Analysis of Impurities in Melatonin by LC/TOF-MS Sharanya Reddy, Shida Shen, Eugene Davidov, Adam Patkin e d lit u H N Ion 50000 p NH H3C O m or A difference calculated CH3 40000 observed H N O OH CH3 NH O H3C O 1 9 0 .0 9 [M+H]+ 249.1234 249.1235 0.4 ppm [M+H-H2O]+ 231.1128 231.1131 1.3 ppm [M+HCH3CONH2] + 190.0863 190.0865 1.1 ppm 100 2 7 1 .1 1 250 m /z 300 350 Peaks E and F are most likely dimers of melatonin based on accurate mass data (Figure 8) 400 Figure 4. MS spectrum and mass accuracy of peaks’ B and C (isomers) from Figure 2 M ass G E N ((0 2 .5 0 1 0 0 :0 2 . 5 3 4 3 3 ) - (0 2 .3 0 1 0 0 :0 2 .3 3 4 3 3 )) 55000 C u rv e 1 50000 Peaks A and D have identical accurate masses, but have different spectra and elution times. We speculate the structure of peak A to be a di-oxidation product of melatonin based on accurate mass of [M+H]+ and its fragments. (Figure 5) G EN 2 0 0 0 0 46 3.2 4 [M+H]+ N 45000 CH3 Ion d e 40000 NH l i t u 35000 H3C O O [M+H]+ calculated observed difference 463.2339 463.2346 1.5 ppm m p 30000 25000 H3C O A Peaks B and C were identified as oxidation products of melatonin based on accurate mass of the [M+H]+ ions and fragment ions (Figure 4). The difference in mass of the fragment ion [M+HCH3CONH2]+ (m/z 190) in peaks B/C to the fragment ion m/z 172 in melatonin spectrum (Figure 3) suggests the peaks B/C are hydroxy products of melatonin and oxidation has occurred at the indole ring or aromatic ring of melatonin. Since the hydroxy products are hydrophilic compared to melatonin, they will elute earlier than melatonin. The results are consistent with previously published work by Williamson, et al (1). NH 20000 O CH3 15000 [M+Na]+ N 10000 48 5.2 2 5000 0 100 150 200 250 300 350 m /z 400 450 500 550 600 Figure 8. Spectrum of peaks E and F Peaks G and H are likely formaldehyde dimers of melatonin (data not shown) . These dimers were also observed by Williamson, et al. (1) who further confirmed their presence by synthesis and NMR spectroscopy. 1. Williamson, B. L., et al. Chemical Research Toxicology, 1998, 11, 234-240 M ass ((0 0 .3 8 4 3 3 :0 0 . 4 6 7 6 6 ) - (0 0 .3 3 4 3 3 :0 0 .3 6 7 6 6 )) 1 8 8 .0 7 C u rv e 1 2 4 7 .1 1 1 7 5 0 0 OH 1 5 0 0 0 p l i t u d e Figure 2 shows the analysis of melatonin tablet in pulse versus trap mode. In trap mode, impurities are detected at higher S/N (3-5 fold higher) than in pulse mode and additional impurity peaks that were not visible in pulse mode were detected. 200 H N OH CH3 1 2 5 0 0 NH O H3C O 2 6 5 .1 2 1 0 0 0 0 A m Pulse vs. Trap Acquisition Mode: The AxIONTM 2 TOF is fitted with a hexapole ion guide that traverses the different vacuum regions of the mass spectrometer for maximum ion transmission. In pulse mode operation, the ion guide solely transmits the ions efficiently into the pulser region. However, in trap mode operation, the hexapole ion guide can be used to trap ions. The ions are trapped by raising the potential of the ion guide exit lens above the ion guide DC offset potential. The trapped ions are then released into the pulser region after a certain delay. As the ions move from ion guide to pulser region, there is some separation of ions based on m/z. By timing the gating and pulsing of the ions, one can selectively accumulate and transmit a defined m/z range of ions into the TOF region drift tube selectively enhancing the intensity of the selected m/z range. Using the AxIONTM 2 TOF in trap mode can significantly increase the S/N of ions 3 to 5 fold in comparison to pulse mode. 150 For these reasons, we speculated the structure of peak D to be N1acetyl-N2-formyl-5-methoxykynurenin (AFMK). We confirmed the structure of peak D by analyzing a synthesized standard of AFMK which matched both the retention time and accurate mass spectrum of peak D (data not shown). 2 3 1 .1 1 2 0 7 .1 2 0 7 5 0 0 Ion calculated observed difference [M+H]+ 265.1183 265.1187 1.5 ppm [M+Na]+ 287.1002 287.1008 2 ppm [M+H-H2O]+ 247.1077 247.1080 1.2 ppm [M+H-H2OCH3CONH2]+ 188.0706 188.0706 0 ppm 4 Summary and Conclusions 2 8 7 .1 0 1 7 6 .0 7 B IC m 4 0 0 0 0 0 / z 5 0 0 0 ( 2 4 5 .0 0 0 : 5 0 0 .0 0 0 ) A m p l i tu d e 3 5 0 0 0 0 A m p lit u d e 3 0 0 0 0 0 2 5 0 0 0 0 2 0 0 0 0 0 impurities 1 5 0 0 0 0 melatonin 2 5 0 0 0 Pulse mode 1 0 0 m/z 240-500 5 0 0 0 0 0 1 2 3 T im e ( m in ) 4 5 6 7 B IC m 4 0 0 0 0 0 / z ( 2 4 5 .0 0 0 : 5 0 0 .0 0 0 ) A m 3 5 0 0 0 0 2 5 0 0 0 0 2 0 0 0 0 0 C 1 5 0 0 0 0 Trap mode melatonin impurities A m p lit u d e 3 0 0 0 0 0 B D A 1 0 0 0 0 0 5 0 0 0 0 m/z 240-500 impurities E F G 1 5 0 2 0 0 2 5 0 m /z 3 0 0 3 5 0 4 0 0 Figure 5. MS spectrum and accurate mass of peak A from Figure 2 1 0 0 0 0 0 H p l i tu d e The structure of peak D was speculated to be a substituted indoline compound by Williamson, et al (1) (Figure 6) who used a triple quadrupole for analysis. The elemental composition (C14H20N2O3) of the indoline structure proposed by Williamson, et al. (1) would have an accurate mass of 265.15467 which is ~140 ppm higher than the mass obtained for peak D by the AxIONTM 2 TOF (Figure 7), thus suggesting the proposed structure may be incorrect. 0 1 2 3 T im e ( m in ) 4 5 6 CH3 M ass C u rv e 1 225000 23 3.1 3 17 4.0 9 H N 200000 CH3 175000 H3C O 125000 100000 Figure 6. Structure of peak D speculated by Williamson, et al. (1). NH O 150000 M ass 174 Ion C u rv e 1 calculated observed difference O 35000 NH 30000 e 75000 G E N ((0 0 .7 8 4 3 3 :0 0 .8 5 1 0 0 ) - (0 0 .7 5 1 0 0 :0 0 .7 5 1 0 0 )) 40000 25000 174.0913 174.0913 1.3 ppm 0.0 ppm lit u d [M+HCH3CONH2]+ 233.1282 2 3 7 .1 3 25000 H3C O 20000 15000 0 100 150 200 m /z 250 300 350 NH O observed difference 265.1183 265.1187 1.5 ppm [M+H-CO]+ 237.1234 237.1235 0.4 ppm [M+H-COCH3CONH2]+ 178.0863 178.0858 -2.8 ppm 10000 5000 Figure 3. melatonin spectrum and mass accuracy calculated [M+H]+ AFMK A 15 9.0 7 Ion CH3 O 2 8 7 .1 0 m 50000 233.1285 H 1 7 8 .0 9 p [M+H]+ 2. The accurate mass measurement capability of the AxION 2 TOF could easily distinguished between elemental compositions C14H20N2O3 (m/z = 265.1547) and C13H16N2O4 (m/z = 265.1183). This provided the confirmation that a previously published structure proposed from data provided by a triple quadrupole MS was incorrect for a specific melatonin impurity. 3. The significant improvement in sensitivity gained from operating the AxIONTM 2 TOF in trap mode along with the mass measurement accuracy allowed the identification of additional melatonin impurities. These included N1-acetyl-N2-formyl-5methoxykynurenin (AFMK) as well as melatonin dimers not observed in previously published work. NH O H3C O G E N ((0 1 .1 6 7 6 6 :0 1 . 3 6 7 6 6 ) - (0 1 .0 0 1 0 0 :0 1 .2 8 4 3 3 )) 1. Operating the TOF MS in trap (vs. pulse) mode provided a 3 to5 fold improvement in sensitivity. The increased sensitivity allowed additional impurities to be detected and measured in an OTC melatonin sample that were not observed in pulse mode. OH H N 7 Figure 2. BICs of melatonin in pulse versus trap mode of operation e Samples and standards: OTC melatonin was obtained from a local grocery store. N1-acetyl-N2-formyl-5-methoxykynurenin (AFMK) was obtained from Cayman Chemicals (Michigan, USA) Sample preparation conditions: Melatonin tablet (containing 1 mg melatonin) was crushed in a mortar and pestle and dissolved in Milli Q water (10 ml). The mixture was shaken vigorously and then centrifuged at 6000 rpm for 10 min. The supernatant was injected on column. LC conditions: LC Pump: PerkinElmer FlexarTM FX-10 LC pump Column: PerkinElmer Brownlee SupraTM column C18 (1.9 µm, 2.1 x 50 mm) Mobile phase A: water containing 0.1% formic acid B: acetonitrile containing 0.1% formic acid Gradient conditions: 0-5 min 20% B to 40%B in a linear gradient 5-7 min 40% B to 70% B Flow rate: 0.4 mL/min Mass Spectrometry conditions: Mass spectrometer: PerkinElmer AxIONTM 2 TOF Ionization source: PerkinElmer UltrasprayTM 2 (Dual ESI source) OH 60000 Results d 2 Experimental conditions 70000 10000 l i t u Figure 1. the structure of melatonin C u rv e 1 2 4 9 .1 3 20000 3 ((0 0 .4 8 4 3 3 :0 0 .6 6 7 6 6 ) - (0 0 .4 3 4 3 3 :0 0 .4 6 7 6 6 )) 80000 30000 p Melatonin is a hormone secreted by the brain to help regulate sleep. Since melatonin is found in certain foods, it can be sold as a dietary “over-the-counter” (OTC) supplement. Unlike pharmaceuticals, OTC drugs are not regulated by agencies such as the Food and Drug Administration (FDA). Hence, impurities present in OTCs are not required to be characterized, quantified, or reported. Impurities found in OTC drugs can be harmful and even cause death. For instance, in 1989 an epidemic referred to as eosinophilia-myalgia syndrome (EMS) resulted in the death of thirty people and affected as many as 1500 people. The epidemic was attributed to consumption of the OTC supplement tryptophan which was manufactured by a single chemical company. The synthesized tryptophan had at least six impurities which could have caused the onset of EMS. Since melatonin has structural similarities to tryptophan we have used the high resolution and accurate mass capability of time-of-flight mass spectrometry to identify and characterize impurities in OTC melatonin tablets. m Introduction 100-700 m/z 100-700 m/z (D7:29, D8:43) Internal calibrant (diluted Agilent tune mix) 25 µL/min through the 2nd ESI sprayer. A 1 Pulse mode: Trap mode: Calibrant: Flow: M a ss G E N 0 100 150 200 250 m /z 300 350 400 Figure 7. MS spectrum of peak D PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com