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INTRODUCTION
Laser Diode Thermal Desorption (LDTD) has
proven to be an ultra-fast sample introduction
technique for MS analysis by atmospheric
pressure chemical ionisation (APCI) with no
chromatographic separation1. The majority of
quantitative data published to date has been
from tandem quadrupole mass spectrometers
acquired in MRM mode2-4. Data showing the high
throughput quantitative analysis of dextrorphan
in protein precipitated human plasma achieved
using a Phytronix 96 well plate LDTD™ interface
(Phytronix Technologies, Quebec, Canada)
coupled to a Xevo G2 QTof (Waters, Milford,
MA) is presented.
The LDTD interface, shown in figure 1 mounted
on a Xevo universal source housing, uses an
infrared laser diode to desorb samples that are
loaded and dried on a 96-well LazWell™ plate.
The desorbed gas phase samples are carried
into a corona discharge region with air
containing ppm levels of water and undergo
APCI.
ULTRA-FAST QUANTITATIVE ANALYSIS OF DEXTROPHAN IN HUMAN PLASMA USING LASER DIODE THERMAL DESORPTION (LDTD) COUPLED TO A XEVO G2 QTOF
Hilary Major
Waters Corporation, Floats Road, Wythenshawe, Manchester, M23 9LZ, UK
Figure 1. Phytronix LDTD interface mounted on a Xevo
universal source housing.
METHODS
Sample Preparation
Stock solution of dextrorphan prepared at 1 mg/mL in
MeOH.
Serial dilution in plasma from 20µg/mL down to 10ng/mL.
50µL of plasma standards diluted with 150µL of 500ng/mL
dextrorphan-d3 internal standard in acetonitrile (protein precipitation).
Vortex for 10seconds.
Centrifuge at 13,000rpm for 10minutes.
Spot 4µL of supernatant onto 96-well LazWell plate (this
compensates for x4 dilution at protein precipitation stage)
Dry at 37°C for 2 minutes
Solvent standards were prepared in the same way omitting
the centrifugation step
LDTD Conditions
Carrier gas flow 3.0L/min (air)
Programmable laser desorption: ramp from 0 to 45%
power, hold for 2sec
The LDTD method editor is shown below
MS Conditions
Source temperature 150°C
Corona current 3µA
Cone voltage 30V
MS, continuum acquisition, m/z 50-600, 5 spectra/sec
Total run time 15 seconds. This was reduced to 10 seconds for
some of the later acquisitions.
RESULTS
Replicate aliquots of the protein precipitated plasma spiked
samples were loaded onto a 96 well LazWell plate. Some of the
samples were analysed immediately after drying then the plate was stored at room temperature for 72 hours before analyzing
the remaining sample wells. The unused sample solutions were stored at –20°C and analysed later.
Linearity
The calibration line generated from the initial analysis of four
replicate loadings is shown in figure 3. This shows >3 orders of linearity over the range 10 to 20,000ng /mL, equivalent to
absolute loadings of 10 to 20,000pg. The linearity and
reproducibility was excellent with a correlation coefficient R2 of 0.999 using a 1/x weighting.
CONCLUSIONS
Pros
LDTD is a high throughput, ultra fast, easy to use
interface
Excellent linearity over more than 3 orders of
magnitude (r2 >0.999) when coupled to a TOF MS
No sample carry over
Good intra and inter plate reproducibility
Minimal sample preparation required
96 well format allows robotic handling of sample
preparation and loading
Samples stored on the plates were stables over 72
hours at room temperature
Reduced environmental impact compared with LC-MS
The high resolution of the TOF compared to a
quadrupole gives improved selectivity
TOF full scan data allows retrospective interrogation
of the data for unexpected metabolites etc.
Cons
No separation therefore some matrix suppression
-minimised by changing laser ramp and hold time
Fewer compounds ionised compared with ESI
Stability and Matrix Effect
The stability of the protein precipitated samples stored on the plate at room temperature for 72 hours was evaluated by
analyzing them against the calibration curve generated previously.
The results are summarised below:
In addition fresh aliquots were spotted onto a new LazWell
plate after storage of the spiked plasma samples at –20°C for 72 hours. These were also analysed using the previously
generated calibration line.
The results are summarised below:
The results in Table 1 and Table 2 show that the mean deviation between the original samples and the samples aged
either dried on the LazWell plate at room temperature or as
solutions at –20°C are within ± 10%. The coefficient of
variation was <10% for the lowest level standards and <3.0% for the highest.
Matrix suppression was evaluated by comparing the response of the standards in the plasma matrix with the response from
pure solvent standards (data not shown). The response for the matrix standards was within acceptable limits being
approximately 85% of that observed for the pure standards.
Figure 2. LDTD method editor
Figure 3. Calibration line for dextrorphan in protein
precipitated plasma over range 10 to 20,000ng/mL
Compound name: Dextrorphan
Correlation coefficient: r = 0.999567, r^2 = 0.999134
Calibration curve: 0.000406821 * x + 0.0084234
Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc-0 2500 5000 7500 10000 12500 15000 17500 20000
Re
sp
on
se
-0.00
2.00
4.00
6.00
8.00
Figure 4. Calibration summary report for dextrorphan
The spectrum from a 2000ng/ml standard in plasma is shown
in figure 6 with the expanded region showing the dextrorphan and the dextrorphan-d3 internal standard. The other major
peaks in the spectrum are from dioctyl phthalate (m/z 391 and 149) and cholestadiene (m/z 369) from cholesterol.
The full summary report is shown in figure 4 and shows that
all the back calculated concentration values are <15% apart from one at 20ng/mL which showed a deviation of 21.7% and
was excluded from the calibration.
Representative extracted exact mass chromatograms for the
20ng/ml dextrorphan standard and dextrorphan-d3 internal standard spiked in plasma are shown in figure 5.
min0.050 0.100 0.150 0.200
%
0
100
TOF MS,AP+
261.2006
LDTD_11Jul11_105 Smooth(Mn,2x2)
20ng/mL dextrorphan in plasma + D3 IS
6.058e+004Dextrorphan D3;0.09;1997.3
min
%
0
100
TOF MS,AP+
258.1828
LDTD_11Jul11_105 Smooth(Mn,2x2)
20ng/mL dextrorphan in plasma + D3 IS
8.986e+002Dextrorphan;0.09;34.9
Table 1. Samples spotted on LazWell plate and analysed after
storage for 72 hours at room temperature
Table 2. Sample solutions stored at –20°C for 72 hours then
spotted on new LazWell plate and analysed
Figure 6. Spectrum from 2000ng/mL dextrorphan standard and
–d3 internal standard in protein precipitated plasma
2000ng/mL dextrorphan in plasma + D3 IS
m/z50 100 150 200 250 300 350 400 450 500
%
0
100
LDTD_11Jul11_131 20 (0.088) TOF MS AP+ 4.30e6149.0226
128.1062
127.0383
391.2848
369.3517
167.0334279.1592
261.2042
244.2632
313.2737
392.2882
dextrorphan
dextrorphan-d3
18.2 89.1 180 929 1922 9237 19945
21.5 96.4 185 952 1974 9517 19722
22.4 94.6 180 901 1845 9339 19626
20.4 87.5 187 965 1968 9344 20508
Mean 20.6 91.9 183 937 1927 9359 19950
Std Dev 1.8 4.3 3.9 28.2 59.5 116 395
%CV 8.8 4.6 2.1 3.0 3.1 1.2 2.0
%Nom conc 103.1 91.9 91.5 93.7 96.4 93.6 99.8
10000 20000Conc ng/mL 20 100 200 1000 2000
25.2 87.7 172 997 2126 9715 21516
21.0 100.3 197 989 2071 10426 22002
19.9 90.0 172 1024 2045 10408 22307
21.1 93.6 184 1009 2038 10002 20627
Mean 21.8 92.9 181 1005 2070 10138 21613
Std Dev 2.02 4.76 10.45 13.27 34.37 297 635
%CV 9.3 5.1 5.8 1.3 1.7 2.9 2.9
%Nom conc 109.0 92.9 90.6 100.5 103.5 101.4 108.1
10000 20000Conc ng/mL 20 100 200 1000 2000
References
1. J. Wu, C. S. Hughes, P. Picard, S. Letarte, M. Gaudreault, J. F. Levesque, D. A. Nicoll-Griffith, K. P. Bateman, High-throughput cytochrome P450 inhibition assays using laser diode thermal desorption-atmospheric pressure chemical ionization–tandem mass spectrometry, Anal. Chem. 79 (2007) 4657–4665.
2. P.B. Fayad, M. Prevost, S. Sauve, Laser diode thermal desorption/atmospheric pressure chemical ionization tandem mass spectrometry analysis of selected steroid hormones in wastewater: method optimization and application, Anal. Chem. 82 (2010) 639–645.
3. P.A. Segura, P. Tremblay, P. Picard, C. Gagnon, S. Sauve, High-throughput quantitation of seven sulfonamide residues in dairy milk using laser diode thermal desorption–negative mode atmospheric pressure chemical ionization tandem mass spectrometry, J. Agric. Food Chem. 58 (2010) 1442–1446.
4. J.G. Swales, R. Gallagher, R.M. Peter, Determination of metformin in mouse, rat, dog and human plasma samples by laser diode thermal desorption/ atmospheric pressure chemical ionization tandem mass spectrometry, J. Pharm. Biomed. Anal. 53 (2010) 740–744.
Figure 5. Extracted exact mass chromatograms showing peak
integration for dextrorphan and the d3 internal standard
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