laser diode thermal desorption ionization source for mass spectrometry
DESCRIPTION
LASER DIODE THERMAL DESORPTION IONIZATION SOURCE FOR MASS SPECTROMETRY. Patrice Tremblay, Ph.D. Motivations. Pharmaceutical, CRO, environmental and food industries need to improve productivity of high throughput screening and analysis. Actual techniques are often limited by : - PowerPoint PPT PresentationTRANSCRIPT
LASER DIODE THERMAL DESORPTION
IONIZATION SOURCE FOR MASS
SPECTROMETRY
Patrice Tremblay, Ph.D.
• Pharmaceutical, CRO, environmental and food industries need to improve productivity of high throughput screening and analysis.
• Actual techniques are often limited by :
Extensive samples preparation;
Risk of cross contamination between samples;
Background noise induced by mobile phase or enhancement matrix;
Analysis time.
• In order to eliminate these problems, a new ionization source has been developed.
• The LDTD technology (Laser Diode Thermal Desorption) coupled to a mass spectrometer offers the same analytical performances as any LC- MS/MS system and is an alternative to the problems encountered with usual techniques.
MotivationsMotivations
LDTD Ionization LDTD Ionization SourceSource
IR Laser Beam
LazWell Sample Plate
Carrier Gas
Corona DischargeNeedle
Mass SpectrometerInlet
Piston
Transfer Tube
Piston head
LDTD Ionization SourceLDTD Ionization Source
• Sample is dried onto the bottom of a well from a standard 96-well plate with a metal sheet insertion.
• Thermal desorption induced by a laser at 980 nm (no photon-sample interactions).
• Gaseous neutral species transferred by a carrier gas.
• Ionization occurs into the corona discharge region.
• The ionization process that occurs in the LDTD source is an Atmospheric pressure chemical (APC) type of ionization without the presence of solvent (no mobile phase or enhancement matrix.)
LC APCI
H2O
O2
N2
H2O+
H2OH3O+
N2
N2+
++++ ++++
e-
e-
e-e-
N4+
N2
H2O N2+
N2+
(H2O)nH+ + Solvent
(Solvent+H)+ transfers charge onto analyte (if
possible)
HVCorona discharge
Theoretical Aspects of the Ionization
Theoretical Aspects of the Ionization
• The ionization process that occurs in the LDTD source is an Atmospheric pressure chemical (APC) type of ionization without the presence of solvent (no mobile phase or enhancement matrix.)
H2O
O2
N2
H2O+
H2OH3O+
N2
N2+
++++ ++++
e-
e-
e-e-
N4+
N2
H2O N2+
N2+
(H2O)nH+ +
LDTD APCI
Analyte
Analyte is forced to react with the
cluster ion
HVCorona discharge
• Low volume sample analysis (1 to 10 µL)
• 96-well plates are designed to be compatible with conventional sample preparation systems.
• No extra sample pre-treatment needed
• The absence of enhance matrix and mobile phase lower the noise signal.
• Elimination of cross contamination due to LC.
• Each well are individually isolated during the thermal desorption.
• The thermal desorption process takes seconds
Key Features
• Plug-and-play device
Key Features
Sciex Source Housing for API 3000, 4000 and 5000
Also available on Thermo, Waters and Agilent systems
• LazSoft and GLP environment :
• Thermo mass spectrometers (Xcalibur)
• LazSoft fully integrated into Xcalibur
• Operated under GLP
• Sciex mass spectrometers (Analyst)
• Actually in discussion with Sciex to have access to the programmation code for LazSoft integration
• Log Book created to trace the launch batch (LazSoft and Analyst)
Key Features
Application – Drugs Application – Drugs Analysis in PlasmaAnalysis in Plasma
ParacetamolParacetamolMifepristoneMifepristoneMidazolamMidazolam
LDTD Method and Plasma Sample LDTD Method and Plasma Sample PreparationPreparation
(Paracetamol analysis)(Paracetamol analysis)
Sample Preparation (crashed plasma)• 100 µL of Human Plasma• Spike Paracetamol and Paracetamol-d4 (40 ng/mL)• 500 µL of acetonitrile (precipitation agent)• Vortex 4 min.• Centrifuge at 14000 RPM for 10 min. • Transfer Manually 4 µL onto LazWell to perform
LDTD-MS/MS analysis
LDTD Parameters• Carrier gas flow : 3 L/min• APCI (+)• Laser Pattern
• Increase laser power to 25 % in 1.0 s• Hold at 25 % for 0.5 sec.• Decrease laser power to 0 %
MS Parameters• MS/MS transition : 152.0 – 110.1 amu
156.0 – 114.1 amu• Collision gas pressure : 1.5 mTorr (Ar)• Collision energy : 16 eV• Scan time : 0.050 s• Q1 width : 0.30 amu• Q3 width : 0.70 amu
M.W. 151.17 g/mol
High-Throughput LDTD-MS/MS High-Throughput LDTD-MS/MS Analysis of Paracetamol in Analysis of Paracetamol in
Human PlasmaHuman PlasmaRT: 0.00 - 7.74 SM: 7G
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NL: 2.18E5TIC F: + c ESI SRM ms2 [email protected] [110.095-110.105] MS Plaquette5_080422103040
NL: 1.79E5TIC F: + c ESI SRM ms2 [email protected] [113.995-114.005] MS Plaquette5_080422103040
RT: 0.08 - 1.00 SM: 13G
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NL: 1.40E5TIC F: + c ESI SRM ms2 [email protected] [110.095-110.105] MS ICIS Batch12B3
NL: 1.38E5TIC F: + c ESI SRM ms2 [email protected] [113.995-114.005] MS ICIS Batch12B3
Analyte Desorption in 1.8 seconds
Paracetamol raw signal
ISTD signal
96-replicates
Robustness and Robustness and RepeatabilityRepeatability
(Paracetamol in Human Plasma)(Paracetamol in Human Plasma)
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0 98 196 294 392 490 588 686 784 882 980
Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Plate 9 Plate 10
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Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 Plate 8 Plate 9 Plate 10
CV of 2.4% over 1008 replicates
Run time of 75 minutes
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LinearityLinearity(Paracetamol in Human Plasma)(Paracetamol in Human Plasma)
ParacetamolY = 0.131127+0.0212371*X R^2 = 0.9944 W: 1/X
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ParacetamolY = 0.14042+0.019*X R^2 = 0.9984 W: 1/X
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0.6 to 160 ng/mL
Concentration (ng/mL)
0.6 to 5000 ng/mL
LDTD Method and Plasma Sample LDTD Method and Plasma Sample PreparationPreparation
(Mifepristone analysis)(Mifepristone analysis)
Sample Preparation (liquid-liquid extraction)• 50 µL of Mouse Heparin Plasma • Spiked Mifepristone (10 to 2000 ng/mL)
• 20 µL IS(d4) + 50 µL NH4OH 4%
• 2 mL of each MTBE and Hexane• Vortex 15 min.• Centrifuge at 2500 RMP for 10 min.• Evaporate organic phase to dryness at 40oC• Reconstitute in 200 µL of Water:ACN:Formic acid (75:25:0.1 v/v/v)• Transfer Manually 2 µL onto LazWell to perform LDTD-MS/MS analysis
LDTD Parameters• Carrier gas flow : 2 L/min• APCI (+)• Laser Pattern
• Increase laser power to 60 % in 3 sec.• Hold at 60 % for 2 sec.• Decrease laser power to 0 %
MS Parameters• MS/MS transition : 430.14 – 372.25 amu• Collision gas pressure : 1.5 mTorr (Ar)• Collision energy : 18 eV• Scan time : 0.02 sec.• Q1 width : 0.70 amu• Q3 width : 0.70 amu
M.W. 429.59 g/mol
Calibration Curve Calibration Curve (Mifepristone (Mifepristone analysis)analysis)
Concentration (ng/mL)
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y = 0,0015x - 0,0205
R2 = 0,9987
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3,5
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w = 1/x
Calibration range : 10 to 2000 ng/mL
Sample-to-sample run time 9 sec.
Back-Calculation and QC’s Back-Calculation and QC’s (Mifepristone analysis)(Mifepristone analysis)
P1 P2 P3 P4 P5 P6 P7 P8 Nominal concentration (ng/mL)
10 20 50 100 200 500 100 2000 Back-calculated conc. 10.7 20.9 50.8 97.3 189.0 472.0 967.1 2072.0
% Nominal Conc. 107 104 102 97 94 94 97 104
• Standard concentrations back-calculated from calibration curve
• QC’s performance
QC 1 QC 2 QC 3 Nominal concentration (ng/mL) 30 300 1600 N 3 3 3 Mean (ng/mL) 30.6 288 1634 CV (%) 7.0 6.2 1.9 % Nominal conc. 102 96 121
LDTD Method and Plasma Sample LDTD Method and Plasma Sample PreparationPreparation(Midazolam)(Midazolam)
Sample Preparation (liquid-liquid extraction)• 100 µL of Human Plasma • Spiked Midazolam (0.5 to 250 ng/mL)
• 10 µL IS(d4) + 50 µL NH4OH 4%
• 3 mL of MTBE and 1 mL of Hexane• Vortex 15 min.• Centrifuge at 2500 RMP for 10 min.• Evaporate organic phase to dryness at 40oC• Reconstitute in 500 µL of Water:ACN:Formic acid (75:25:0.1 v/v/v)• Transfer Manually 2 µL onto LazWell to perform LDTD-MS/MS analysis
LDTD Parameters• Carrier gas flow : 2 L/min• APCI (+)• Laser Pattern
• Increase laser power to 40 % in 2 sec.• Hold at 40 % for 2 sec.• Decrease laser power to 0 %
MS Parameters• MS/MS transition : 430.14 – 372.25 amu• Collision gas pressure : 1.5 mTorr (Ar)• Collision energy : 18 eV• Scan time : 0.02 sec.• Q1 width : 0.70 amu• Q3 width : 0.70 amu
M.W. 325.78 g/mol
Calibration Curve Calibration Curve (Midazolam (Midazolam analysis)analysis)midazolam
Y = 0.000762841+0.0110162*X R^2 = 0.9940 W: 1/X
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Concentration (ng/mL)
Calibration range : 0.5 to 250 ng/mL
Sample-to-sample run time 8 sec.
Back-Calculation, QC’s and Back-Calculation, QC’s and Unknown Unknown
(Midazolam analysis)(Midazolam analysis)• Standard concentration back-calculated from calibration curve
• QC’s performance
P1 P2 P3 P4 P5 P6 P7 P8 P9 Nominal concentration (ng/mL)
0.5 1.0 2.5 5.0 10.0 25.0 50.0 100.0 200.0 Back-calculated conc. 0.46 1.01 2.46 4.62 11.32 26.79 48.98 97.57 250.80
% Nominal Conc. 92 101 98 93 113 107 98 98 100
QC 1 QC 2 QC 3 Nominal concentration (ng/mL)
1.5 15 200 N 3 3 3 Mean (ng/mL) 1.42 14.3 190.0 CV (%) 10.9 5.9 1.3 % Nominal Conc. 95 95 95
• Unknown : LDTD-MS/MS vs LC-MS/MS
* Calculated as LC-MS/MS provides the true values
LC-MS/MS LDTD-MS/MS % Difference* ng/mL
2.54 2.14 -15.7 2.56 2.71 5.8 4.68 5.00 6.8 4.83 5.37 11.2 6.28 6.36 1.3 6.37 6.15 -3.5
* Calculated as if LC-MS/MS provides the true values…
Application – Drugs Application – Drugs Analysis in Dried Analysis in Dried
Blood SpotBlood Spot
ParacetamolParacetamol
LDTD Method and Blood Spot Sample LDTD Method and Blood Spot Sample PreparationPreparation
(Paracetamol analysis)(Paracetamol analysis)
Sample Preparation• Dried blood spot (with Paracetamol)• Punch out a 3 mm disc
• 100 µL 50/50 Meoh/H2O + 250 ng/mL IS(D4)
• Vortex for 20 sec.• Allow to stand for 30 min.• Centrifuge at 14000 RPM for 1 min• Transfer Manually 2 µL onto LazWell to perform LDTD-MS/MS analysis
LDTD Parameters• Carrier gas flow : 2 L/min• APCI (+)• Laser Pattern
• Increase laser power to 25 % in 2.0 sec.• Hold at 25 % for 2 sec.• Decrease laser power to 0 %
MS Parameters• MS/MS transition : 152.0 – 110.15 amu• Collision gas pressure : 1.5 mTorr (Ar)• Collision energy : 16 eV• Scan time : 0.02 s• Q1 width : 0.70 amu• Q3 width : 0.70 amu
M.W. 151.17 g/mol
Calibration Curve : Paracetamol in Calibration Curve : Paracetamol in bloodblood
Concentration (ng/mL)
ParacetamolY = 0.0858798+0.0439791*X R^2 = 0.9980 W: 1/X
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Calibration range : 3.6 to 909 ng/mL
4 replicates
CV lower then 4.6 %
Sample-to-sample run time 8 sec.
LDTD-MS/MS SignalLDTD-MS/MS SignalParaCurve01 - TIC - SM: 3 RT: 0.01 - 0.25 NL: 2.57E4F: + c ESI SRM ms2 [email protected] [ 109.750-110.450]
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AA: 26063
ParaCurve03 - TIC - SM: 3 RT: 0.00 - 0.25 NL: 6.04E4F: + c ESI SRM ms2 [email protected] [ 109.750-110.450]
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AA: 93111
ParaCurve44 - TIC - SM: 3 RT: 0.00 - 0.25 NL: 1.99E7F: + c ESI SRM ms2 [email protected] [ 109.750-110.450]
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AA: 25308818
Limit of detection : 2.6 ng/mL (3-times the blank value)
Blank
3.6 ng/mL 909 ng/mL
Application – Application – Sulfonamide Residues Sulfonamide Residues in Dairy Milk Analysisin Dairy Milk Analysis
How to obtain specificity How to obtain specificity without LCwithout LC
SulfonamidesSulfonamidesIsomers and related structures• APCI(+), isomers show the same MRM transitions• APCI (-), specific MRM transitions• Specificity achieve by :
• Right APCI mode• MRM mode
Sulfacetamide
Sulfadiazine
Sulfathiazole
Sulfapyridine
Sulfamerazine
Sulfamethazine
Sulfamethizole
Sulfamethoxazole
Sulfachloropyridazine
Sulfachlorpyridazine
Sulfaquinoxaline
Sulfisoxazole
Sulfadimethoxine
Sulfadoxine
Sulfamethoxypyridazine
Sulfaethoxypyridazine
LDTD-MS/MS SpecificityLDTD-MS/MS Specificity• No chromatographic separation to analyze 16 compounds in 10 seconds
• Specificity achieve using MRM in (-)APCI
Sulfadoxine
309 251
Sulfadimethoxine
309 131
C:\Xcalibur\data\03 avril 07\P990034 07 4/3/2007 10:02:58 AM sulfa 310 isomerems/ms negatifRT: 0.00 - 0.75 SM: 7G
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NL:3.34E8TIC MS Genesis P990034 07
RT: 0.00 - 0.75 SM: 7G
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NL:1.28E7m/z= 250.87-251.07 MS Genesis P990034 07
RT: 0.00 - 0.75 SM: 7G
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NL:7.92E6m/z= 130.80-131.00 MS Genesis P990034 07
TIC• Isomer analysis without chromatographic separation
• 2 extracted samples with Sulfadoxine or Sulfadimethoxine (isomers)
• TIC signal and extract signal for 2 MS/MS transitions
• Excellent specificity
LDTD Method and Dairy Milk LDTD Method and Dairy Milk Sample PreparationSample Preparation
Sample Preparation• 100 µL of Whole dairy milk• Spiked 16 sulfonamides (2 ng/mL to 1000 ng/mL)• Add Indapamide as internal standard• 500 µL of acetonitrile (precipitation agent)• Vortex 4 min.• Centrifuge at 14000 RPM for 10 min.• Filtrate supernatant on Nanosep 0.2 µm• Transfer 2 µL onto LazWell to perform
LDTD-MS/MS analysis
LDTD Parameters• Carrier gas flow : 2 L/min• APCI (-)• Laser Pattern
• Increase laser power to 25 % in 2 sec.• Hold at 25 % for 3 sec.• Decrease laser power to 0 %
MS Parameters• Collision gas pressure : 1.5 mTorr (Ar)• Scan time : 0.02 sec.• Q1 width : 0.70 amu• Q3 width : 0.70 amu• MS/MS transition and Collision energy :
Compound Q1 Q3 Collision Energy
(m/z) (V) Sulfacetamide 213 170 25 Sulfadiazine 249 185 25 Sulfathiazole 254 156 22 Sulfapyridine 248 184 25 Sulfamerazine 263 199 26 Sulfamethazine 277 122 28 Sulfamethizole 269 196 28 Sulfamethoxazole 252 156 28 Sulfachloropyridazine 283 128 34 Sulfachlorpyridazine 283 107 34 Sulfaquinoxaline 299 144 28 Sulfisoxazole 266 171 28 Sulfadimethoxine 309 131 34 Sulfadoxine 309 251 34 Sulfamethoxypyridazine 279 156 25 Sulfaethoxypyridazine 293 156 25 Indapamide (internal standard) 364 190 26
Analytical PerformanceAnalytical Performance
• Excellent linearity for all sulfonamides (> 0.99)
• Extraction recovery from 85 % to 100 %
• LOD of 2 ng/mL (4 pg loaded into well) from blank analysis
R T: 0 .0 0 - 2 .36 SM : 7 G
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NL : 2 .14 E5T IC F : + c ES I SR M m s2 2 54 .0 04 @ c id -3 0.00 [9 1.65 0-92 .3 50 ] M S G e ne sis s ulfon am id e0 2
R2 = 0,9954
R2 = 0,9979
R2 = 0,9983
R2 = 0,9987
R2 = 0,9986
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Sulfamethoxine
Sulfamethozaxole
Sulfamerazine
Sulfapyridine
Sulfamethazine
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311 - 92
265 - 92
250 - 156
279 - 124
254 - 92
Concentration (ng/mL)
Blank
1000 ng/mL
Application – Phase I Application – Phase I and Phase II and Phase II
Metabolite Back Metabolite Back Conversion EvaluationConversion Evaluation
Back-ConversionBack-Conversion• LDTD-MS/MS does not have any chromatographic separation
• All sample constituent may thermally desorbed, ionized and be introduced into the MS
• Phase I and Phase II metabolites may back-convert (thermally or in the APCI region) into the corresponding drugs which may affect the quantification
• Experiment
• Sample containing high metabolite quantities
• LDTD setup at the drug operating conditions
• Monitoring the MS/MS transition of the drug and the metabolite
• Evaluation of the back-conversion of the metabolite
OH-Midazolam Back-ConversionOH-Midazolam Back-ConversionRT: 0.99 - 1.41
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AA: 78882AA: 80390
AA: 44885137
AA: 44090203
NL: 3.54E4TIC F: + c ESI SRM ms2 [email protected] [290.690-291.390] MS Genesis BackconvertHydro
NL: 2.43E7TIC F: + c ESI SRM ms2 [email protected] [304.650-305.350] MS Genesis BackconvertHydro
MS/MS Midazolam signal
326.04 – 291.04 amu
MS/MS OH-Midazolam signal
2.5 µg/mL solution
340.0 – 305.0 amu
Observed back-conversion of 0.2 %
Testosterone and Testosterone Testosterone and Testosterone Glucuronide Glucuronide
• Experiment :
•100 µL Stripped Human EDTA Plasma
• 500 µL Ethyl Acetate
• Vortex agitation for 4 min.
• Spiked supernatant with Testosterone (3 ng/mL)
• Spiked supernatant with Testosterone glucuronide (200 ng/mL)
• Analyze 2 µL in LDTD-MS/MS, following Testosterone transition (289.26 – 109.19 uma, 21 eV)
• From Peng et al. Clinical Chemistry, 46:4, 515-522 (2000)
•Testosterone glucuronide in healthy Caucasian subject lower then 5 nM
•Testosterone oral dose of 120-mg
•1 hour after administration blood Testosterone glucuronide increase at 310 nM
Testosterone and Testosterone Testosterone and Testosterone GlucuronideGlucuronide
• Real-life (Peng et al.) [5 nM of Testosterone glucuronide in blood]
• Liquid-liquid extraction with organic hydrophobic solvent
• Testosterone extracted in organic phase
• Testosterone glucuronide stays in aqueous phase
• 0.25 % of back-conversion will be negligible on the Testosterone signal (less then 0.01 ng/mL)
• Results
• Extract with Testosterone and no Testosterone glucuronide : 23254 count
• Extract with Testosterone + Testosterone glucuronide : 27021 count
• Back-conversion : 0.25 %
Human Liver Human Liver MicrosomesMicrosomes
CYP3A4 inhibition studyCYP3A4 inhibition study(Midazolam signal)(Midazolam signal)
Inhibition 1
Inhibition 2
Inhibition 3
T-30T-20 T-10 T-0T-30T-20 T-10 T-0 T-30T-20 T-10 T-0
Inhibition 1
Inhibition 2
Inhibition 3
T-30T-20 T-10 T-0T-30T-20 T-10 T-0T-30T-20 T-10 T-0T-30T-20 T-10 T-0 T-30T-20 T-10 T-0T-30T-20 T-10 T-0
• 3 Inhibition studies
• 4 replicates
• 2 µL directly spotted into well
• Sample-to-sample run time of 10 seconds
• No internal standard correction
CYP3A4 inhibition studyCYP3A4 inhibition study(Midazolam signal)(Midazolam signal)
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CYP3A4 inhibition studyCYP3A4 inhibition study(OH-Midazolam signal) (OH-Midazolam signal)
• No internal standard correction• CV lower then 15 %
CYP3A4 : OH-MidazolamCYP3A4 : OH-Midazolam(with ISTD) (with ISTD)
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CYP2D6 : OH-BufurololCYP2D6 : OH-Bufurolol(with ISTD)(with ISTD)
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CYP2C9 : OH-DiclofenacCYP2C9 : OH-Diclofenac(with ISTD)(with ISTD)
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Sample number
Pooled-CYP and CYP-Cocktail SamplesPooled-CYP and CYP-Cocktail Samples
Isozyme Metabolite
CYP3A4 OH-Testosterone OH-Midazolam Oxidized Nifedine
CYP1A2 Acetaminophene
CYP2C9 OH-Diclofenac OH-Tolbutamide
CYP2C19 Mephynetoin CYP2E1 OH-Chloroxazone
CYP2D6 OH-Bufurolol Dextrophan
• LDTD allows to analyzed Pooled-CYP and CYP-cocktail samples
• Run-time of 9 seconds per samples• List of CYP studies available until now :
• Thermal desorption in induced indirectly by laser diode.
• No photon-sample interactions
• There is no need for an enhancement matrix.
• There is no liquid mobile phase.
• Ionization is produced by corona discharge.
• Sample-to-sample run time as low as 4.5 seconds.
CONCLUSIONSCONCLUSIONS
• Picogram sensitivity can be achieved using 2-5 μL of sample.
• No carryover or memory effect is observed during the process of 960 samples batch (and more).
• Excellent linearity and accuracy achieve with the LDTD-MS/MS system.
• Comparable performance to LC-MS/MS with higher throughput.
CONCLUSIONSCONCLUSIONS
QUESTIONSQUESTIONS