Unknown Constituent Identification in Topical …...Unknown Constituent Identification in Topical...
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Unknown Constituent Identification in Topical Preparation using a Q-TOF Mass Spectrometer Evelyn H. Wang, Helen Hao, Jeffrey H. Dahl, Jennifer C. Davis, Priyanka Chitranshi, Katie Pryor, Christopher T. Gilles Shimadzu Scientific Instruments. Columbia, Maryland 2. Introduction Skin is the most exposed organ in the human body and is susceptible to various diseases and disorders. Topical preparations are often used to provide treatment for skin since it is easily administered. Although most topical preparations are meant to provide treatment locally, a small amount of those drugs, including the active compounds and other constituents, can be absorbed and lead to systemic effect. To avoid undesirable effects caused by the unknown constituents in the topical preparation, an LCMS method utilizing a Shimadzu LCMS- 9030 Q-TOF was created to analyze topical preparation products. A typical workflow on data analysis was developed for conducting unknown constituent analysis in topical preparation. 4-2. LC-MS/MS MS/MS fragment patterns and assignments results exhibit the similarity between the main ingredient and the unknown compound (similar fragments highlighted in green). The results also suggest the differences between the unknown constituent and the main ingredient are expected to be on the two functional groups circled in red and purple on the diflorasone diacetate structure. 5. Conclusions The Shimadzu Q-TOF mass spectrometer with the Shimadzu Formula Prediction and Assign software provide a simple and straightforward qualitative analysis workflow. In this experiment, LCMS-9030 demonstrated the excellent capabilities to identify low level unknowns in a complex extraction sample. TP-587 Disclaimer: The products and applications in this presentation are intended for Research Use Only (RUO). Not for use in diagnostic procedures. 1. Overview Using a high-resolution mass spectrometer and a new qualitative analysis software (Assign) to identify low level unknowns in topical preparations. 3. Method A topical preparation was commercially obtained and evaluated for unknown constituent analysis. Qualitative analysis of the unknown constituent was performed on a Shimadzu LCMS-9030 Q- TOF mass spectrometer. Both the LC and MS parameters are outlined in Table 1. This poster will explore the typical workflow for conducting qualitative analysis of an unknown constituent. (Figure 1) LC -30AD LCMS-9030 Column: Shimadzu ODS-III column (150 x 2.1 mm) Nebulizing Gas: 2 L/min Mobile phase: A: 0.1 % Formic acid in water; B: 0.1 % Formic acid in Acetonitrile Heating Gas: 10 L/min Flow Rate: 0.4 mL/min Interface Temp.: 300 °C Oven Temp.: 40°C DL Temp.: 250 °C Injection Vol.: 1 μL Heat Block Temp.: 400 °C UV wavelength: 237 nm Drying Gas: 10 L/min Scan range (m/z): 100-1000 Table 1 LC-MS conditions and parameters Step 1: Obtain accurate mass of a prominent ion under the UV unknown peak . (LabSolutions) Step 3: Possible structures of the predicted formula can be found in open source databases such as Pubchem and Metlin. Step 4: With a proposed structure and the MS/MS data, Insight Assign software provides possible fragmentation assignments to the obtained fragment ions. Step 5: Unknown tentatively identified Step 2: Set element selection range and use Formula Predictor (LabSolutions Insight) to identify compounds according to the accurate mass and isotopic pattern Qualitative Analysis Workflow Figure 1 A typical workflow for analysing an unknown constituent using a Q-TOF mass spectrometer. 4. Results 4-1. LC-MS To mimic a standard pharmaceutical unknown analysis, tandem UV and MS data were acquired. The unknown preparation contains an additional peak (Rt=15 min) that does not exist in the control preparation. Accurate masses and the MS/MS fragments of the main ingredient, diflorasone diacetate (Rt=13 min; m/z 495.2186) and the prominent ion (Rt=15 min; m/z 485.1907) under the unknown peak were acquired. (Figure 2) 1.60e5 1:MS(+) RT:[14.982-15.217]-[14.065-16.687] 485.19071 485.36694 486.19397 486.33593 487.18850 487.35514 488.19143 488.31864 489.19447 m/z 484 485 486 487 488 489 490 491 492 493 494 0.0e0 2.0e4 4.0e4 6.0e4 8.0e4 1.0e5 1.2e5 1.4e5 1.6e5 2.87e5 1:MS(+) RT:[12.880-13.152]-[11.257-14.577] 495.21860 495.22907 495.25629 495.31043 496.02528 496.22298 496.39485 496.79552 497.22555 497.36816 498.22886 499.23658 m/z 494.0 494.5 495.0 495.5 496.0 496.5 497.0 497.5 498.0 498.5 499.0 499.5 500.0 500.5 501.0 501.5 502.0 0.0e0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 Unknown m/z 485.1907 Main m/z 495.2186 Main Unknown A) B) C) Figure 2 A) TIC of the unknown sample showing both the main and the unknown peaks. B) Mass spectrum of the main ingredient, diflorasone diacetate, with m/z 495.2186, and C) mass spectrum of the unknown ion, m/z 485.1907. Distinct isotopic distributions and ratio were observed. 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z 0.0 1.0 2.0 3.0 4.0 Inten.(x10,000) 317.1531 289.1582 275.1426 121.0643 335.1637 159.0800 245.0957 221.0959 203.0861 353.1742 373.1804 395.1847 115.0538 91.0538 261.1269 415.1910 435.1972 MS/MS of main m/z 495.2186 Control Sample Main compound: Diflorasone diacetate Figure 3 Fragment structure assignment results from the Shimadzu Insight Assign software. MS/MS data obtained by the Q-TOF on the main ingredient, ion m/z 495.2186, and the known structure of diflorasone diacetate were used for the prediction. 4-3. Formula prediction and fragment assignment results of the unknown constituent Based on the clues provided by the accurate mass, isotopic distribution, and MS/MS fragmentation patterns of the unknown and main ingredient, a proper range of possible elements for formula prediction was set. Sixteen potential formulas were predicted for the unknown ion. (Table 2) The best matched formula according to accurate mass, isotopic distribution, and isotopic ratio was C 25 H 31 O 5 F 2 Cl. Two possible structures, Halobetasol Propionate and 11-Propionate 21-chlorodiflorasone, were matched to the predicted formula of the unknown in an open source database (Metlin). Shimadzu Insight Assign software was then used to assign the MS/MS fragments of the unknown against the possible fragments of the two possible structures. (Figure 4) Halobetasol Propionate had a closer match (82.7%) in fragment assignment of the unknown’s fragmentation spectrum. Therefore, Halobetasol Propionate was tentatively identified as the unknown compound. # Score Pred. (M) Pred. m/z Meas. m/z Diff. (mDa) Formula (M) Ion Diff. (ppm) Iso Score DBE 1 96.98 484.18281 485.19008 485.19072 0.64 C25 H31 O5 F2 Cl [M+H]+ 1.310 97.35 9.0 2 91.14 484.18395 485.19123 485.19072 -0.51 C22 H32 O6 F3 Cl [M+H]+ -1.046 90.69 5.0 3 88.45 462.20205 485.19127 485.19072 -0.55 C22 H35 O8 Cl [M+Na]+ -1.126 87.75 5.0 4 86.80 462.20179 485.19101 485.19072 -0.29 C26 H29 O4 F3 [M+Na]+ -0.607 96.72 11.0 5 62.00 484.18167 485.18894 485.19072 1.78 C28 H30 O4 F Cl [M+H]+ 3.665 60.88 13.0 6 57.52 462.20294 485.19216 485.19072 -1.44 C23 H30 O5 F4 [M+Na]+ -2.963 66.03 7.0 7 56.01 462.20065 485.18987 485.19072 0.85 C29 H28 O3 F2 [M+Na]+ 1.748 63.24 15.0 8 54.22 484.18256 485.18983 485.19072 0.89 C29 H25 O F5 [M+H]+ 1.829 61.31 15.0 9 48.90 462.19960 485.18882 485.19072 1.90 C20 H34 O6 F3 Cl [M+Na]+ 3.913 46.80 2.0 10 46.97 484.18386 485.19113 485.19072 -0.41 C34 H25 O2 F [M+H]+ -0.855 52.61 22.0 11 45.48 484.18509 485.19237 485.19072 -1.65 C19 H33 O7 F4 Cl [M+H]+ -3.401 42.10 1.0 12 44.70 462.20319 485.19241 485.19072 -1.69 C19 H36 O9 F Cl [M+Na]+ -3.482 41.35 1.0 13 42.97 484.18272 485.18999 485.19072 0.73 C37 H24 O [M+H]+ 1.501 48.57 26.0 14 29.60 484.18500 485.19228 485.19072 -1.56 C31 H26 O3 F2 [M+H]+ -3.210 35.31 18.0 15 21.77 462.19951 485.18873 485.19072 1.99 C32 H27 O2 F [M+Na]+ 4.104 28.20 19.0 16 21.49 484.18141 485.18869 485.19072 2.03 C32 H24 F4 [M+H]+ 4.184 28.11 19.0 Table 2 Formula prediction results of the unknown ion (m/z 485.1907). 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z 0.0 2.5 5.0 7.5 Inten.(x1,000) 261.127 4 121.064 6 353.1304 171.0802 246.1039 317.153 5 299.1431 159.0802 221.096 1 203.086 3 373.1366 335.1195 115.0541 91.0540 276.1508 371.1408 391.1473 343.1458 Halobetasol propionate 11 - Propionate 21 - chloro diflorasone 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z 0.0 2.5 5.0 7.5 Inten.(x1,000) 261.1274 121.0646 353.1304 171.0802 246.1039 317.153 5 299.143 1 159.0802 221.0961 203.086 3 373.1366 335.1195 115.0541 91.0540 276.1508 371.1408 391.1473 343.1458 Figure 4 Fragment structure assignment results of the two possible unknown structures, Halobetasol Propionate and 11-Propionate 21-chlorodiflorasone, from the Shimadzu Insight Assign software. C 25 H 31 O 5 F 2 Cl Halobetasol propionate Proposed molecule for unknown ion m/z 485 . 1907 ( Rt = 15 min) in the unknown topical preparation sample
Transcript of Unknown Constituent Identification in Topical …...Unknown Constituent Identification in Topical...
Unknown Constituent Identification in Topical Preparation using a Q-TOF Mass SpectrometerEvelyn H. Wang, Helen Hao, Jeffrey H. Dahl, Jennifer C. Davis, Priyanka Chitranshi, Katie Pryor, Christopher T. Gilles
Shimadzu Scientific Instruments. Columbia, Maryland
2. Introduction
Skin is the most exposed organ in the human
body and is susceptible to various diseases and
disorders. Topical preparations are often used to
provide treatment for skin since it is easily
administered. Although most topical
preparations are meant to provide treatment
locally, a small amount of those drugs, including
the active compounds and other constituents,
can be absorbed and lead to systemic effect. To
avoid undesirable effects caused by the
unknown constituents in the topical preparation,
an LCMS method utilizing a Shimadzu LCMS-
9030 Q-TOF was created to analyze topical
preparation products. A typical workflow on data
analysis was developed for conducting unknown
constituent analysis in topical preparation.
4-2. LC-MS/MSMS/MS fragment patterns and assignments results exhibit the similarity between the main ingredient
and the unknown compound (similar fragments highlighted in green). The results also suggest the
differences between the unknown constituent and the main ingredient are expected to be on the two
functional groups circled in red and purple on the diflorasone diacetate structure.
5. ConclusionsThe Shimadzu Q-TOF mass spectrometer with the Shimadzu Formula Prediction and Assign software provide a simple and
straightforward qualitative analysis workflow. In this experiment, LCMS-9030 demonstrated the excellent capabilities to
identify low level unknowns in a complex extraction sample.
TP-587
Disclaimer: The products and applications in this presentation are intended for Research Use Only (RUO). Not for use in diagnostic procedures.
1. Overview Using a high-resolution mass spectrometer and
a new qualitative analysis software (Assign) to
identify low level unknowns in topical
preparations.
3. Method
A topical preparation was commercially obtained
and evaluated for unknown constituent analysis.
Qualitative analysis of the unknown constituent
was performed on a Shimadzu LCMS-9030 Q-
TOF mass spectrometer. Both the LC and MS
parameters are outlined in Table 1. This poster
will explore the typical workflow for conducting
qualitative analysis of an unknown constituent.
(Figure 1)
LC -30AD LCMS-9030
Column:Shimadzu ODS-III column
(150 x 2.1 mm)Nebulizing Gas: 2 L/min
Mobile
phase:
A: 0.1 % Formic acid in
water;
B: 0.1 % Formic acid in
Acetonitrile
Heating Gas: 10 L/min
Flow Rate: 0.4 mL/min Interface Temp.: 300 °C
Oven Temp.: 40°C DL Temp.: 250 °C
Injection
Vol.:1 µL Heat Block Temp.: 400 °C
UV
wavelength:237 nm Drying Gas: 10 L/min
Scan range (m/z): 100-1000
Table 1 LC-MS conditions and parameters
Step 1:Obtain accurate mass of a prominent ion under the UV unknown peak . (LabSolutions)
Step 3:Possible structures of the predicted formula can be found in open source databases such as Pubchem and Metlin.
Step 4:With a proposed structure and the MS/MS data, Insight Assign software provides possible fragmentation assignments to the obtained fragment ions.
Step 5:Unknown tentatively identified
Step 2:Set element selection range and use Formula Predictor (LabSolutions Insight) to identify compounds according to the accurate mass and isotopic pattern
Qualitative Analysis Workflow
Figure 1 A typical workflow for analysing an
unknown constituent using a Q-TOF mass
spectrometer.
4. Results
4-1. LC-MSTo mimic a standard pharmaceutical unknown analysis,
tandem UV and MS data were acquired. The unknown
preparation contains an additional peak (Rt=15 min)
that does not exist in the control preparation. Accurate
masses and the MS/MS fragments of the main
ingredient, diflorasone diacetate (Rt=13 min; m/z
495.2186) and the prominent ion (Rt=15 min; m/z
485.1907) under the unknown peak were acquired.
(Figure 2)
1.60e51:MS(+) RT:[14.982-15.217]-[14.065-16.687]
485.19071
485.36694
486.19397
486.33593
487.18850
487.35514
488.19143
488.31864 489.19447
m/z
484 485 486 487 488 489 490 491 492 493 4940.0e0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
2.87e51:MS(+) RT:[12.880-13.152]-[11.257-14.577]
495.21860
495.22907
495.25629
495.31043496.02528
496.22298
496.39485 496.79552
497.22555
497.36816 498.22886 499.23658
m/z
494.0 494.5 495.0 495.5 496.0 496.5 497.0 497.5 498.0 498.5 499.0 499.5 500.0 500.5 501.0 501.5 502.00.0e0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
Unknownm/z 485.1907
Mainm/z 495.2186
Main
Unknown
A)
B)
C)
Figure 2 A) TIC of the unknown sample showing both the main and the unknown peaks.
B) Mass spectrum of the main ingredient, diflorasone diacetate, with m/z 495.2186, and
C) mass spectrum of the unknown ion, m/z 485.1907. Distinct isotopic distributions and
ratio were observed.
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0
1.0
2.0
3.0
4.0Inten.(x10,000)
317.1531289.1582
275.1426
121.0643 335.1637
159.0800 245.0957221.0959
203.0861
353.1742
373.1804
395.1847115.053891.0538
261.1269
415.1910
435.1972
MS/MS of main m/z 495.2186
Control SampleMain compound:
Diflorasone diacetate
Figure 3 Fragment structure assignment results from the Shimadzu Insight Assign
software. MS/MS data obtained by the Q-TOF on the main ingredient, ion m/z 495.2186,
and the known structure of diflorasone diacetate were used for the prediction.
4-3. Formula prediction and fragment assignment results of the unknown
constituentBased on the clues provided by the accurate mass, isotopic distribution, and MS/MS fragmentation patterns of the unknown and
main ingredient, a proper range of possible elements for formula prediction was set. Sixteen potential formulas were predicted for
the unknown ion. (Table 2) The best matched formula according to accurate mass, isotopic distribution, and isotopic ratio was
C25H31O5F2Cl. Two possible structures, Halobetasol Propionate and 11-Propionate 21-chlorodiflorasone, were matched to the
predicted formula of the unknown in an open source database (Metlin). Shimadzu Insight Assign software was then used to assign
the MS/MS fragments of the unknown against the possible fragments of the two possible structures. (Figure 4) Halobetasol
Propionate had a closer match (82.7%) in fragment assignment of the unknown’s fragmentation spectrum. Therefore, Halobetasol
Propionate was tentatively identified as the unknown compound.
# Score Pred. (M) Pred. m/z Meas. m/z Diff. (mDa) Formula (M) Ion Diff. (ppm) Iso Score DBE
1 96.98 484.18281 485.19008 485.19072 0.64 C25 H31 O5 F2 Cl [M+H]+ 1.310 97.35 9.02 91.14 484.18395 485.19123 485.19072 -0.51 C22 H32 O6 F3 Cl [M+H]+ -1.046 90.69 5.0
3 88.45 462.20205 485.19127 485.19072 -0.55 C22 H35 O8 Cl [M+Na]+ -1.126 87.75 5.0
4 86.80 462.20179 485.19101 485.19072 -0.29 C26 H29 O4 F3 [M+Na]+ -0.607 96.72 11.0
5 62.00 484.18167 485.18894 485.19072 1.78 C28 H30 O4 F Cl [M+H]+ 3.665 60.88 13.0
6 57.52 462.20294 485.19216 485.19072 -1.44 C23 H30 O5 F4 [M+Na]+ -2.963 66.03 7.0
7 56.01 462.20065 485.18987 485.19072 0.85 C29 H28 O3 F2 [M+Na]+ 1.748 63.24 15.0
8 54.22 484.18256 485.18983 485.19072 0.89 C29 H25 O F5 [M+H]+ 1.829 61.31 15.0
9 48.90 462.19960 485.18882 485.19072 1.90 C20 H34 O6 F3 Cl [M+Na]+ 3.913 46.80 2.0
10 46.97 484.18386 485.19113 485.19072 -0.41 C34 H25 O2 F [M+H]+ -0.855 52.61 22.0
11 45.48 484.18509 485.19237 485.19072 -1.65 C19 H33 O7 F4 Cl [M+H]+ -3.401 42.10 1.0
12 44.70 462.20319 485.19241 485.19072 -1.69 C19 H36 O9 F Cl [M+Na]+ -3.482 41.35 1.0
13 42.97 484.18272 485.18999 485.19072 0.73 C37 H24 O [M+H]+ 1.501 48.57 26.0
14 29.60 484.18500 485.19228 485.19072 -1.56 C31 H26 O3 F2 [M+H]+ -3.210 35.31 18.0
15 21.77 462.19951 485.18873 485.19072 1.99 C32 H27 O2 F [M+Na]+ 4.104 28.20 19.0
16 21.49 484.18141 485.18869 485.19072 2.03 C32 H24 F4 [M+H]+ 4.184 28.11 19.0
Table 2 Formula prediction results of the unknown ion (m/z 485.1907).
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0
2.5
5.0
7.5
Inten.(x1,000)261.127
4
121.064
6 353.1304171.0802 246.1039 317.153
5299.1431159.0802 221.096
1203.086
3
373.1366
335.1195115.054191.0540
276.1508
371.1408391.1473343.1458
Halobetasol propionate
11-Propionate 21-chloro diflorasone
50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0
2.5
5.0
7.5
Inten.(x1,000)261.1274
121.0646
353.1304171.0802 246.1039 317.153
5299.143
1159.0802 221.0961203.086
3
373.1366
335.1195115.054191.0540
276.1508
371.1408391.1473343.1458
Figure 4 Fragment structure assignment results of the two possible unknown structures, Halobetasol
Propionate and 11-Propionate 21-chlorodiflorasone, from the Shimadzu Insight Assign software.
C25H31O5F2Cl
Halobetasol propionate
Proposed molecule for unknown ion m/z 485.1907 (Rt = 15 min) in the unknown
topical preparation sample
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