Institute of Toxicology Clinical Toxicology and Pharmacology LOGO Berliner Betrieb für Zentrale Gesundheitliche Aufgaben, Berlin

Rapid Quantification of , 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 introduction Tilidine

O CH3

 Synthetic O CH3 N

CH3  Strong analgesic effect of its main metabolite (+)-tilidine nortilidine [2].

 Hepatically N-demethylation of tilidine to nortilidine O CH3 O

and bisnortilidine (CYP3A4, CYP2C19, CYP2D6) [1] NH

CH3 (+)-nortilidine  In commercially available preparations tilidine is combined with the µ-opioid-receptor antagonist

preventing opioid abuse [2]. HO O CH3

O O NH N 2 OH CH2 O (+)-bisnortilidine naloxone (1S,5 R,13 R,17 S)

Source: [1] Clin. Chem. 24/2, 692-697 (1978) 2 [2] Fachinfo Valorone N®, Fa. Pfizer; 2008 introduction Aims

 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 introduction Online-SPE-LC

Highly automated Chromatography sample preparation Analytical column: Automatization: Luna phenyl-hexyl 50x2 mm 5 µm 1. IS-mixing procedure Mobile phase: 2. Complete SPE procedure 0.2% FA + MeOH (gradient elution ) Total run time: 3.5 min

Symbiosis Pico™ Spark Holland

Online-SPE LC

4 SPE method development SPE Optimization

Mix setup: 100 µL Aqueous analyte solution of tilidine, nortilidine and naloxone (c = 50 µg/L)

+ 100 µL 100 mM NH 4Ac-solution (pH 6); IS PCP-D5 (c=100 ng/mL)

SPE sorbent: OASIS WCX 10x1mm

Generic SPE procedure: volume flow rate SPE step solvent (µl) (ml/min) Conditioning MeOH 1500 5.0 Equilibration 1000 5.0

Loading 10 mM NH 4Ac-solution pH 6 500 0.5 Clean up 1000 1.0 Elution Mobile phase 1050 0.3

5 SPE method development SPE Optimization

Mix setup: 100 µL Aqueous analyte solution of tilidine, nortilidine and naloxone (c = 50 µg/L)

+ 100 µL 100 mM NH 4Ac-solution (pH 6); IS PCP-D5 (c=100 ng/mL)

SPE sorbent: OASIS WCX 10x1mm

Generic SPE procedure: volume flow rate SPE step solvent (µl) (ml/min) Conditioning MeOH 1500 5.0 Equilibration 1000 5.0 10 mM NH 4Ac-solution pH 6 Loading 500 0.5 Clean up ?? 1.0 Elution Mobile phase ? 0.3

6 SPE method development Results (wash solution)

 Wash solution: 10 mM NH 4Ac-solution pH 6 + 0/10/15/20/40% MeOH

100

80

60 tilidine 40 nortilidine

recovery (%) (%) recovery recovery naloxone 20 IS 0 0 10 20 30 40 MeOH in wash solution (%)

 Conclusion: 10% MeOH

7 SPE method development Results (wash volume)

 Wash solution: 10 mM NH 4Ac-solution pH 6 + 10% MeOH  Wash volume: 500/1000/1500 µL

100

80

60 tilidine nortilidine 40 naloxone

recovery (%) (%) recovery recovery IS 20

0 0 500 1000 1500 wash volume ( µl)

 Conclusion: 500 µL ≙≙≙ 60 bed volumes

8 SPE method development Results (elution volume)

 Wash solution: 10 mM NH 4Ac-solution pH 6 + 10% MeOH  Elution volume: 150/300/600/900/1200 µL

100

80

60 tilidine nortilidine 40

recovery (%) (%) recovery recovery naloxone 20 IS

0 0 200 400 600 800 1000 1200 elution volume (µL)

 Conclusion: 600 µL ≙ 2 min elution time

9 SPE method development Final SPE method

Mix setup: 100 L sample (urine)

+ 100 L 100 mM NH 4Ac-solution (pH 6); c(PCP-D5)=100 ng/mL

SPE sorbent: OASIS WCX 10x1mm

Generic SPE procedure: volume flow rate SPE step solvent (µl) (ml/min) Conditioning MeOH 1500 5.0 Equilibration 1000 5.0 10 mM NH 4Ac-solution pH 6 Loading 500 0.5 10 mM NH Ac-solution pH 6 + Clean up 4 500 (1000) 1.0 10% MeOH Elution Mobile phase 600 (1050) 0.3

10 Validation and routine work Validation data of the new method

Calibration curves of the analytes

Untitled 18 (Tilidin 1): "Linear Through Zero" Regr ession ("No" weighting): y = 0.0101 x (r = 0.9993)  Selectivity: 1.00 0.95 0.90 0.85 no interference by matrix peaks 0.80 0.75 0.70 tilidine 0.65 0.60 0.55  Ionsuppression: 0.50 0.45 0.40 Analyte Area / IS Area IS / Area Analyte 0.35 < 10% for all analytes 0.30 R = 0.9993 0.25 0.20 0.15 0.10  Recovery: 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. > 80% for all analytes and IS Untitled 18 (Nortilidin 1): "Linear Through Zero" R egression ("No" weighting): y = 0.00911 x (r = 0.99 92) 0.90 0.85 0.80  Intra -assay precision (n=6 per conc .): 0.75 0.70 0.65 0.60 nortilidine coefficient of variation: ≤ 6% 0.55 0.50 0.45 0.40  Inter-assay precision (n=6 per conc.): Area IS / Area Analyte 0.35 0.30 R = 0.9992 0.25 0.20 coefficient of variation: ≤ 7% 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  LLOQ : Analyte Conc. / IS Conc. Untitled 18 (Bisnortilidin 1): "Linear Through Zero " Regression ("No" weighting): y = 0.00635 x (r = 0 .9996)

0.63 1.0 µg/L for all analytes 0.60 0.55

0.50  Linearity : 0.45 0.40 bisnortilidine

0.35 1.0-100 µg/L 0.30

Analyte Area / IS Area IS / Area Analyte 0.25

0.20 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 11 Toxichem Krimtech 2009; 76 (3): 185 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) or c ( nortilidine ) and c ( bisnortilidine ) > 1.0 µg/L (cutoff)

 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 Tilidine Urinary Excretion of 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)

Drug detection window g Analyte (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 Concentration: N/A Concentration: N/A Calculated Conc: 14.5 ng/mL Calculated Conc: 1.66 ng/mL 1.00e5 2.0e4 Tilidine (c=7.63 g/L) Bisnortilidine (c=96.5 g/L) 8.00e4 1.5e4 → Proc. Algorithm: Analyst Classic m/z 246.1 →155.1 Proc. Algorithm: Analyst Classic m/z 274.2 155.1 6.00e4

1.0e4

Intensity, cps Intensity, 4.00e4 Intensity, cps Intensity,

5000.0 2.00e4

0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min Time, min 1.81 Concentration: N/A Concentration: N/A 6.0e4 Calculated Conc: 1.80 ng/mL Calculated Conc: 14.7 ng/mL 2.05 7000 Bisnortilidine Tilidine 5.0e4 6000 → 4.0e4 m/z 246.1 229.2 5000 Proc. Algorithm: Analyst Classic m/z 274.2 →115.1 Proc. Algorithm: Analyst Classic 4000 3.0e4

3000 Intensity, cps Intensity, Intensity, cps Intensity, 2.0e4 2000 1.0e4 1000

0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min Time, min 1.81 Concentration: N/A Calculated Conc: 5.44 ng/mL 6.0e4

5.0e4 Nortilidine (c=36.9 g/L)

Proc. Algorithm: Analyst Classic 4.0e4 m/z 260.2 →155.1 3.0e4

Intensity, cps Intensity, 2.0e4

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 1.94 4000 Concentration: 1.00 ng/mL 7.0e5 Nortilidine Calculated Conc: N/A IS -D5 (c=33.33 g/L) 6.0e5 3000 m/z 249.3 →96.1 Proc. Algorithm: Analyst Classic m/z 260.2 →115.1 5.0e5 Proc. Algorithm: Analyst Classic 4.0e5 2000

Intensity, cps Intensity, 3.0e5 Intensity, cps Intensity, 1000 2.0e5 1.0e5 0 0.0 2.13 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 Time, min Time, min

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

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