Analysis of Impurities in Melatonin by LC/TOF-MS

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
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