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!