examples of toxicology applications - arc sciences apps and ppt… · •no photon interactions...

Principle of Operation

Key Features

Key Applications in Toxicology

Examples of Toxicology Applications

Technical Documentation

Agenda

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)

LDTD: Laser Diode Thermal Desorption

What is Laser Diode Thermal Desorption ?

IR Laser Beam

Plate

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

Principle of operation

IR Laser Beam

LazWell Sample Plate with metal insert Carrier Gas

Corona Discharge Needle

Mass Spectrometer Inlet

Piston

Transfer Tube

Piston head


Ion Transfer Process:

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

H2O

O2

N2

H2O+

H2O

H3O+

N2

N2+

+ + + + + + + +

e-

e-

e- e-

N4+

N2

H2O N2+

N2+

(H2O)nH+ + Analyte

Analyte is forced to react with the cluster ion producing a complete ionization

High Voltage

Corona Discharge

Carrier Gas

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)

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

Key Features

Key Features

Plug-and-play device

Full software compatible with MassLynx™, Xcalibur™, Analyst™

No need to modify the actual MS

96-Well and 384-Well LDTD Devices available

Thermo

Agilent Waters

AB Sciex

Applications

PHARMACEUTICAL

CRO

TOXICOLOGY

FOOD

ENVIRONMENT

CLINICAL

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

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)

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

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-018 + JWH-073) both OH and COOH metabolites

Benzodiazepines and Barbiturates

Street drugs such as

Amphetamines, Cocaine, Ecstasy (MDA, MDMA,MDEA) …

Toxicology Application

High-Throughput Screening of Synthetic Cannabinoids

using LDTD-TripleTOF

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)

High Resolution Quantitation

5-hydroxylpentyl-JWH-018 4-hydroxylbutyl-JWH-073 5-carboxylbutyl-JWH-018

• 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

4-carboxylbutyl-JWH-073


Drug Nominal Mass (amu) High Resolution Fragment (amu)

4-hydroxybutyl-JWH-073 344.3 155.0410 – 155.0510

5-hydroxypentyl-JWH-018 358.3 230.1104 – 230.1154

4-carboxylbutyl-JWH-073 358.3 230.0740 – 230.0790

5-carboxylpentyl-JWH-018 372.3 155.0410 – 155.0510

Specific Fragments

• 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


Drug LOD (ng/mL) LOQ (ng/mL)

4-hydroxybutyl-JWH-073 0.22 0.73

5-hydroxypentyl-JWH-018 0.01 0.04

4-carboxylbutyl-JWH-073 0.36 1.2

5-carboxylpentyl-JWH-018 0.36 1.2

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 (cross-reactivity)

LDTD-TripleTOF™5600

• Fast System (5 seconds / sample)

• Unlimited number of compounds for detection

• Possible feedback actions during analysis

Generic Multi-Drug HR Quantitation

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

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

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.

Generic Sample Preparation

There are currently no Immunoassays available for the detection of synthetic cannabinoids.


Example of a mass spectrum analysis from a basic extraction

Estazolam

Codeine

Chlordiazepoxide

Temazepam

Oxymorphone

Methadone

Oxycodone

Lorazepam

5 Second Runtime

Toxicology Analysis

Methadone and EDDP confirmation in urine

samples

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

Methadone

EDDP

Calibration Curves

• 15 to 9600 ng/mL (r2>0.998)

• Accuracy (back-calculation) from 84 to 117 %

Methadone and EDDP

Intra-run (n=24) on 1 specimen sample

Inter-run: Over 40 specimens run in 3 different batches; CV’s between 0.7 and 19.1 % (EDDP being more variable)

Methadone EDDP

Target Concentration (ng/mL) 2132 2956

Average Concentration (ng/mL) 2247 2735

SD (ng/mL) 66 130

CV (%) 3.0 5.0

* LDTD has withstood Joint Commission (JCAHO) and CAP audits.

Results

Method Accuracy

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)

Clinical Application

MASCL 2012

Selectivity Enhancement in High Throughput Analysis of

Testosterone using Differential Ion Mobility to

LDTD-MS/MS

Testosterone

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

Effect of Ion Mobility

SeleXionTM

Results

High sensitivity with LOQ at 0.1 ng/ml (50 femtogram on plate)

Excellent linearity, with r2 = 0.99972, 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

More Publications

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

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

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

Please visit www.phytronix.com 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:

Technical Documentation

@Phytronix

Global Sales and Marketing

Alex Birsan, Research Scientist

[email protected]

Questions

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


Real Patient sample (unknown concentrations) evaluated for JWH-018 (OH-Metabolite) M.W. 357.4 g/mol - HR-TOF : 358.18016 amu

Blank No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10

Fixed limit of detection : 10 ng/mL

Screening Example


Real Patient sample (unknown concentrations) evaluated for JWH-250 (OH-Metabolite) M.W. 351.2 g/mol - HR-TOF : 352.19072 amu

Screening Example

Blank No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10

Fixed limit of detection : 10 ng/mL

Propoxyphene and Norpropoxyphene

confirmation in urine samples

Toxicology Analysis

Propoxyphene / Norpropoxyphene

Sample Preparation

• 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

Propoxyphene

Norpropoxyphene

Calibration Curves

• 25 to 12800 ng/mL (r2>0.99)

• Accuracy (back-calculation) from 90.9 to 114 %


Intra-run (n=15) for 3 specimens

PPX NPPX

Sample 1

Average Concentration

(ng/mL) 182.9 1729.2

SD (ng/mL) 11.9 33.7

CV (%) 6.5 1.9

Sample 2


(ng/mL) 459.9 12.600

SD (ng/mL) 12.8 233.5 CV (%) 2.8 1.9

Sample 3


(ng/mL) 3395.8 21,733.3

SD (ng/mL) 71 465.8 CV (%) 2.1 2.1

Inter-run: Over 40 specimens run in 3 different batches; CV’s below 6.3 %


Method Accuracy

40 specimens run in LDTD-MS/MS and in GC-MS (reference lab)

• LDTD-MS/MS calibration range 25-12800 ng/mL

• GC-MS calibration range 50-4000 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)

Wide Infrared Laser Beam at 3 mm diameter

LDTD

Metal Sheet

Carrier Gas

Differences between LDTD and MALDI

•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

Neutral Ion

Laser beam

•Matrix needed for ionizaiton •Pulsed Laser reacting with the sample •Important matrix effects and adducts •Sampling of the surface

MALDI

Is it like MALDI?

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

Market Differentiation

High-Throughput LDTD-MS/MS analysis

RT: 0.00 - 7.74 SM: 7G

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TIC F: + c ESI SRM ms2 [email protected] [110.095-110.105] MS Plaquette5_080422103040

NL: 1.79E5

TIC 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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TIC F: + c ESI SRM ms2 [email protected] [113.995-114.005] MS ICIS Batch12B3

Analyte Desorption

in 1.8 seconds

96-Replicates

examples of toxicology applications - arc sciences apps and ppt… · •no photon interactions...

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