1 / 19

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 ,

matty
Télécharger la présentation

Asimina A. Papanastasiou , Alexandros G. Asimakopoulos ,

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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!

More Related