Transcript Διαφάνεια 1 - Archives Of Analytical Chemistry

Asimina A. Papanastasiou, Alexandros G. Asimakopoulos,
Viola L. Borova and Nikolaos S. Thomaidis
WHY A MULTI-ANALYTE SCREENING?
 Use of illicit drugs is widespread
 Absence of multi - residual methods for drug-use surveillance programs
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


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
1. only common abused drugs can be determined
2. immunoassays are applied for only one drug each time
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
Literature review
Number of compounds
Matrix
LC-MS System
LOQs, LOIs, Linear range
97 Drugs/DoA/metabolites
Plasma
References
LC-MS/MS (QTRAP)
LOI: 5-1000 μg/L
62 Drugs/DoA/metabolites LC-HRMS (LTQ-Orbitrap)
Urine
LLOQs: 0.1 - 50 ng/mL
LC-MS (Ion Trap)
19 DoA
Urine
Linear range: 5-1600 ng/mL
Viette et al.,
Clinical Biochem. 2011
6 groups of DoAs (27)
Urine
LC-MS/MS (QTRAP)
Linear range: 0-900 ng/mL
de Jager et al., JCB 2011
13 DoAs
Urine
LC-MS/MS (QqQ)
LOQs: 0.3 - 20 ng/mL
Lin et al., JCB 2013
11 DoAs
Plasma
LC-MS/MS (QTRAP)
Linear range: 1-250 ng/mL
Maralikova et al.,
JCB 2004
19 DoAs
Urine
LC-MS/MS (QqQ)
Linear range: 1-1200 ng/mL
Chen et al.,
Talanta 2009
Li et al., JCA 2013
Cheng et sl.,
Forensic Sci. Int., 2006
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
Hybrid SPE-PPT Cartridge
 Opiates – Opioids (8)
72 Target Analytes
 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)
Optimization
Precipitating agents tested:
1.
Methanol
2.
Methanol (1% v/v formic acid)
3.
Methanol (1% w/v ammonium formate)
4.
Acetonitrile
5.
Acetonitrile (1% v/v formic acid)
6.
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
Method Protocols
Hybrid SPE-PPT
Ούρα (300 μL)+ IS +
ACN 1% FA
(1200 μL)
Vortex
Φυγοκέντρηση
10 min, 4000rpm
Hybrid SPE-PPT με
ενζυματική αποσύζευξη
β- Γλυκορουνιδάση/
Επώαση στους 37 °C
για 24 hours
Υπερκείμενο στο
Hybrid SPE-PPT
cartridge
Εκχύλισμα στα
1500 μL,
LC/MS-MS
*β-Glucuronidase Type HP-2
(Helix Pomatia)
Validation (1/3)
Hybrid SPE-PPT with enzymatic
deconjugation
Hybrid SPE-PPT
 LLOQ
 LLOQ
0.05 ng/mL (EDDP) – 25 ng/mL (EME)
0.25 ng/mL (Lidocaine/EDDP) - 25 ng/mL
(Phenytoin)
 Linear Range
 Linear Range
0.05 (EDDP) – 500 ng/mL
 R2 > 0.99
0.25 (EDDP) – 250 ng/mL
 R2 > 0.99
EDDP transitions (spike 0.05 ng/mL)
Validation (2/3)
Hybrid SPE-PPT
Hybrid SPE-PPT with enzymatic
deconjugation
Recovery tests
Recovery tests
Low level (25 ng/mL):
Low level (25 ng/mL):
63.3 (Amphetamine) - 111 (Morphine)
68.5 (Flunitrazepam) – 100 (Codeine)
Medium level (100 ng/mL):
Medium level (100 ng/mL):
60.1 (Benzylpiperazine) - 109 (Primidone)
62.9 (Δ9-THC) - 119 (Lamotrigine)
High level (500 ng/mL):
High level (250 ng/mL):
67.9 (Benzylpiperazine) – 109 (Morphine)
70.7 (Phenobarbital) – 109 (OH-LSD)
Validation (3/3)
Hybrid SPE-PPT
Hybrid SPE-PPT with enzymatic
deconjugation
Intermediate Precision (%RSDs)
Intermediate Precision (%RSDs)
Low level (25 ng/mL):
Low level (25 ng/mL):
4.6 (Benzylpiperazine) – 19 (Phenyntoin)
4.2 (Sertraline) – 19 (Carbamazepine)
Medium level (100 ng/mL):
Medium level (100 ng/mL):
4.0 (MDMA) – 19 (Pentobarbital)
5.6 (Risperidone) – 19 (Heroin)
High level (500 ng/mL):
High level (250 ng/mL):
4.8 (Codeine) – 19 (Fluoxetine)
4.7 (Diazepam) – 17 (Fluoxetine)
Matrix effects
Opiates & Opioids
100
Hallucinogens
(-)
0
80
-5
60
LSD
0
-20
-20
-25
-40
-30
-60
-35
-80
-40
Cocaine Compounds
0
BECG
Cocaine
Amphetamines
EME
0
-5
-10
-15
-60
-20
-80
-100
Δ9-THCA
-15
20
-40
Δ9-THC
-10
40
-20
OH-LSD
(-)
-25
-30
Matrix effects
Anesthetics
Antipsychotics
10
0
(-)
0
-10
-10
-20
-20
-30
-30
-40
-40
-50
-50
-60
-60
Sympathomimetics, Hypnotics, Barbiturates
Antiepileptics
(-)
10
80
0
60
-10
40
-20
20
-30
0
-40
-20
-50
-60
-70
-80
-40
-60
-80
(-)
(-)
Antidepressants (TCAs & TeCAs)
0
Antidepressants (SSRIs & SNRIs)
0
-10
-10
-20
-20
-30
-30
-40
-50
-40
-60
-50
-70
-80
-60
Benzodiazepines
30
New Designer Drugs
30
20
10
20
0
10
-10
-20
-30
-40
0
-10
-20
-50
-60
-30
-70
-40
Benzodiazepines
22.0
30
20
10
0
-10
-20
-30
-40
-50
-60
-70
23.0
16.7
21.6
20.1
18.3
21.2
20.9
21.5
17.7
20.9
21.7
22.0
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) -25 ng/mL (EME)
Method with hybrid SPE-PPT with enzymatic deconjugation: 0.25 (EDDP) –
25 ng/mL (Phenyntoin)
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)