LOGO Institute of Toxicology Clinical Toxicology and Pharmacology Berliner Betrieb für Zentrale Gesundheitliche Aufgaben, Berlin Rapid Quantification of Tilidine, Nortilidine and Bisnortilidine in Urine by automated online-spe-lc-ms/ms Christoph Köhler Thomas Grobosch Torsten Binscheck 48th Annual Meeting of the International Association of Forensic Toxicologists Conference in Bonn August 29 - September 2, 2010
Tilidine introduction Synthetic opioid analgesic Strong analgesic effect of its main metabolite nortilidine [2]. Hepatically N-demethylation of tilidine to nortilidine and bisnortilidine (CYP3A4, CYP2C19, CYP2D6) [1] In commercially available preparations tilidine is combined with the µ-opioid-receptor antagonist naloxone preventing opioid abuse [2]. HO (+)-tilidine O CH 3 O CH 3 N CH 3 O CH 3 O NH CH 3 (+)-nortilidine O CH 3 O O N OH naloxone (1S,5R,13R,17S) CH 2 O NH 2 (+)-bisnortilidine Source: [1] Clin. Chem. 24/2, 692-697 (1978) [2] Fachinfo Valorone N, Fa. Pfizer; 2008 2
Aims introduction Sensitive quantification of tilidine and its metabolites in urine samples No immunoassay for the detection of tilidine abuse is available! Offline-LLE LC QTrap Increasing number of samples (max. 100 samples/d!) consumed much time and human resources for the LLE (max. 180 min/d!). Online-SPE LC QTrap 3
Online-SPE-LC introduction Highly automated sample preparation Automatization: 1. IS-mixing procedure 2. Complete SPE procedure Chromatography Analytical column: Luna phenyl-hexyl 50x2 mm 5 µm Mobile phase: 0.2% FA + MeOH (gradient elution) Total run time: 3.5 min Symbiosis Pico Spark Holland Online-SPE LC 4
SPE Optimization SPE method development Mix setup: SPE sorbent: 100 µl Aqueous analyte solution of tilidine, nortilidine and naloxone (c = 50 µg/l) + 100µL 100 mm NH 4 Ac-solution (ph 6); IS PCP-D 5 (c=100 ng/ml) OASIS WCX 10x1mm Generic SPE procedure: SPE step solvent volume (µl) flow rate (ml/min) Conditioning MeOH 1500 5.0 Equilibration 1000 5.0 Loading 10 mm NH 4 Ac-solution ph 6 500 0.5 Clean up 1000 1.0 Elution Mobile phase 1050 0.3 5
SPE Optimization SPE method development Mix setup: SPE sorbent: 100 µl Aqueous analyte solution of tilidine, nortilidine and naloxone (c = 50 µg/l) + 100µL 100 mm NH 4 Ac-solution (ph 6); IS PCP-D 5 (c=100 ng/ml) OASIS WCX 10x1mm Generic SPE procedure: SPE step solvent volume (µl) flow rate (ml/min) Conditioning MeOH 1500 5.0 Equilibration 1000 5.0 10 mm NH 4 Ac-solution ph 6 Loading 500 0.5 Clean up?? 1.0 Elution Mobile phase? 0.3 6
Results (wash solution) SPE method development Wash solution: 10 mm NH 4 Ac-solution ph 6 + 0/10/15/20/40% MeOH 100 recovery (%) 80 60 40 20 0 0 10 20 30 40 tilidine nortilidine naloxone IS MeOH in wash solution (%) Conclusion: 10% MeOH 7
Results (wash volume) SPE method development Wash solution: 10 mm NH 4 Ac-solution ph 6 + 10% MeOH Wash volume: 500/1000/1500 µl 100 %) recovery ( 80 60 40 20 tilidine nortilidine naloxone IS 0 0 500 1000 1500 wash volume (µl) Conclusion: 500 µl 60 bed volumes 8
Results (elution volume) SPE method development Wash solution: 10 mm NH 4 Ac-solution ph 6 + 10% MeOH Elution volume: 150/300/600/900/1200 µl 100 recovery (% %) 80 60 40 20 tilidine nortilidine naloxone IS 0 0 200 400 600 800 1000 1200 elution volume (µl) Conclusion: 600 µl 2 min elution time 9
Final SPE method SPE method development Mix setup: SPE sorbent: 100 µl sample (urine) + 100µL 100 mm NH 4 Ac-solution (ph 6); c(pcp-d 5 )=100 ng/ml OASIS WCX 10x1mm Generic SPE procedure: SPE step solvent volume (µl) flow rate (ml/min) Conditioning MeOH 1500 5.0 Equilibration 1000 5.0 10 mm NH4 Ac-solution ph 6 Loading 500 0.5 Clean up 10 mm NH 4 Ac-solution ph 6 + 10% MeOH 500 (1000) 1.0 Elution Mobile phase 600 (1050) 0.3 10
Untitled 18 (Nortilidin 1): "Linear Through Zero" Regression ("No" weighting): y = 0.00911 x (r = 0.9992) Validation data of the new method Validation and routine work Selectivity: no interference by matrix peaks Ionsuppression: < 10% for all analytes Recovery: > 80% for all analytes and IS Intra-assay precision (n=6 per conc.): coefficient of variation: 6% Inter-assay precision (n=6 per conc.): coefficient of variation: 7% LLOQ: 1.0 µg/l for all analytes Linearity: 1.0-100 µg/l Analyte Area / IS Area Analyte Area / IS Area Analyte Area / IS Area Untitled 18 (Tilidin 1): "Linear Through Zero" Regression ("No" weighting): y = 0.0101 x (r = 0.9993) 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 Calibration curves of the analytes tilidine R = 0.9993 0.25 0.20 0.15 0.10 0.05 0.00 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Analyte Conc. / IS Conc. 0.90 0.85 0.80 0.75 0.70 0.65 0.60 nortilidine 0.55 0.50 0.45 0.40 0.35 0.30 R = 0.9992 0.25 0.20 0.15 0.10 0.05 0.00 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Analyte Conc. / IS Conc. Untitled 18 (Bisnortilidin 1): "Linear Through Zero" Regression ("No" weighting): y = 0.00635 x (r = 0.9996) 0.63 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 bisnortilidine R = 0.9996 0.15 0.10 0.05 0.00 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Analyte Conc. / IS Conc. Society of Toxicological and Forensic Chemistry (GTFCH) Guidance in forensic quality terms Toxichem Krimtech 2009; 76 (3): 185 11
Implementation in routine Reduction of the manual preparation time by the factor 10 Positive result: c (tilidine) and c (nortilidine) > 1.0 µg/l (cutoff) c (nortilidine) and c (bisnortilidine) > 1.0 µg/l (cutoff) or Analysis of 3665 samples from correctional facilities: - 55 positive results (1.5 %) - positive samples: c (bisnortilidine) > c (nortilidine) could the detection of bisnortilidine increase the drug detection window? 12
Urinary Excretion of Tilidine Tilidine A single oral dose of Valoron N solution (50 mg T-HCl and 4 mg NX-HCl) was administered to a male volunteer (age: 28 years; body height: 175 cm; body weight: 72 kg) g Analyte Drug detection window (d) - ß-glucuronidase + ß-glucuronidase Tilidine 4.5 4.5 Nortilidine 5.5 5.5 Bisnortilidine 6.8 7.8 positive result 5.5 5.5 13
Chromatograms 1.81 L Intensity, cps 2.0e4 1.5e4 1.0e4 Tilidine (c=7.63 µg/l) m/z 274.2 155.1 L Intensity, cps 1.00e5 8.00e4 6.00e4 4.00e4 Bisnortilidine (c=96.5 µg/l) m/z 246.1 155.1 5000.0 2.00e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min 0.00 0.5 1.0 1.5 2.0 2.5 3.0 Time, min L Intensity, cps 7000 6000 5000 4000 3000 2000 1000 Tilidine m/z 274.2 115.1 L Intensity, cps 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 Bisnortilidine m/z 246.1 229.2 1.81 2.05 0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min Concentration: N/A Calculated Conc: 5.44 ng/ml Proc. Algorithm: Analyst Classic Intensity, cps 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 Nortilidine (c=36.9 µg/l) m/z 260.2 155.1 1.81 1.0e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min Concentration: N/A Calculated Conc: 5.17 ng/ml Proc. Algorithm: Analyst Classic Intensity, cps 4000 3000 2000 1000 Nortilidine m/z 260.2 115.1 Concentration: 1.00 ng/ml Calculated Conc: N/A Proc. Algorithm: Analyst Classic Intensity, cps 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.94 IS Phencyclidine-D 5 (c=33.33 µg/l) m/z 249.3 96.1 1.0e5 0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min 2.13 14
Conclusion An highly sensitive method for the determination of tilidine, nortilidine and bisnortilidine in urine was developed and validated on an online- SPE-LC-QTrap Due to the automatization of the online-spe the manual preparation time of the new method is 20 min/100 samples. 3665 authentic samples were analyzed resulting in 55 positive results. The LLOQ of 1 µg/l allowed detecting a single oral dose of a tilidine preparation up to 5 days after administration. 15
Aknowledgements Martin Sibum(Spark Holland) for technical support Dr. Goebel (Medizinisches Labor Bremen) for delivering Bisnortilidine Thank you for your attention 16