Post on 21-Jul-2020
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
Generic SPE procedure:
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 methoddevelopment
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 step solventvolume
(µl)flow rate (ml/min)
Conditioning MeOH 1500 5.0
Equilibration10 mM NH4Ac-solution pH 6
1000 5.0
Loading 500 0.5
Clean up ? ? 1.0
Elution Mobile phase ? 0.3
6
Generic SPE procedure:
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
(%
)
SPE method development
� 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
SPE method development
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
SPE methoddevelopment
Final SPE method
Mix setup: 100 µL sample (urine)+ 100 µL 100 mM NH4Ac-solution (pH 6); c(PCP-D5)=100 ng/mL
SPE sorbent: OASIS WCX 10x1mm
Generic SPE procedure:
SPE step solventvolume
(µl)flow rate (ml/min)
Conditioning MeOH 1500 5.0
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
Generic SPE procedure:
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 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
0.63
Analyte
Are
a / IS A
rea
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
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
Calculated Conc: 14.5 ng/mL
Proc. Algorithm: Analyst Classic
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
Calculated Conc: 1.80 ng/mL
Proc. Algorithm: Analyst Classic
3000
4000
5000
6000
7000
Inte
nsity, cps
Concentration: N/A
Calculated Conc: 1.66 ng/mL
Proc. Algorithm: Analyst Classic
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
Calculated Conc: 5.17 ng/mL
Proc. Algorithm: Analyst Classic
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
Calculated Conc: 5.44 ng/mL
Proc. Algorithm: Analyst Classic
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
Proc. Algorithm: Analyst Classic
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