Evaluation of Sample Preparation Methods for

Evaluation of Sample Preparation
Methods for Semi-Quantitative,
Ultra-High Throughput Urine Forensic
Screening Using an LDTD HRAM
MS Technique
Marta Kozak, Kristine Van Natta
Thermo Fisher Scientific, San Jose, CA
Overview
Purpose: Evaluation of sample preparation procedures for a laser diode thermal
desorption (LDTD) ion source coupled to a high-resolution, accurate-mass (HRAM)
mass spectrometer for ultrafast quantitative screening of drugs of abuse in urine, for
forensic toxicology.
Methods: Samples were prepared by either liquid-liquid extraction (LLE) or urine
dilution and then plated onto a 96--well LazWell™ plate, and introduced into the HRAM
MS using a Phytronix™ LDTD™ ion source.
Results: With adequate sample preparation, LDTD can achieve the limits of detection
required for forensic screening of urine samples.
Introduction
The new technique – laser diode thermal desorption (LDTD), high-resolution accuratemass (HRAM) mass spectrometry – allows for rapid screening of compounds in
biological matricies. Because of the short 10 s analysis time of LDTD and high
selectivity provided by HRAM MS, the method meets ultra-high throughput
requirements of forensic toxicology labs. Since there is no chromatographic separation
between matrix components and analytes, the sample preparation method has to be
carefully selected to minimize matrix effects and to obtain the required limit of
quantitation (LOQ). Here we investigated two sample preparation methods – LLE and
multiple dilutions – to evaluate matrix effects and LOQ for eight representative
compounds, including three each benzodiazepines (alprazolam, lorazepam, and
nordiazepam) and amphetamines (amphetamine, methamphetamine, and MDMA) and
two opioids (fentanyl and oxycodone), in urine samples.
LDTD
The LDTD carrier gas flow was
from 0 to 65% in six seconds w
(Figure 1). The total acquisition
shown in Figure 2.
Mass Spectrometry
The Q Exactive mass spectrom
parent ions were selected with
fragmentation data was collect
of data obtained from this scan
was 1 m/z and fragmentation s
were selected for quantitation a
reconstructed with mass accur
settings are listed in Table 1. Ta
FIGURE 2. Schematic showin
Methods
Sample Preparation
Samples were prepared by enzymatic hydrolysis followed by liquid-liquid extraction or
urine dilution at 1x, 4x, 10x, 20x, 40x, or 80x. Calibration standards (1–500 ng/mL)
were prepared in negative urine. Briefly, 0.2 mL of urine (spiked calibrator) was spiked
with internal standard and incubated with 0.1••mL of 10,000 U/mL beta-glucuronidase
enzyme in pH 5.5 buffer for 60 min at 60 °C. For LLE, a 0.1 mL aliquot of the resulting
mixture was basified with 0.05 mL of 1 M sodium carbonate and extracted with 0.3 mL
of ethylacetate/hexane (1:1). For urine dilution, the incubation mixture was diluted with
water by the factors given above. A 96-well LazWell plate was prepared by depositing
5 µL of a 20 µg/mL EDTA solution in methanol/water/ammonium hydroxide (75:20:5)
onto the plate and drying. Then 5 µL of either the LLE organic supernatant or the
diluted urine was aliquoted onto the same plate and again dried. Samples were
introduced into the Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap™
mass spectrometer by thermal desorption from the plate using a Phytronix LDTD
source.
FIGURE 1. LDTD method
FIGURE 3. Targeted-MS2 data
Parent ion was selected by q
cell. Full high resolution-acc
from the Orbitrap mass analy
quantification and confirmati
with m/z accuracy of 5 ppm.
Relative Abundance Relative Abundance Relative Abundance Relative Abundance
C:\TraceFinderData\...\Data\500-LLE
RT:0.00 - 0.17
100
80
60
40
20
0
100
80
60
40
Fra
2
20
0
100
80
60
40
Fra
2
20
0
100
80
60
40
Re
20
0
0.00
2 Evaluation of Sample Preparation Methods for Semi-Quantitative, Ultra-High Throughput Urine Forensic Screening Using an LDTD HRAM MS Technique
0.05
0.10
Time (min)
0.15
for a laser diode thermal
on, accurate-mass (HRAM)
f drugs of abuse in urine, for
extraction (LLE) or urine
and introduced into the HRAM
achieve the limits of detection
DTD), high-resolution accurateeening of compounds in
time of LDTD and high
ra-high throughput
no chromatographic separation
reparation method has to be
n the required limit of
eparation methods – LLE and
or eight representative
azolam, lorazepam, and
amphetamine, and MDMA) and
LDTD
Data Analysis
The LDTD carrier gas flow was set at 3 L/min, and the laser pattern was an energy ramp
from 0 to 65% in six seconds with a short hold followed by an immediate return to zero
(Figure 1). The total acquisition time was ten seconds. Details of the LDTD process are
shown in Figure 2.
Data were acquired and processed w
version 3.2.
Mass Spectrometry
The Q Exactive mass spectrometer was operated in Targeted-MS2 mode. In this mode,
parent ions were selected with the quadrupole filter and then high-resolution
fragmentation data was collected in the Orbitrap mass analyzer. An example of the type
of data obtained from this scan mode is shown in Figure 3. Parent ion isolation window
was 1 m/z and fragmentation spectra were collected at 17,500 resolution. Two fragments
were selected for quantitation and confirmation and related chronograms were
reconstructed with mass accuracy of 5 ppm. Other Q Exactive MS source parameter
settings are listed in Table 1. Targeted-SRM parameter are shown in Figure 4.
FIGURE 2. Schematic showing LDTD process
Calibration ranges, LODs, and LOQs
accuracy; back-calculated concentrat
Matrix effects were determined by co
in multiple lots of urine to the respons
without beta-glucuronidase enzyme.
TABLE 1. Source parameters for
Q Exactive MS
Parameter
Value
Sheath Gas
0
Aux Gas
0
Sweep Gas
0
Discharge Current (µA)
3
Capillary Temp (°C)
300
RF-Lens Level
50
Vaporizer Temp (°C)
0 (off)
Results
ed by liquid-liquid extraction or
n standards (1–500 ng/mL)
(spiked calibrator) was spiked
00 U/mL beta-glucuronidase
0.1 mL aliquot of the resulting
nate and extracted with 0.3 mL
bation mixture was diluted with
e was prepared by depositing
monium hydroxide (75:20:5)
rganic supernatant or the
ain dried. Samples were
brid quadrupole-Orbitrap™
e using a Phytronix LDTD
Limits of Quantitation
For limits of quantitation, results were
opioids gave their lowest limits of qua
Amphetamines responded best with 4
Table 2. Figure 5 shows representativ
and oxycodone. Figure 6 shows chro
Matrix Effects
FIGURE 3. Targeted-MS2 data obtained from Q Exactive MS for alprazolam.
Parent ion was selected by quadrupole mass filter and then fragmented in HCD
cell. Full high resolution-accurate mass fragmentation spectra were obtained
from the Orbitrap mass analyzer. Appropriate fragments were then selected for
quantification and confirmation. Fragment chronograms were reconstructed
with m/z accuracy of 5 ppm.
Based on the data obtained earlier fo
samples processed by LLE and at 1x
the LOQs, class dependent. Benzodi
fewest matrix effects with LLE proces
sample preparation scheme was orig
also responded best with LLE. Oxyco
Amphetamines showed matrix enhan
dilution. The wide range of matrix effe
for analysis by LDTD. Results are sh
Relative Abundance Relative Abundance Relative Abundance Relative Abundance
C:\TraceFinderData\...\Data\500-LLE
500-LLE
#45 RT:0.06 AV:1 NL:3.50E6
NL: 1.78E7
F: FTMS + c APCI corona Full ms2 [email protected] [50.00-335.00]
281.0713
TIC F: FTMS + c APCI 100
corona Full ms2
[email protected]
95
[50.00-335.00] MS
500-LLE
90
RT:0.00 - 0.17
100
quantifier
80
60
40
TIC
20
60
40
Fragment m/z
281.0712
NL: 8.24E5
20
0
100
55
m/z= 274.1199-274.122750
F: FTMS + c APCI corona
45
Full ms2
[email protected]
[50.00-335.00] MS
40
500-LLE
35
80
60
40
Fragment m/z
274.1213
NL: 1.14E6
20
0
100
60
40
20
0.05
0.10
Time (min)
Remaining
Parent
0.15
parent
309.0901
30
25
m/z= 309.0886-309.0916
F: FTMS + c APCI corona
20
Full ms2
[email protected]
15
[50.00-335.00] MS
500-LLE
10
80
0
0.00
Relative Abundance
80
LLE
85
80
NL: 3.50E6
m/z= 281.0698-281.072675
F: FTMS + c APCI corona
Full ms2
70
[email protected]
[50.00-335.00] MS
65
500-LLE
60
0
100
TABLE 2. Limits of quantitation in
5
0
qualifier
274.1213
241.0527
165.0216
205.0755
138.0106
191.0880 224.0262 251.0371
149.0600172.0870
150
200
m/z
250
1x
Amphetamine
ND
10
MDMA
200
100
Methamphetamine
ND
NA
Alprazolam
2
ND
Lorazepam
5
500
Nordiazepam
1
0
Fentanyl
5
5
Oxycodone
5
500
294.1029
300
Thermo Scientific Poster Note • PN-64128-ASMS-EN-0614S 3
Data Analysis
he laser pattern was an energy ramp
wed by an immediate return to zero
ds. Details of the LDTD process are
Targeted-MS2 mode. In this mode,
and then high-resolution
ss analyzer. An example of the type
gure 3. Parent ion isolation window
at 17,500 resolution. Two fragments
related chronograms were
Q Exactive MS source parameter
ter are shown in Figure 4.
Calibration ranges, LODs, and LOQs were evaluated based on concentration
accuracy; back-calculated concentrations had to be within 30%.
Matrix effects were determined by comparing the peak response of samples prepared
in multiple lots of urine to the response of samples prepared in water both with and
without beta-glucuronidase enzyme.
TABLE 1. Source parameters for
Q Exactive MS
Parameter
Value
Sheath Gas
0
Aux Gas
0
Sweep Gas
0
Discharge Current (µA)
3
Capillary Temp (°C)
300
RF-Lens Level
50
Vaporizer Temp (°C)
FIGURE 4. Targeted-MS2 scan settings
with inclusion list
0 (off)
Results
Limits of Quantitation
For limits of quantitation, results were compound class specific. Benzodiazepines and
opioids gave their lowest limits of quantitation with LLE sample processing.
Amphetamines responded best with 4x urine dilution. These results are summarized in
Table 2. Figure 5 shows representative calibration curves for MDMA, nordiazepam,
and oxycodone. Figure 6 shows chronograms for the same compounds at their LOQs.
FIGURE 6. Representative chro
showing quantifier and confirm
Matrix Effects
xactive MS for alprazolam.
er and then fragmented in HCD
tation spectra were obtained
gments were then selected for
nograms were reconstructed
0.06 AV:1 NL:3.50E6
PCI corona Full ms2 [email protected] [50.00-335.00]
281.0713
quantifier
parent
309.0901
qualifier
274.1213
241.0527
165.0216
205.0755
106
191.0880 224.0262 251.0371
49.0600172.0870
150
Data were acquired and processed with Thermo Scientific™ TraceFinder™ software
version 3.2.
FIGURE 5. Representative cali
oxycodone obtained using LD
200
m/z
250
Based on the data obtained earlier for LOQs, matrix effects were determined in
samples processed by LLE and at 1x, 4x, and 40x dilution. Matrix effects were, as for
the LOQs, class dependent. Benzodiazepines showed the best signal response and
fewest matrix effects with LLE processing. This is to be expected since this particular
sample preparation scheme was originally optimized for benzodiazepines. Fentanyl
also responded best with LLE. Oxycodone showed the fewest effects at 4x dilution.
Amphetamines showed matrix enhancement with LLE and the fewest effects at 40x
dilution. The wide range of matrix effects indicate that sample processing will be critical
for analysis by LDTD. Results are show in Figures 7 and 8.
TABLE 2. Limits of quantitation in ng/mL for compounds tested
LLE
1x
4x
10x
20x
40x
Amphetamine
ND
10
5
50
100
50
50
MDMA
200
100
1
1
20
50
50
Methamphetamine
ND
NA
5
10
50
100
200
2
ND
10
5
20
1
5
Lorazepam
5
500
100
200
50
20
50
Nordiazepam
1
0
200
5
20
2
5
Fentanyl
5
5
10
NA
ND
NA
NA
Oxycodone
5
500
5
5
20
20
10
Alprazolam
80x
294.1029
300
4 Evaluation of Sample Preparation Methods for Semi-Quantitative, Ultra-High Throughput Urine Forensic Screening Using an LDTD HRAM MS Technique
ntific™ TraceFinder™ software
FIGURE 5. Representative calibration curves for MDMA, nordiazepam, and
oxycodone obtained using LDTD-MS
based on concentration
within 30%.
FIGURE 7. Matrix effects. Peak
sample preparation technique
water with beta-glucuronidase
242%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
k response of samples prepared
epared in water both with and
RE 4. Targeted-MS2 scan settings
nclusion list
347%
312%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
FIGURE 8. Detail of matrix effe
processing schemes. Matrix e
There was no significant diffe
40x dilution and with LLE proc
areas for nordiazepam.
effects were determined in
ution. Matrix effects were, as for
d the best signal response and
be expected since this particular
for benzodiazepines. Fentanyl
e fewest effects at 4x dilution.
E and the fewest effects at 40x
sample processing will be critical
and 8.
pounds tested
0x
20x
40x
80x
50
100
50
50
100000000
FIGURE 6. Representative chronograms for MDMA, nordiazepam, and oxycodone
showing quantifier and confirming ions
Water w/ Water
enzyme enzy
10000000
Peak Area
s specific. Benzodiazepines and
E sample processing.
These results are summarized in
rves for MDMA, nordiazepam,
same compounds at their LOQs.
urine
1000000
100000
10000
1x
Conclusion

Sample preparation is crit

For best results, compoun
preparation can be suitab

With adequate sample pre
required for forensic scree
1
20
50
50
10
50
100
200
5
20
1
5
200
50
20
50
We would like to thank Dr. Pierre
Technologies Inc., for his assista
5
20
2
5
NA
ND
NA
NA
For forensic use only.
5
20
20
10
Acknowledgeme
Phytronix, LDTD, and LazWell are a trade
property of Thermo Fisher Scientific and it
This information is not intended to encour
intellectual property rights of others.
Thermo Scientific Poster Note • PN-64128-ASMS-EN-0614S 5
MDMA, nordiazepam, and
FIGURE 7. Matrix effects. Peak response for compounds tested using different
sample preparation techniques. Graphs are normalized to samples prepared in
water with beta-glucuronidase enzyme.
605%
242%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
601%
347%
LLE
Lots
H2O+ß
H2O-ß
312%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
4x
Lots
H2O+ß
H2O-ß
431%
769% 251% 478% 479%
455%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
200%
180%
160%
140%
120%
100%
80%
60%
40%
20%
0%
1x
Lots
H2O+ß
H2O-ß
512%
40x
Lots
H2O+ß
H2O-ß
FIGURE 8. Detail of matrix effects for nordiazepam under different sample
processing schemes. Matrix effects were greatest at 1x and then 4x dilution.
There was no significant difference between matrix and non-matrix samples at
40x dilution and with LLE processing; however, LLE gave the highest peak
areas for nordiazepam.
100000000
MA, nordiazepam, and oxycodone
urine
Water w/ Water w/o
enzyme enzyme
urine
urine
10000000
Peak Area
urine
W+ß W-ß
W+ß W-ß
W+ß W-ß
1000000
100000
10000
1x
4x
40x
Sample Processing Scheme
LLE
Conclusion

Sample preparation is critical for successful analysis by LDTD.

For best results, compounds should be analyzed in classes, so sample
preparation can be suitably tailored.

With adequate sample preparation, LDTD can achieve the limits of detection
required for forensic screening of urine samples.
Acknowledgements
We would like to thank Dr. Pierre Picard, Vice President of R&D at Phytronix
Technologies Inc., for his assistance in setting up the LDTD source.
For forensic use only.
Phytronix, LDTD, and LazWell are a trademarks of Phytronix Technologies, Inc. All other trademarks are the
property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
PO64128-EN 0614S
6 Evaluation of Sample Preparation Methods for Semi-Quantitative, Ultra-High Throughput Urine Forensic Screening Using an LDTD HRAM MS Technique
For forensic toxicology use only.
www.thermoscientific.com
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