MH+ ) 265 ion (peak [3], Figure 1) from the HPLC/ESI-MS/MS analysis of an extract from commercially available melatonin. HPLC conditions are as for Figure 1. Interpretation of the spectra is inset into the figure. -H indicates the additional loss of one hydrogen.

minutes before melatonin on a C-18 reversed-phase column is expected, as observed. The product ion spectrum of peak [3] (MH+ ) 265, 25:30 min, Figure 1) was very different from that of melatonin (shown in Figure 4). The numerous product ions produced suggested that the structure of this compound is not based around a substituted indole ring. It is likely that this compound is a substituted indoline compound (as illustrated in Figure 4), as the increased fragmentation could be accounted for by the loss in aromaticity of the fivemembered ring. The most abundant product ion at m/z 164 could be a result of a concomitant loss of CH2OH and CH2NHC(O)CH3 as derived from the structure shown in Figure 4. The product ion spectral data are consistent

Contaminants in Commercial Melatonin

Figure 5. Product ion MS/MS spectra of MH+ ) 477 ions (peaks [5] and [6], Figure 1) from the HPLC/ESI-MS/MS analysis of an extract from commercially available melatonin. HPLC conditions are as for Figure 1.

with a formaldehyde addition product and are chemically feasible based on indole ring chemistry of parent melatonin. The LC/MS/MS analysis of peaks [5]-[8] (MH+ ) 477, 48:50, 49:45, 50:30, and 51:00 min, Figure 1) all produced virtually identical spectra and suggested these are all structural isomers of melatonin-formaldehyde condensation products. The product ion spectra of peaks [5] and [6] are shown in Figure 5, with the dominant fragmentation (m/z 245) resulting from the loss of 232 mass units, most likely the loss of a melatonin moiety from the molecular species. Other fragmentation processes would appear to involve the loss of a melatonin moiety together with fragmentation of the side chain of melatonin. To confirm the origin of the m/z 245 ion, precursor ions were subjected to collisions in the electrospray source region of the mass spectrometer and CID experiments were performed on product ions produced (MS3 experiments). MS3 experiments on the m/z 245 fragment ions from peaks [5] and [6] resulted in very similar spectra (Figure 6). The main product ion in both spectra was the m/z 186 ion which would result from the loss of NH2C(O)CH3 from a melatonin-formaldehyde moiety. The presence of ions at m/z 203 (the loss of CH2dCdO) and 160 [the loss of CH2dCHNHC(O)CH3] clearly identifies the m/z 245 ion as a melatonin formaldehyde product (as illustrated in Figure 6) and can be attributed to the condensation reaction occurring at differing positions of the melatonin molecule. The MS3 experiments on the m/z 245 ion from peaks [7] and [8] produced similar MS3 spectra. The MS/MS and MS3 data from peaks [5]-[8] are therefore consistent with melatonin-formaldehyde condensation isomers. Different intensities of product ions between the spectra can be attributed to the condensation reaction occurring at either the ring or sidechain amine groups of the melatonin molecules. Synthesis of these melatonin-formaldehyde condensation compounds was attempted in order to conclusively

Chem. Res. Toxicol., Vol. 11, No. 3, 1998 237

Figure 6. MS3 spectra of MH+ ) 245 ions from peaks [5] and [6]. The MH+ ) 245 ion was generated by inducing fragmentation of the MH+ ) 477 ions in the skimmer region of the electrospray source to produce MH+ ) 245 ions. This was achieved by increasing the voltage on the heated capillary and tube lens in the electrospray source by 30 V. The MH+ ) 245 ions were then subjected to CID in the normal way. The HPLC conditions are as for Figure 1. Interpretation of the data is inset into the figure. -H indicates the additional loss of one hydrogen. +H indicates the additional gain of one hydrogen.

determine the structure of the detected melatonin contaminants. Melatonin was reacted with formaldehyde under acidic conditions. Analysis of this reaction mixture using the HPLC/MS conditions described for the melatonin studies revealed five main peaks (Figure 7). The most intense peak exhibited a retention time of 31:00 min. This was identified as unreacted melatonin and was characterized by a pseudomolecular ion at MH+ ) 233. The four other peaks observed between 48:30 and 51:30 min had MH+ ions at m/z 477 and had identical retention times to that of the MH+ ) 477 ions observed in the HPLC/MS analysis of commercial melatonin. LC/MS/MS spectra obtained for each of the MH+ ) 477 ions observed in the reaction mixture are inset into Figure 7. These spectra are identical to the LC/MS/MS spectra of the MH+ ) 477 ions found in the melatonin preparations (Figure 5). MS3 data for the m/z 245 product ion were also consistent with that observed from commercial melatonin indicating these compounds are indeed melatonin-formaldehyde condensation products. To further structurally elucidate peaks [5] and [6], these compounds were fraction-collected from a HPLC analysis of the reaction mixture, lyophilized, and subjected to proton NMR analysis. Synthetic melatonin was first analyzed by proton NMR in order to more accurately interpret the data from peaks [5] and [6]. Proton NMR resonances from melatonin dissolved in DMSO-d6 are shown in Table 1. The peak positions are relative to the solvent DMSO-d6 peak at 2.49 ppm. The observed proton splittings in the onedimensional NMR spectra, along with the two-dimensional NMR double-quantum-filtered correlated spectroscopy (DFCOSY) and nuclear Overhauser effect spec-

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Williamson et al.

Table 1. Proton NMR Data at 25 °C from the Analysis of Synthetic Melatonin and Peaks [5] and [6] Isolated from the Reaction between Melatonin and Formaldehyde

melatonin dissolved in CD3OD (500 MHz) observed proton at 1H 1′H 2H 5,5′H 7,7′H 8,8′H side chain NH N-CH2-C N-CH2-N -OCH3 a CH2 b CH2 -C(CO)CH3

ppm not seen 7.02 7.04 6.74 7.20 8.55 3.82 2.89 3.44 1.91

J (Hz)

melatonin dissolved in DMSO-d6 (300 MHz) ppm 10.61 none 7.08 7.00 6.70 7.20 7.91 none none 3.75 2.75 3.28 1.79

Figure 7. Reconstructed ion chromatogram from the HPLC/ ESI-MS analysis of the formaldehyde-melatonin reaction mixture (see Materials and Methods for reaction conditions). The HPLC/ESI-MS conditions are as for Figure 1. The HPLC/MS/ MS spectra for each of MH+ ) 477 peaks are inset into the figure.

troscopy (NOESY) at a mixing time of 1000 ms, aided in unambiguously assigning the proton NMR peaks to the protons of the melatonin molecule (data not shown). It is interesting to note from the observed NOESY spectrum that the ring -OCH3 group is situated close to the body

J (Hz)

J ) 2.2 ) 2.4 ) 8.7 4J ) 2.4 3J ) 8.7 4J 3J

peak [5] dissolved in DMSO-d6 (300 MHz) ppm none 10.72 7.16 7.02 6.75, 6.68 7.40, 7.16 7.92 5.10 none 3.73 2.87, 2.73 3.24 1.76

peak [6] dissolved in DMSO-d6 (300 MHz)

J (Hz)

) 2.5 ) 8.9, 8.8 4J ) 2.4, 2.4 3J ) 8.8 4J 3J

ppm none none 7.40 6.98 6.77 7.61 7.87 none 6.40 3.72 2.71 3.24 1.76

J (Hz)

singlet ) 2.4 ) 8.8 4J ) 2.4 3J ) 8.9 4J 3J

singlet

of the ring and the side chain extends out as a tail. The protons on the side chain show the most changes in the peak positions in going from one solvent (CD3OD) to the other (DMSO-d6). Peaks [5] and [6] were dissolved separately in DMSOd6, and the 300-MHz proton spectra are illustrated in Table 1. Observation of the peaks in the aromatic region, the peak integral areas, and the peak splittings aided in assigning the peaks to two different molecular structures. Significantly, peak [6] produced a proton NMR spectrum in which the two melatonin adducts were identical. The occurrence of a peak at 6.40 ppm corresponding to a N-CH2-N group and the loss of the indole N-H peak at 10.72 ppm allowed easy assignment (Table 1). The loss of symmetry in peak [5] due to nonequivalence of corresponding protons in two adducts gave rise to more NMR observed peaks. The appearance of an indole N-H peak at 10.72 ppm equivalent to one proton together with a one-proton strong signal at 7.40 ppm allowed assignment of peak [5] (Table 1). It is likely that peak [7] contains the methyl bridge between the amine group on the side chain of one melatonin moiety and the 2-position of the indole ring. Peak [8] would therefore contain the methyl bridge between the amine group on the side chain of one melatonin moiety and the amine group on the indole ring of the second melatonin moiety.

Discussion Following the outbreak of EMS in the United States in 1989, analysis of EMS-implicated Trp revealed the presence of many contaminants. Six of these contaminants were identified as being associated with EMS (22). These case-related contaminants were labeled as peak

Contaminants in Commercial Melatonin

C, UV-5, E, 200, FF, and AAA. Peak UV-5 was subsequently identified as 3-(phenylamino)alanine (28), and peak E was identified as 1,1′-ethylidenebis(tryptophan) (29) (see below for structure). It was originally believed that peak E was the contaminant responsible for the EMS outbreak (27). However, peak E has never been shown to cause EMS in any laboratory animal, and therefore other case-related contaminants were investigated. Peak 200 was identified as 2-(3-indolylmethyl)-L-tryptophan (24). Peak C was hypothesized to be a positional isomer of 5-hydroxytryptophan (22) (see below for structure). However, 8 years later the contaminant(s) responsible for the outbreak has still not been determined. Reports that melatonin, a structurally similar, indolebased food supplement, had previously induced eosinophilia (13) gave cause for concern. In particular, given that a number of unknown impurities had been reported to be present in commercial preparations of melatonin (11, 16), we investigated the structure of the contaminants.

Analysis of commercial preparations of melatonin by LC/ESI-MS enabled the detection and molecular weight assignment of seven contaminants. Tandem mass spectrometry identified two of these contaminants as hydroxymelatonin isomers (peaks [1] and [2]). This is a significant finding as these are melatonin structural analogues of peak C which was identified as a contaminant in EMS-implicated Trp. In addition tandem mass spectrometry together with MS3 experiments suggested peaks [5]-[8] were formaldehyde condensation products. We confirmed this observation by synthesis of the formaldehyde condensation products of melatonin and subsequent analysis of these compounds by tandem mass spectrometry and NMR analysis. This is an even more significant observation as these melatonin-formaldehyde condensation products found in commercial melatonin are structural analogues of peak E, also present as a caseimplicated compound in EMS-related Trp. These formaldehyde condensation products are present in amounts of ∼0.1-0.5% of unmodified melatonin in all three of the commercial preparations that were analyzed. This compares with the occurrence of peak E which is present in much lower quantities of case-related Trp (∼0.05-0.1% of parent Trp). It is important to note that 15 mg/day melatonin administered for 4 weeks to cancer patients induced eosinophilia (13, 14). It is not clear if commercial or synthetically pure melatonin was used in this study. Although no cases of EMS-like diseases have been reported from other subjects ingesting melatonin, it is perhaps significant that a typical dose regime of melatonin to alleviate jetlag is ∼5 mg/day for 7 days. This contrasts with the reported intake of ∼0.5-4.0 g/day Trp for up to several months for patients who contracted

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EMS. This represents an ∼100-800-fold decrease intake of contaminants, and this may be why, to date, we know of no new EMS outbreak that has been associated with melatonin consumption. However, since daily intake of dietary supplements is not medically supervised, the probability of increased doses of melatonin being consumed is very real. This study highlights the need for tighter controls on nutritional supplements sold and used as drugs.

Acknowledgment. This work was supported in part by NIH Grant AI31155, Showa Denko K.K., Finnigan MAT, and Mayo Foundation.

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