Rapid Quantification of Tilidine, Nortilidine and ...applications.sparkholland.com/pdf/838.pdfin...

LOGO

Rapid Quantification of Tilidine, Nortilidine and Bisnortilidine

in Urine by automated online-SPE-LC-MS/MS

Institute of Toxicology

Clinical Toxicology and Pharmacology

Berliner Betrieb für Zentrale Gesundheitliche Aufgaben, Berlin

Christoph Köhler

Thomas Grobosch

Torsten Binscheck

48th Annual Meeting of the International Association of Forensic Toxicologists Conference in Bonn

August 29 - September 2, 2010

� Synthetic opioid analgesic

� Strong analgesic effect of its main metabolite nortilidine [2].

� Hepatically N-demethylation of tilidine to nortilidine

Tilidine

introduction

N

CH3

O

O CH3

CH3

O CH3

(+)-tilidine

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

� In commercially available preparations tilidine iscombined with the µ-opioid-receptor antagonist naloxone preventing opioid abuse [2].

Source: [1] Clin. Chem. 24/2, 692-697 (1978)

[2] Fachinfo Valorone N®, Fa. Pfizer; 2008

OH

O

O

NCH2OH

naloxone(1S,5R,13R,17S)

2

NH

CH3

O

NH2

O

O CH3

(+)-nortilidine

(+)-bisnortilidine

Aims

� Sensitive quantification of tilidine and its metabolites in urine samples

� No immunoassay for the detection of tilidine abuse is available!

introduction

3

� Increasing number of samples (max. 100 samples/d!) consumedmuch time and human resources for the LLE (max. 180 min/d!).

Offline-LLE QTrapLC

Online-SPE QTrapLC

Online-SPE-LC

Automatization:

1. IS-mixing procedure

2. Complete SPE procedure

Analytical column: Luna phenyl-hexyl 50x2 mm 5 µm

Mobile phase: 0.2% FA + MeOH (gradient elution)

Highly automatedsample preparation

Chromatography

introduction

2. Complete SPE procedure

Symbiosis Pico™Spark Holland

Online-SPE LC

0.2% FA + MeOH (gradient elution)

Total run time:3.5 min

4

SPE Optimization

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

+ 100 µL 100 mM NH4Ac-solution (pH 6); IS PCP-D5 (c=100 ng/mL)

SPE sorbent: OASIS WCX 10x1mm

Generic SPE procedure:

SPE methoddevelopment


SPE step solventvolume

(µl)flow rate (ml/min)

Conditioning MeOH 1500 5.0

Equilibration

10 mM NH4Ac-solution pH 6

1000 5.0

Loading 500 0.5

Clean up 1000 1.0

Elution Mobile phase 1050 0.3

5


SPE Optimization

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

+ 100 µL 100 mM NH4Ac-solution (pH 6); IS PCP-D5 (c=100 ng/mL)






Equilibration10 mM NH4Ac-solution pH 6

1000 5.0

Loading 500 0.5

Clean up ? ? 1.0

Elution Mobile phase ? 0.3

6


Results (wash solution)

� Wash solution: 10 mM NH4Ac-solution pH 6 + 0/10/15/20/40% MeOH

60

80

100

reco

very

(%

)

SPE method development

� Conclusion: 10% MeOH

0

20

40

60

0 10 20 30 40

reco

very

(%

)

MeOH in wash solution (%)

tilidine

nortilidine

naloxone

IS

7

Results (wash volume)

� Wash solution: 10 mM NH4Ac-solution pH 6 + 10% MeOH� Wash volume: 500/1000/1500 µL

80

100

reco

very

(%

)


� Conclusion: 500 µL ≙ ≙ ≙ ≙ 60 bed volumes

0

20

40

60

0 500 1000 1500

reco

very

(%

)

wash volume (µl)

tilidinenortilidinenaloxoneIS

8

80

100

reco

very

(%

)

� Wash solution: 10 mM NH4Ac-solution pH 6 + 10% MeOH� Elution volume: 150/300/600/900/1200 µL


Results (elution volume)

0

20

40

60

0 200 400 600 800 1000 1200

reco

very

(%

)

elution volume (µL)

tilidine

nortilidine

naloxone

IS

� Conclusion: 600 µL ≙ 2 min elution time

9


Final SPE method

Mix setup: 100 µL sample (urine)+ 100 µL 100 mM NH4Ac-solution (pH 6); c(PCP-D5)=100 ng/mL






Equilibration10 mM NH4Ac-solution pH 6

1000 5.0

Loading 500 0.5

Clean up10 mM NH4Ac-solution pH 6 +

10% MeOH500 (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)

0.85

0.90

Untitled 18 (Tilidin 1): "Linear Through Zero" Regression ("No" weighting): y = 0.0101 x (r = 0.9993)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Analyte Conc. / IS Conc.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

Analyte

Are

a / IS A

rea

Validation data of the new method

� 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.):

tilidine

R = 0.9993

Calibration curves of the analytes

Validation androutine work

Untitled 18 (Bisnortilidin 1): "Linear Through Zero" Regression ("No" weighting): y = 0.00635 x (r = 0.9996)


0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.63

Analyte

Are

a / IS A

rea


0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

Analyte

Are

a / IS A

rea

� 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

R = 0.9992

nortilidine

bisnortilidine

R = 0.9996

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)

or

c (nortilidine) and c (bisnortilidine) > 1.0 µg/L (cutoff)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 bisnortilidineincrease 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)

Analyte

Drug detection window(d)

- ß-glucuronidase + ß-glucuronidase

g

13

- 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

Concentration: N/A

Calculated Conc: 14.7 ng/mL

Proc. Algorithm: Analyst Classic

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

Inte

nsity, cps

1.81

2.05

Concentration: N/A



0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.00

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

Inte

nsity, cps

1.81

Concentration: N/A



3000

4000

5000

6000

7000

Inte

nsity, cps

Concentration: N/A



0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

Inte

nsity, cps

Chromatograms

Tilidine (c=7.63 µg/L)

m/z 274.2→155.1

Tilidinem/z 274.2→115.1

Bisnortilidine (c=96.5 µg/L)

m/z 246.1→155.1

Bisnortilidinem/z 246.1→229.2

0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.0

1.0e4

Concentration: N/A



0.5 1.0 1.5 2.0 2.5 3.0Time, min

0

1000

2000

3000

4000

Inte

nsi

ty, cp

s

Concentration: N/A



0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

Inte

nsity, cps

1.81

0.5 1.0 1.5 2.0 2.5 3.0Time, min

0

1000

2000

14

Concentration: 1.00 ng/mL

Calculated Conc: N/A


0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

Inte

nsity

, cp

s

1.94

2.13

Nortilidine (c=36.9 µg/L)

m/z 260.2→155.1

Nortilidinem/z 260.2→115.1

IS Phencyclidine-D5 (c=33.33 µg/L)

m/z 249.3→96.1

Conclusion

� An highly sensitive method for the determination of tilidine, nortilidineand bisnortilidine in urine was developed and validated on an online-SPE-LC-QTrap

� Due to the automatization of the online-SPE the manual preparation � 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 tilidinepreparation up to 5 days after administration.

15

Aknowledgements

� Martin Sibum (Spark Holland) for technical support

� Dr. Goebel (Medizinisches Labor Bremen) for

delivering Bisnortilidinedelivering Bisnortilidine

16

Thank you for your attention

Rapid Quantification of Tilidine, Nortilidine and ...applications.sparkholland.com/pdf/838.pdfin...

Documents

Transcript of Rapid Quantification of Tilidine, Nortilidine and ...applications.sparkholland.com/pdf/838.pdfin...