Supporting information
Coupling of Ultrafast LC with Mass Spectrometry by DESI
Yi Cai1, Yong Liu2, Roy Helmy2 and Hao Chen1,3*
1
Center for Intelligent Chemical Instrumentation, Department of Chemistry and
Biochemistry; 3Edison Biotechnology Institute, Ohio University, Athens, OH, USA 45701
2
Merck & Co., Inc. One Merck Drive, Whitehouse Station, New Jersey, USA 08889
1. LC/DESI-MS experimental details
Chemicals
HPLC-grade methanol and acetonitrile were purchased from Fisher Chemicals
(Pittsburgh, PA) and from EMD Chemicals Inc. (Billerica, MA), respectively. Dopamine
hydrochloride, 3-methoxytramine hydrochloride (≥95.5%), L-kynurenine, L-tryptophan
(≥98%), codeine, cocaine hydrochloride, flunitrazepam, ketoprofen (≥98%), ibuprofen (≥98%)
and fenoprofen calcium salt hydrate were all purchased from Sigma-Aldrich (St. Louis, MO).
Cola was Pepsi purchased from Walmart.
LC/DESI-MS apparatus and LC separation conditions
In all monolithic column LC experiments, the mobile phase elution flow rate was
kept at 3.0 mL/min and a 20 μL injection loop was used for sample loading. With such a flow
rate and 100 μm i.d. orifice being used, the splitting ratio was measured to be ca. 4:96, which
is subject to small change depending on the composition of mobile phase solvent (probably
due to viscosity change from varying solvent composition). For example, elution using 5%
and 95% MeOH at the flow rate of 3.0 mL/min led to splitting ratios of 4.5:95.5 and 3.9:96.1,
respectively.
1
Phenomenex Onyx Monolithic C18 column (100×4.6 mm) was employed for the
separation of dopamine, 3-methoxytyramine, L-tryptophan and L-kynurenine in urine. The
urine sample doped with these compounds was diluted 1:1 (v/v) with water to have the final
concentration of 60 μM for each species, and then filtered with 0.2 μm Nylon Membrane for
removing possible particulates before LC injection. Solvent A was 0.1% FA in H2O, and
solvent B was 0.1% FA in ACN with elution program as such: 0-1min, 1% B; 1-3min, 1% B
was ramped to 50%.
For analysis of acidic anti-inflammatory drug mixture consisting of ketoprofen,
fenoprofen and iburopfen (100 μM each), Phenomenex Onyx Monolithic C18 column
(100×4.6 mm) was used again. The isocratic elution program was used H2O:MeOH:FA
(30:70:0.05, by volume).
For the UPLC experiments, ACQUITY UPLC® BEH C18 column (50×2.1 mm) was
employed for the separation. A cola sample spiked with codeine, cocaine and flunitrazepam
(150 μM each) was diluted 10 times by water before injection for UPLC/DESI-MS analysis.
The mobile phase consisted of A: 0.1% FA in H2O and B: 0.1% FA in ACN. A linear
gradient program ran from 23% to 90% solvent B in 3 min. With the mobile phase elution
flow rate kept at 0.3 mL/min, the splitting ratio using the PEEK capillary tube carrying the
100 μm i.d. orifice was 1:1. For the high temperature (80 oC) separation of the drug mixture
in cola, a gradient program held 23% B for 0.3 min then ramped to 90% B in 0.7 min with the
flow rate of 1.0 mL/min. The splitting ratio was measured to be ca. 3:7.
2
2. Additional supporting data
DESI spray
probe
N2
. .
...
Monolithic or UPLC column
Mixture
sample
DESI
spray
PEEK
tubing
UV
IR
NMR
MS
....
... orifice
Structure
identification
Outlet
PEEK
tubing
Figure 1S. Apparatus of LC/DESI-MS using an orifice (i.d. 50 or 100 μm) drilled on PEEK
capillary tubing for splitting
LC/ESI-MS
100
a) EIC of dopamine
NL: 2.40E4
Relative Abundance
0
100
b) EIC of 3-methoxytyramine
NL: 1.77E5
0
100
c) EIC of L-kynurenine
NL: 4.49E4
0
100
d) EIC of L-tryptophan
NL: 3.53E4
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Time (min)
Figure 2S. EICs of a) dopamine (m/z 154), b) 3-methoxytyramine (m/z 168), c) L-kynurenine
(m/z 209) and d) L-tryptophan (m/z 204) acquired by LC/ESI-MS
3
Table S1. Data showing LC/DESI-MS sensitivity for the urine sample analysis
Injection
Compounds
concentrations ng to MS Signal/Noise (S/N)*
0.36
dopamine
450 ng/mL
5
0.20
3-methoxytyramine
250 ng/mL
7
0.50
L-tryptophan
620 ng/mL
10
L-kynureine
600 ng/mL
0.48
14
* The S/N was measured based on the ratio of the signal height (SIM signal in our
experiment) to the standard deviation of blank signal height (noise).
NMR, UV and IR of collected 3-methoxytyramine
With 3-methoxytyramine collected at hand via MS-directed purification after LC
separation, 1H NMR, UV and IR spectra of the collected explicitly were acquired, confirming
its structure. In the 1H NMR spectrum, the methoxyl hydrogens -OCH3 give rise to a singlet
peak at δ=3.861 ppm; CH2-CH2 hydrogens produce two triplet peaks at δ=3.141 ppm and at
δ=2.865 pp, respectively. Three peaks at 6.695-6.846 ppm correspond to the three aromatic
hydrogens. The maximum UV absorption of 3-methoxytyramine occurs at 282 nm. In the IR
spectrum, major peaks observed include 3141 cm-1 (O-H stretch), 1525 cm-1 (aromatic ring
C-C stretch), 1156 cm-1 (C-O stretch), 1278 cm-1(C-N stretch) and 799 cm-1 (N-H wag).
Improving the recovery yield of the MS-directed purification
In this study, with further reducing the orifice i.d. from 100 μm down to 50 μm, the
splitting ratio of 0.9:99.1 (approximately 1:99) was obtained when the mobile phase elution
flow rate was kept at 3.0 mL/min. The use of the smaller orifice would improve the sample
collection yield up to 99% with only 1% sample being consumed for DESI-MS detection.
Experiment was conducted to testify this hypothesis and the mixture of dopamine,
3-methoxytyramine, L-tryptophan and L-kynurenine was again chosen as the test sample.
4
Using the smaller orifice (50 μm i.d.), the four samples were successfully detected online by
DESI-MS
(data
not
shown).
During
the
run
of
LC/DESI-MS,
we
collected
3-methoxytyramine from the PEEK capillary tube outlet and quantified the collection yield
using UV absorption spectroscopy.
LC with a UV detector (detection wavelength was selected at 210 nm) was used for
the quantification of the DESI-MS-directed collection yield. 3-Methoxytyramine standard
stock solutions were diluted into the standard solutions with five different concentrations (1,
2, 5, 10, and 20 μM), which were analyzed three times by LC/UV using the same LC
separation condition described in text using monolithic C18 column to establish the calibration
curve (Figure 3S). The collected 3-methoxytyramine solution was also injected for LC/UV
analysis in triplicate measurements. The peak areas of collected 3-methoxytyramine solutions
were brought into the regression equation of the calibration curve to get the concentrations of
the collected 3-methoxytyramine solutions (Table 2S). Then, based on the collected volume,
the moles of collected samples can be obtained to compare with the original amount of
injected 3-methoxytyramine for LC separation to obtain the recovery yield of 98.6±0.6% on
average (Table 2S). This collection yield is fairly close to the actual splitting ratio value of
99.1%.
5
100000
90000
y = 4381.8x - 2146.8
R² = 0.9963
80000
Peak Area
70000
60000
50000
40000
30000
20000
10000
0
0
5
10
15
20
25
3-MT Concentration (μM)
Figure 3S. Calibration curves of 3-methoxytyramine (UV absorption peak area vs.
3-methoxytyramine solution concentration)
Table 2S. Quantification results for the collection yield
Replicate #1
Collected
Concentration Volume
(μM)
(μL)
3-MT
3.295
360
Replicate #2
Yielda
Concentration
(μM)
Collected
Volume
(μL)
98.85
%
3.299
360
Replicate #3
Yield
98.97
%
Concentration
(μM)
Collected
Volume
(μL)
3.266
360
Yield
97.98
%
Average
Yield
98.6%
3-MT stands for “3-methoxytyramine”.
UPLC/DESI-MS
Relative Abundance
100
a) EIC of Codeine in Pepsi
Absolute intensity : 5.10E3
0
100
b) EIC of Cocaine in Pepsi
Absolute intensity : 7.34E3
0
100
0
c) EIC of Flunitrazepam in Pepsi
Absolute intensity: 2.63E4
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20
Time (min)
Figure 4S. EICs of a) codeine (m/z 300), b) cocaine (m/z 304) and c) flunitrazepam (m/z 314)
acquired by UPLC/DESI-MS
6
UPLC/ESI-MS
Relative Abundance
100
a) EIC of Codeine in Pepsi
Absolute intensity : 4.85E3
0
100
b) EIC of Cocaine in Pepsi
Absolute intensity : 1.14E4
0
100
0
c) EIC of Flunitrazepam in Pepsi
Absolute intensity : 2.41E4
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20
Time (min)
Figure 5S. EICs of a) codeine (m/z 300), b) cocaine (m/z 304) and c) flunitrazepam (m/z 314)
acquired by UPLC/ESI-MS
When elevated column temperature (80 oC) was used, the elution flow rate for UPLC
separation was increased to 1.0 mL/min and the separation was completed in 45 s (Figure 3).
In addition, the separated drugs were also online collected with the aid of DESI-MS detection
and re-analysis of the collected drugs was performed using ESI-MS. The ESI-MS spectra
shown in Figure 6S confirm that purified individual drugs were collected.
7
100
300
a) Re-analysis of codeine
Relative Abundance
0
304
100
b) Re-analysis of cocaine
0
314.
100
0
295
c) Re-analysis of flunitrazepam
300
305
310
315
320
m/z
Figure 6S. ESI-MS spectra of a) collected codeine, b) collected cocaine and c) collected
flunitrazepam after high temperature UPLC separation
8