Agenda. Principle of Operation Key Features Key Applications in Toxicology Examples of Toxicology Applications Technical Documentation
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2 Agenda Principle of Operation Key Features Key Applications in Toxicology Examples of Toxicology Applications Technical Documentation
3 LDTD: Laser Diode Thermal Desorption High-Throughput Ion source Plug-and-play front end solution 4-6 seconds sample-to-sample analysis time Can be combined with AB Sciex Ion Mobility Source (SeleXion ) for separation of Isobaric compounds Sensitivity, accuracy, linearity and reproducibility equivalent or superior to LC-ESI and APCI Used for Small molecule analysis (< 1200 amu)
4 Principle of operation What is Laser Diode Thermal Desorption? Plate IR Laser Beam Metal Sheet Sample dried into the well cavity No photon interactions with the sample Continuous laser (no pulse) Ultra fast heating transfer Quantitative sample desorption
5 Principle of operation Ion Transfer Process: LazWell Sample Plate with metal insert Carrier Gas IR Laser Beam Transfer Tube Mass Spectrometer Inlet Piston head Piston Corona Discharge Needle
6 Principle of operation Ionization Process for LDTD : The Atmospheric Pressure Chemical Ionization (APCI) of the LDTD is exempt of solvent and mobile phase: Reinvented APCI Water molecules are the sole source of protons (H + ) Low temperature process, 30⁰ Celsius Carrier Gas H 2 O O 2 N 2 Corona Discharge H 2 O H 2 O + H 3 O + N + 2 N 2 e - e High Voltage H 2 O N 2 + N + 2 e - N 2 N + e - 4 (H 2 O) n H + + Analyte Analyte is forced to react with the cluster ion producing a complete ionization
7 Market Differentiation There are two ways to improve the speed of analysis on a LC-MS/MS system Work with the LC Multiplexing TLX/LX (Thermo) UPLC (Waters, Shimadzu, etc) Shotgun approach No chromatography LDTD-Phytronix Biocius RapidFire (Agilent)
8 Key Features The thermal desorption process takes seconds Low volume sample analysis (1 to 10 µl) 96-well plates are designed to be compatible with conventional sample preparation systems Elimination of carry over due to LC (needle wash) Each well is individually isolated during the thermal desorption Plug-and-play device with all Mass Spec OEM Low maintenance system Small molecule analysis only
9 Key Features Plug-and-play device Full software compatible with MassLynx, Xcalibur, Analyst Thermo AB Sciex No need to modify the actual MS 96-Well and 384-Well LDTD Devices available Agilent Waters
10 Applications TOXICOLOGY CRO FOOD PHARMACEUTICAL ENVIRONMENT CLINICAL
11 Various Applications: Toxicology and Forensics (Drugs of abuse screening): Urine screening Urine confirmation Opiates (SeleXion) Environmental, Personal Health Care products in water Emerging contaminants in waste water Cyanotoxin Food analysis, antibiotic detection and quantification Antibiotics in milk, honey, shrimp, etc. Melamine testing Clinical analysis Testosterone in plasma (Combined with AB Sciex - SeleXion ) Vitamin D
12 Drug Confirmation on LDTD-MS Gold standard related to drug class GC-MS (Trap) and LC-MS/MS Time-consuming system : run can take up to 30 minutes GC-MS often requires a chemical derivatization LDTD-MS/MS MS/MS allows high specificity Sensitivity down to low ng/ml Runtime : 10 seconds per sample Allows multi-analyte confirmation including metabolite(s)
13 Drug Confirmation on LDTD-MS LDTD-Q-TOF Exact mass measurement (qualitative information) Possible retro-action on the results Very fast system : 10 sec / sample Large number of drugs can be targeted LDTD-Triple-TOF Accurate mass measurement meets MS/MS specificity Comparable sensitivity to a triple-quad system (down to low ng/ml) Runtime : 10 seconds per sample Allows multi-analyte confirmation including analytes and metabolites
14 Drug Coverage on LDTD Analgesics and Opiates such as Natural: Morphine, Codeine, Oxymorphone, Oxycodone, Hydrocodone Synthetics: Tramadol, Demerol, Fentanyl, Methadone, Darvon, Heroin Cannabinoids Natural: THCA Synthetics: K2/SPICE (JWH JWH-073) both OH and COOH metabolites Benzodiazepines and Barbiturates Street drugs such as Amphetamines, Cocaine, Ecstasy (MDA, MDMA,MDEA)
15 Toxicology Application High-Throughput Screening of Synthetic Cannabinoids using LDTD-TripleTOF
16 LDTD-TripleTOF 5600 System Synthetic cannabinoids have similar biologic activities as natural cannabis, THC. 5 compounds are already considered illegal in the United States (Controlled Substances Act March 1, 2011 Federal Register) Drugs are constantly changing in structure to bypass state legislation. We are therefore dealing with a moving target. To properly identify and quantify these drugs, we need : A fast, selective, and sensitive quantitative method: LDTD- HR-TOF MS/MS A system that allows for a fast screening of samples: LDTD-HR-TOF A way to select and target the best ions to extract MS/MS spectra during a sample screening run (Information Dependant Acquisition)
17 High Resolution Quantitation 5-hydroxylpentyl-JWH hydroxylbutyl-JWH carboxylbutyl-JWH carboxylbutyl-JWH-073 Range of drug concentrations in urine: 0.2 to 1000 ng/ml Sample Preparation: 100µL Urine 100µL Methanol/HCl (0.5N) [1:1v/v] with IS (Clomiphene) 300µL 1-Chlorobutane Vortex and Centrifuge 200µL from organic phase is evaporated Reconstitute with 20µL of Methanol/Water [75:25 v/v] 2µL used for LDTD analysis with a runtime of 8 seconds/sample
18 High Resolution Quantitation Drug Nominal Mass (amu) High Resolution Fragment (amu) 4-hydroxybutyl-JWH hydroxypentyl-JWH carboxylbutyl-JWH Specific Fragments 5-carboxylpentyl-JWH High Resolution permits the quantitation of 2 molecules that have the same Nominal Mass and structure. Dynamic range in the 3.5 order of magnitude
19 High Resolution Quantitation Drug LOD (ng/ml) LOQ (ng/ml) 4-hydroxybutyl-JWH hydroxypentyl-JWH carboxylbutyl-JWH carboxylpentyl-JWH
20 Conclusion The specific sample preparation method provided allows for a high quantitation range of 2 to 4000 ng/ml of JWH compounds on LDTD-TOF. This analysis mode can simultaneously detect multiple drugs. It is possible to perform a screening and quantification during the same analysis. To expand the range of compounds able to be detected in a sample, it may be preferable to use a generic extraction method. Immunoassay Fast System (10-30 seconds / sample) Expensive reagents Limited number of reagents available More or less accurate response (crossreactivity) LDTD-TripleTOF 5600 Fast System (5 seconds / sample) Unlimited number of compounds for detection Possible feedback actions during analysis
21 Generic Multi-Drug HR Quantitation Generic Sample Preparation Liquid-liquid (Acid) Extraction 50 µl Urine 50 µl HCl 0.2 N with 50ng/mL Clomiphene (IS) 200 µl 1-chlorobutane 2 µl of organic phase analyzed on LDTD-TripleTOF 5600 Liquid-Liquid (Basic) Extraction 50 µl Urine 50 µl NaOH 0.2 N with 50ng/mL Clomiphene (IS) 200 µl Ethyl Acetate 2 µl of organic phase analyzed on LDTD-TripleTOF 5600 Urine Samples spiked with: Fentanyl/Norfentanyl, Propoxyphene/Norpropoxyphene, Methadone/EDDP, Codeine, Morphine, Oxycodone, Oxymorphone, Diazepam, Estazolam, Hydroalprazolam, Hydroethylflurazepam, Hydromidazolam, Hydroxytriazolam, Lorazepam, Nordiazepam, Oxazepam, Temazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, Chlorodiazepoxide, Meperidine/Normeperidine, 5-hydroxypentyl-JWH-018 and 4-hydroxybutyl-JWH-073 Concentrations vary between 25 et 600 ng/ml, depending on the typical immunoassay cutoff range for each of the drugs. There are currently no Immunoassays available for the detection of synthetic cannabinoids.
22 Generic Multi-Drug HR Quantitation Estazolam Methadone Oxycodone Chlordiazepoxide Temazepam 5 Second Runtime Codeine Oxymorphone Lorazepam Example of a mass spectrum analysis from a basic extraction
23 Toxicology Analysis Methadone and EDDP confirmation in urine samples
24 Methadone and EDDP Sample Preparation Labelled internal standard used Solid Phase Extraction using a basic elution 2µL of extract were used on LDTD-MS/MS Run-time : 8 seconds / sample Calibration Curves 15 to 9600 ng/ml (r 2 >0.998) Accuracy (back-calculation) from 84 to 117 % Methadone EDDP
25 Methadone and EDDP Intra-run (n=24) on 1 specimen sample Methadone EDDP Target Concentration (ng/ml) Average Concentration (ng/ml) SD (ng/ml) CV (%) Inter-run: Over 40 specimens run in 3 different batches; CV s between 0.7 and 19.1 % (EDDP being more variable) * LDTD has withstood Joint Commission (JCAHO) and CAP audits.
26 Method Accuracy Results 40 specimens run in LDTD-MS/MS and in GC-MS (reference lab) No bias between both methods (within ± 20 %) 2 samples from the College of American Pathologists (CAP) were run and gave results within 1SD of the CAP survey results Sample stability, Carry-over and Interferences LazWell plate stability : within 3-days Carry-over not observed No interferences observed (Phenylpanolamine, Pseudo-ephedrine, Ibuprofen, Lidocaine, Procaine, Ephedrine, Caffeine and Acetaminophen)
27 Clinical Application Selectivity Enhancement in High Throughput Analysis of Testosterone using Differential Ion Mobility to LDTD-MS/MS MASCL 2012
28 Testosterone
29 Sample Preparation for Analysis Gain in Specificity with DMS allows a simple preparation: Spike standard curve in stripped plasma Liquid-liquid extraction with MTBE 1:4 v/v ratio Vortex 10 seconds 2 µl of the upper layer directly spotted onto the Lazwell plate Dry at room temperature MS/MS
30 Effect of Ion Mobility
31 SeleXionTM
32 Results High sensitivity with LOQ at 0.1 ng/ml (50 femtogram on plate) Excellent linearity, with r 2 = , over 5 orders of magnitude Accuracy and reproducibility were within the accepted values as shown in the table below. The sample-to-sample run time was only 7 seconds. In comparison, the equivalent analysis using conventional LC-MS/MS would typically require approximately 3-5 minutes per sample
33 More Publications
34 Conclusions 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 5 seconds
35 Conclusions Picogram sensitivity can be achieved using 2-5 μl of sample Added benefits using AB Sciex SeleXion source for Isobaric compounds No carryover or memory effect Comparable performance to LC-MS/MS with higher throughput
36 Comparison Procedure GC/MS LC/MS/MS LDTD/MS/MS Applicability to target screening Medium High High Effort on sample preparation High Low/Medium Low/Medium Risk of biological matrix effect Low High Medium Risk of carry-over High High (extensive needle wash) Very Low Analysis specificity Low/Medium High High Sensitivity Medium High High Thermolabile compounds analysis Low to medium Medium/High Medium/High Quantification power Medium/High High High System maintenance Medium High Very Low Analytical speed Low Medium (Higher UPLC) Very High
37 Technical Documentation Please visit for application notes, poster and presentations. Complete References of scientific papers are available on the web site. Specific applications and presentations available on demand to the marketing team. For more information, please contact: Global Sales and Marketing Alex Birsan, Research
38 Questions
39 LDTD and Mass Spectrometry The LDTD-APCI ion source can be adapted to : Triple quadrupole MS Ion trap MS Q-TOF systems Triple-TOF system (AB Sciex 5600) The LDTD allows : Fast qualitative screening Fast quantitative screening Fast confirmation results
40 Generic Multi-Drug HR Quantitation Screening Example Fixed limit of detection : 10 ng/ml Blank No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 Real Patient sample (unknown concentrations) evaluated for JWH-018 (OH- Metabolite) M.W g/mol - HR-TOF : amu
41 Generic Multi-Drug HR Quantitation Screening Example Fixed limit of detection : 10 ng/ml Blank No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 Real Patient sample (unknown concentrations) evaluated for JWH-250 (OH- Metabolite) M.W g/mol - HR-TOF : amu
42 Toxicology Analysis Propoxyphene and Norpropoxyphene confirmation in urine samples
43 Sample Preparation Propoxyphene / Norpropoxyphene Labelled internal standard used Solid Phase Extraction using a basic elution 2 µl of extract were used to run LDTD-MS/MS analysis Run-time : 14 seconds / sample Calibration Curves 25 to ng/ml (r 2 >0.99) Accuracy (back-calculation) from 90.9 to 114 % Propoxyphene Norpropoxyphene
44 Propoxyphene / Norpropoxyphene Intra-run (n=15) for 3 specimens Sample 1 Sample 2 Sample 3 Average Concentration (ng/ml) PPX NPPX SD (ng/ml) CV (%) Average Concentration (ng/ml) SD (ng/ml) CV (%) Average Concentration (ng/ml) ,733.3 SD (ng/ml) CV (%) Inter-run: Over 40 specimens run in 3 different batches; CV s below 6.3 %
45 Method Accuracy Propoxyphene / Norpropoxyphene 40 specimens run in LDTD-MS/MS and in GC-MS (reference lab) LDTD-MS/MS calibration range ng/ml GC-MS calibration range ng/ml No bias between both methods (within ± 20 %) Sample stability, Carry-over and Interferences LazWell plate stability : within 3-days Carry-over not observed No interferences observed (Phenylpanolamine, Pseudo-ephedrine, Ibuprofen, Lidocaine, Procaine, Ephedrine, Caffeine and Acetaminophen)
46 Is it like MALDI? Differences between LDTD and MALDI Wide Infrared Laser Beam at 3 mm diameter LDTD Carrier Gas Laser beam MALDI Metal Sheet Neutral Ion High Efficiency APCI Ionization NO interaction photon-molecule Dry sample in the bottom of the well Thermal Vaporisation by fast and precise heat transfert Complete Vaporisation of the sample Matrix needed for ionizaiton Pulsed Laser reacting with the sample Important matrix effects and adducts Sampling of the surface
47 Market Differentiation Differences between LDTD and RapidFire Dried sample thermally desorbed every 5 seconds Highly resistant to Ionic suppression and matrix effect APCI based ionization No CarryOver Plug and Play device, easy to use Generic method for most of compounds Much cleaner, less MS inlet maintenance and shut down Liquid sample by switching valve every 5 seconds No separation High ionic suppression and matrix effect High Carry Over ESI based ionization SPE online sample preparation multiple cartridges tuning method necessary Complex, Expensive maintenance cost
48 Relative Abundance Relative Abundance High-Throughput LDTD-MS/MS analysis RT: SM: 13G NL: 1.40E5 TIC F: + c ESI SRM ms @cid16.00 [ ] MS ICIS Batch12B3 RT: SM: 7G 96-Replicates NL: 2.18E5 TIC F: + c ESI SRM ms @cid16.00 [ ] MS Plaquette5_ NL: 1.38E5 TIC F: + c ESI SRM ms @cid16.00 [ ] MS ICIS Batch12B NL: 1.79E5 TIC F: + c ESI SRM ms @cid16.00 [ ] MS Plaquette5_ Time (min) Analyte Desorption in 1.8 seconds Time (min)
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