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Asimina A. Papanastasiou, Alexandros G. Asimakopoulos, Viola L. Borova and Nikolaos S. Thomaidis.

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Presentation on theme: "Asimina A. Papanastasiou, Alexandros G. Asimakopoulos, Viola L. Borova and Nikolaos S. Thomaidis."— Presentation transcript:

1 Asimina A. Papanastasiou, Alexandros G. Asimakopoulos, Viola L. Borova and Nikolaos S. Thomaidis

2 WHY A MULTI-ANALYTE SCREENING? Use of illicit drugs is widespread Absence of multi - residual methods for drug-use surveillance programs for prisoners and probationers pre-employment screening for employees screening for abuse of crime offenders and of their victims doping control and clinical screening for treatment of patients Inadequate application of immunoassay 1. only common abused drugs can be determined 2. immunoassays are applied for only one drug each time

3 Urine matrix Urine is a simple aqueous matrix which has the following advantages in the drug analysis: Concentration of drugs and their metabolites are remarkably high Urine can be easily sampled Testing is non-invasive Volume of the sample is adequate Urine test provides long detection windows for drug use

4 Literature review Number of compounds Matrix LC-MS System LOQs, LOIs, Linear range References 97 Drugs/DoA/metabolites Plasma LC-MS/MS (QTRAP) LOI: μg/L Viette et al., Clinical Biochem Drugs/DoA/metabolites Urine LC-HRMS (LTQ-Orbitrap) LLOQs: ng/mL Li et al., JCA DoA Urine LC-MS (Ion Trap) Linear range: ng/mL Cheng et sl., Forensic Sci. Int., groups of DoAs (27) Urine LC-MS/MS (QTRAP) Linear range: ng/mL de Jager et al., JCB DoAs Urine LC-MS/MS (QqQ) LOQs: ng/mL Lin et al., JCB DoAs Plasma LC-MS/MS (QTRAP) Linear range: ng/mL Maralikova et al., JCB DoAs Urine LC-MS/MS (QqQ) Linear range: ng/mL Chen et al., Talanta 2009

5 Aims of the study The development of a  multi-analyte  sensitive  accurate and  fast  screening method for the simultaneous determination of 72 licit and illicit drugs, and their metabolites belonging to different groups with a generic sample preparation in urine samples

6 Hybrid SPE-PPT Cartridge

7 72 Target Analytes Opiates – Opioids (8) Cocaine Compounds (3) Amphetamines (5) Hallucinogens ( Cannabinoids (2), LSD (2) ) Benzodiazepines (13) Barbiturates (2) Antipsychotics (5) Anesthetics (6) Antiepileptics (6) Antidepressants ( TCAs (5), TeCAs (2), SSRIs (4), SNRIs (1) ) Hypnotics (1) Sympathomimetics (2) New Designer Drugs (5)

8 Optimization Precipitating agents tested: 1.Methanol 2.Methanol (1% v/v formic acid) 3.Methanol (1% w/v ammonium formate) 4.Acetonitrile 5.Acetonitrile (1% v/v formic acid) 6.Acetonitrile / Methanol 1/1 (1% w/v ammonium formate) Best precipitating agent Acetonitrile (1% v/v formic acid) *Mobile Phase, ESI and MS/MS parameters were optimized in previous studies

9 Method Protocols Ούρα (300 μL)+ IS + ACN 1% FA (1200 μL) Vortex Φυγοκέντρηση 10 min, 4000rpm Υπερκείμενο στο Hybrid SPE-PPT cartridge Εκχύλισμα στα 1500 μL, LC/MS-MS β- Γλυκορουνιδάση/ Επώαση στους 37 °C για 24 hours Hybrid SPE-PPTHybrid SPE-PPT με ενζυματική αποσύζευξη *β-Glucuronidase Type HP-2 (Helix Pomatia)

10 Validation (1/3) Hybrid SPE-PPT Hybrid SPE-PPT with enzymatic deconjugation LLOQ 0.05 ng/mL (EDDP) – 25 ng/mL (EME) Linear Range 0.05 (EDDP) – 500 ng/mL R 2 > 0.99 LLOQ 0.25 ng/mL (Lidocaine/EDDP) - 25 ng/mL (Phenytoin) Linear Range 0.25 (EDDP) – 250 ng/mL R 2 > 0.99 EDDP transitions (spike 0.05 ng/mL)

11 Validation (2/3) Hybrid SPE-PPT Hybrid SPE-PPT with enzymatic deconjugation Recovery tests Low level (25 ng/mL): 63.3 (Amphetamine) (Morphine) Medium level (100 ng/mL): 60.1 (Benzylpiperazine) (Primidone) High level (500 ng/mL): 67.9 (Benzylpiperazine) – 109 (Morphine) Recovery tests Low level (25 ng/mL): 68.5 (Flunitrazepam) – 100 (Codeine) Medium level (100 ng/mL): 62.9 (Δ9-THC) (Lamotrigine) High level (250 ng/mL): 70.7 (Phenobarbital) – 109 (OH-LSD)

12 Intermediate Precision (%RSDs) Low level (25 ng/mL): 4.6 (Benzylpiperazine) – 19 (Phenyntoin) Medium level (100 ng/mL): 4.0 (MDMA) – 19 (Pentobarbital) High level (500 ng/mL): 4.8 (Codeine) – 19 (Fluoxetine) Intermediate Precision (%RSDs) Low level (25 ng/mL): 4.2 (Sertraline) – 19 (Carbamazepine) Medium level (100 ng/mL): 5.6 (Risperidone) – 19 (Heroin) High level (250 ng/mL): 4.7 (Diazepam) – 17 (Fluoxetine ) Validation (3/3) Hybrid SPE-PPT Hybrid SPE-PPT with enzymatic deconjugation

13 Matrix effects (-)

14 Matrix effects

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17 Conclusions (1/2) The method developed has the following characteristics: Multi-analyte (72 licit & illicit drugs, belonging to 13 different groups) Confirmatory (both confirmation and quantification ions were monitored) Efficient Sensitive LLOQs achieved: Method with only hybrid SPE-PPT: 0.05 (EDDP) -25 ng/mL (EME) Method with hybrid SPE-PPT with enzymatic deconjugation: 0.25 (EDDP) – 25 ng/mL (Phenyntoin)

18 Conclusions (2/2) Satisfactory recoveries Method with hybrid SPE-PPT >60.0 % Method with hybrid SPE-PPT with enzymatic deconjugation > 63.0 % Fast and generic sample preparation Simultaneous extraction of polar, non-polar and medium polarity compounds Novel clean-up step using Hybrid SPE-PPT cartridges No evaporation step Relatively fast and sensitive chromatographic separation (analysis time: 28 min)

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