Download - Asimina A. Papanastasiou, Alexandros G. Asimakopoulos, Viola L. Borova and Nikolaos S. Thomaidis
Simultaneous determination of 72 drugs of abuse in human urine by hybrid solid phase extraction –
protein precipitation technique (Hybrid SPE-PPT) tailored to Liquid Chromatography-Tandem Mass
Spectrometry (LC-MS/MS)
Asimina A. Papanastasiou, Alexandros G. Asimakopoulos, Viola L. Borova and Nikolaos S. Thomaidis
WHY A MULTI-ANALYTE SCREENING?
Use of illicit drugs is widespreadAbsence of multi - residual methods for drug-use surveillance programs
for prisoners and probationerspre-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 determined2. 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 highUrine can be easily sampledTesting is non-invasiveVolume of the sample is adequateUrine test provides long detection windows for drug use
Literature reviewNumber of compoundsMatrix
LC-MS SystemLOQs, LOIs, Linear range
References
97 Drugs/DoA/metabolitesPlasma
LC-MS/MS (QTRAP)LOI: 5-1000 μg/L
Viette et al., Clinical Biochem. 2011
62 Drugs/DoA/metabolitesUrine
LC-HRMS (LTQ-Orbitrap)LLOQs: 0.1 - 50 ng/mL
Li et al., JCA 2013
19 DoAUrine
LC-MS (Ion Trap)Linear range: 5-1600 ng/mL
Cheng et sl., Forensic Sci. Int., 2006
6 groups of DoAs (27)Urine
LC-MS/MS (QTRAP)Linear range: 0-900 ng/mL
de Jager et al., JCB 2011
13 DoAsUrine
LC-MS/MS (QqQ)LOQs: 0.3 - 20 ng/mL
Lin et al., JCB 2013
11 DoAsPlasma
LC-MS/MS (QTRAP)Linear range: 1-250 ng/mL
Maralikova et al., JCB 2004
19 DoAsUrine
LC-MS/MS (QqQ)Linear range: 1-1200 ng/mL
Chen et al., Talanta 2009
Aims of the study
The development of amulti-analytesensitiveaccurate and fast screening methodfor 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
72 Target AnalytesOpiates – Opioids (8)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)
OptimizationPrecipitating 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 ProtocolsΟύρα (300 μL)+ IS +
ACN 1% FA (1200 μL)
VortexΦυγοκέντρηση
10 min, 4000rpm
Υπερκείμενο στο Hybrid SPE-PPT
cartridge
Εκχύλισμα στα 1500 μL,
LC/MS-MS
β- Γλυκορουνιδάση/Επώαση στους 37 °C
για 24 hours
Hybrid SPE-PPT Hybrid SPE-PPT με ενζυματική αποσύζευξη
*β-Glucuronidase Type HP-2 (Helix Pomatia)
Validation (1/3)Hybrid SPE-PPT Hybrid SPE-PPT with enzymatic
deconjugationLLOQ 0.05 ng/mL (EDDP) – 25 ng/mL (EME)
Linear Range0.05 (EDDP) – 500 ng/mL R2 > 0.99
LLOQ0.25 ng/mL (Lidocaine/EDDP) - 25 ng/mL
(Phenytoin) Linear Range0.25 (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
Low level (25 ng/mL): 63.3 (Amphetamine) - 111 (Morphine)
Medium level (100 ng/mL):60.1 (Benzylpiperazine) - 109 (Primidone)
High level (500 ng/mL):67.9 (Benzylpiperazine) – 109 (Morphine)
Recovery tests
Low level (25 ng/mL): 68.5 (Flunitrazepam) – 100 (Codeine)
Medium level (100 ng/mL):62.9 (Δ9-THC) - 119 (Lamotrigine)
High level (250 ng/mL):70.7 (Phenobarbital) – 109 (OH-LSD)
Intermediate Precision (%RSDs)
Low level (25 ng/mL): 4.6 (Benzylpiperazine) – 19 (Phenyntoin)
Medium level (100 ng/mL):4.0 (MDMA) – 19 (Pentobarbital)
High level (500 ng/mL):4.8 (Codeine) – 19 (Fluoxetine)
Intermediate Precision (%RSDs)
Low level (25 ng/mL): 4.2 (Sertraline) – 19 (Carbamazepine)
Medium level (100 ng/mL):5.6 (Risperidone) – 19 (Heroin)
High level (250 ng/mL):
4.7 (Diazepam) – 17 (Fluoxetine)
Validation (3/3)Hybrid SPE-PPT Hybrid SPE-PPT with enzymatic
deconjugation
Matrix effects
(-)(-)
6-M
AM
Bupro
norp
hine
Codeine
EDDP
Her
oin
Met
hado
ne
Mor
phin
e
Oxyco
done
-80
-60
-40
-20
0
20
40
60
80
100
Opiates & Opioids
LSD OH-LSD Δ9-THC Δ9-THCA
-40
-35
-30
-25
-20
-15
-10
-5
0
Hallucinogens
BECG Cocaine EME
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
Cocaine Compounds
Am-phetami
ne
MA MDA MDEA MDMA
-30
-25
-20
-15
-10
-5
0
Amphetamines
(-)
(-)
(-)
9-OH
-Rispe
rido
ne
Chlor
prom
azin
e
Cloza
pine
Nor
cloz
apin
e
Rispe
rido
ne
-60
-50
-40
-30
-20
-10
0
Antipsychotics
Fent
anyl
Ketam
ine
Lido
cain
e
Nor
fent
anyl
Nor
keta
min
e
Thiop
enta
l
-60
-50
-40
-30
-20
-10
0
10
Anesthetics
Carba
maz
epin
e
Lam
otrigi
ne
Leve
tirac
etam
Prim
idon
e
Topiram
ate
Pheny
toin
-80
-70
-60
-50
-40
-30
-20
-10
0
10
Antiepileptics
Ephed
rine
Nor
ephe
drin
e
Zopi
clon
e
Pento
barb
ital
Pheno
barb
ital
-80
-60
-40
-20
0
20
40
60
80
Sympathomimetics, Hypnotics, Barbiturates
(-)
Matrix effects
7-am
ine-flu
nitraz
epam
Alpra
zolam
Brom
azep
am
Chlor
diaz
epox
ide
Cloba
zam
Diaze
pam
Flun
itraz
epam
Lora
zepa
m
Mid
azol
am
Nitr
azep
am
Nor
diaz
epam
Oxaze
pam
Temaz
epam
-70
-60
-50
-40
-30
-20
-10
0
10
20
30
Benzodiazepines
8-OH
mirta
zapi
ne
Amitr
ipty
lline
Clom
ipra
min
e
Doxep
ine
Imip
ram
ine
Mirta
zapi
ne
Nor
tryp
tilin
e
-60
-50
-40
-30
-20
-10
0
Antidepressants (TCAs & TeCAs)
Citalopram Fluoxetine Paroxetine Sertraline Venlafaxine
-80
-70
-60
-50
-40
-30
-20
-10
0
Antidepressants (SSRIs & SNRIs)
Benzy
lpip
eraz
ine
JWH-0
18
JWH-0
73
Mef
edro
ne
MPPP
-40
-30
-20
-10
0
10
20
30
New Designer Drugs
7-am
ine-flu
nitraz
epam
Alpra
zolam
Brom
azep
am
Chlor
diaz
epox
ide
Cloba
zam
Diaze
pam
Flun
itraz
epam
Lora
zepa
m
Mid
azol
am
Nitr
azep
am
Nor
diaz
epam
Oxaze
pam
Temaz
epam
-70
-60
-50
-40
-30
-20
-10
0
10
20
30
Benzodiazepines
16.7 20.9
22.0
17.721.520.9
23.0
21.218.320.121.6
22.021.7
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)EfficientSensitive 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 preparationSimultaneous extraction of polar, non-polar and medium polarity compoundsNovel clean-up step using Hybrid SPE-PPT cartridgesNo evaporation step Relatively fast and sensitive chromatographic separation
(analysis time: 28 min)
Thank you very much for your attention!