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Asimina A. Papanastasiou , Alexandros G. Asimakopoulos ,

Simultaneous determination of 72 drugs of abuse in human urine by hybrid solid phase extraction – protein precipitation technique (Hybrid SPE-PPT) tailored to Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Asimina A. Papanastasiou , Alexandros G. Asimakopoulos ,

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Asimina A. Papanastasiou , Alexandros G. Asimakopoulos ,

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  1. Simultaneous determination of 72 drugs of abuse in human urine by hybrid solid phase extraction – protein precipitation technique (Hybrid SPE-PPT) tailored to Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) 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 • only common abused drugs can be determined • 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

  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: • Methanol • Methanol (1% v/v formic acid) • Methanol (1% w/v ammonium formate) • Acetonitrile • Acetonitrile (1% v/v formic acid) • 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 Hybrid SPE-PPT Hybrid SPE-PPT με ενζυματική αποσύζευξη Ούρα (300 μL)+ IS + ACN 1% FA (1200 μL) β- Γλυκορουνιδάση/ Επώαση στους 37 °C για 24 hours Vortex Φυγοκέντρηση 10 min, 4000rpm Υπερκείμενο στο Hybrid SPE-PPT cartridge Εκχύλισμα στα 1500 μL, LC/MS-MS *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 • R2> 0.99 • LLOQ 0.25 ng/mL (Lidocaine/EDDP) - 25 ng/mL (Phenytoin) • Linear Range 0.25 (EDDP) – 250 ng/mL • R2> 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) - 111 (Morphine) Medium level (100 ng/mL): 60.1 (Benzylpiperazine) - 109 (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) - 119 (Lamotrigine) High level (250 ng/mL): 70.7 (Phenobarbital) – 109 (OH-LSD)

  12. Validation (3/3) Hybrid SPE-PPT Hybrid SPE-PPT with enzymatic deconjugation 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)

  13. Matrix effects (-) (-)

  14. Matrix effects (-) (-) (-) (-)

  15. 22.0 21.7 22.0 23.0 16.7 21.6 21.2 20.1 18.3 20.9 21.5 17.7 20.9

  16. 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) -25ng/mL (EME) Method with hybrid SPE-PPT with enzymatic deconjugation: 0.25 (EDDP) – 25ng/mL (Phenyntoin)

  17. 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)

  18. Thank you very much for your attention!

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