ultra-fast quantitative analysis of dextrophan in …figure 4. calibration summary report for...

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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