Jennifer P. Pascali, PhD

dtoLABS, Resana (TV)

Agilent Technologies Users Meeting, 20 May 2014

The project dtoLABS

dtoLABS is an analytical research centre

founded by two leading companies in the

production and marketing

of analytical instrumentation and solutions,

Agilent Technologies and DTO Servizi.

dtoLABS belongs to the "Advanced Partner

Laboratory" network of Agilent

Technologies.

dtoLABS

The project - dtoLABS

dtoLABS European Analytical Exellence Center Agilent

Main activities:

• European Demo Center

• Training Center

• Solutions Center

• MAPS (Markets & Applications Programs Solutions

for your analytical business )

• Beta Tester Center ( Instrumentation &

Consumables )

Outline

Introduction

• Hair analysis & drug incorporation

• Alcohol -Ethylglucuronide

Materials & method

• Sample preparation

• Instrumentation

Results

Method validation & application

1. Introduction

Hair analysis

The toxicologist takes it as a

method for retrospective

detection of illegal and

therapeutic drug exposure.

The medical doctor presumes the health state of a person

by measuring mineral ion concentrations in hair.

A hairdresser understands by hair analysis the

characterisation of the hair status as a starting

point for decision about a fitting hair style and

hair cosmetics.

What does it mean?

Drug incorporation

Possible route of drug entry include diffusion from blood, sweat, sebum, skin and entry from the environment. Evidence is reviewed regarding the importance of each of these routes as possible contributors to drug deposition in hair.

Many licit and illicit drugs can be found in hair matrix in different amounts, usually in the range of ng/mg or pg/mg.

Alcohol & Alcoholism

According WHO, chronic excessive alcohol drinking corresponds to a consumption higher than 60 g/day for several months

The direct determination of ethanol itself in hair is not possible due to its volatility and its potential absorption from external sources.

Alcohol is the most widely consumed psychoactive substance and is becoming a problematic addiction issue in millions of people worldwide.

Ethylglucuronide (EtG)

After absorption, a small fraction of ethanol (<0.1%) is conjugated with glucuronic acid during phase II metabolism to form EtG

Literature

Currently available techniques:

1. IA

2. LC-MS/MS (ESI)

3. GC-MS/MS (EI/NCI)

Guidelines from Society of Hair Testing (SoHT)

ETG < 30 pg/mg to distinguish from moderate and heavy alcohol consumption

ETG < 7 pg/mg alcohol abstinence

Measured in the 0-3 cm proximal segment (or segmental analysis).

2. Materials & methods

Sample preparation

1-3 cm hair segments samples were:

- decontaminated (CH2Cl2 + CH3OH)

- dried at room temperature

- cut into small pieces (2-5 mm)

- weighted 45-55 mg and transferred into glass tubes

- added of 500 ml of deionized water

- added of 10 pg/mg of ETG-D5

- centrifuged (3000 rpm for 10 minutes)

- incubated overnight

- ultrasonicated

Finally surnatant was taken to dryness under a gentle air stream at 60°C for 80 minutes. Samples were reconstructed in MSTFA and DMF and incubated for 40 minutes.

One microliter was finally injected into the GC-

MSMS.

Decontamination

Fragmentation

Add of IS

Hydrolysis

Derivatization

Injection

Instrumentation

GC Agilent Technologies 7890A

Triple-quad MS Agilent Technologies 7000

Method & temperatures

Injection port @ 250°C

Column: HP-5 (5% phenyl, methylsilicone)

12 m X 0.2mm X 0.33mm

Oven program:

1’@100°C; 30°C/min to 200°C; 15°C/min to 290°C; 5’@290°C.

MRM acquisition, dwell time 50ms

3. Results: validation & method application

Results 1:

Selectivity (n=7)

Figure: blank hair sample added of IS.

NO INTERFERENCES WERE OBSERVED at RT 5.3

Figure: spiked hair sample at the concentration of 30 pg/mg.

TIC

IS

ETG

TIC

IS 266 73

ETG 261 73

ETG 261 143

Results 2: Linearity

Fig. Seven points calibration curve

2.5- 100 pg/mg

Linear, no weight R2= 0.99789

LOQ: 2.5 pg/mg (RSD accuracy% < 20%) [LOD: 1.3 pg/mg (S/N=5)]

Results 3:

Precision & accuracy

Quality control (QC) samples at the concentration of 2.5 (LOQ), 10, 30 and 100 pg/mg, five replicates each on five non consecutive days. Accuracy was calculated as bias in the difference between expected concentration and measured concentration. Precision and accuracy data are all summarized in the table below.

Results 4:

Method application (n=8) Subjects with a well-known drinking behaviour, plus known CDT value. The ETG concentrations varied between < LOD and 7.5 pg/mg for moderate drinkers (n=4, CDT concentrations< 1.8%). No ETG was determined in tee-to-tallers hair samples (n=2, CDT concentrations <1.0 %), who declared no use of any alcohol containing food or medicaments or of hair cosmetics potentially containing ETG.

@ 39 pg/mg

Case 7 A single heavy drinker (daily alcohol intake: ten units, proved by a CDT value 6.0%) showed an ETG concentration of 39 pg/mg.

Concluding remarks

1. Hair analysis for ethyl glucuronide has been

increasingly employed for diagnosing

chronic excessive drinking or for

monitoring abstinence in both clinical and

forensic fields.

2. From the analytical point of view, the literature

had traditionally been focused on methods

based on LC-MS/MS mainly because of the

limited sample treatment procedure and very

low detection limits.

3. The present application clearly shows the

applicability of the GC-tripleQ technique as a

valuable alternative to LC-MS/MS in terms of

specificity, sensitivity and total analysis time

(at lower initial costs of investment).

Acknowledgements

Prof Franco Tagliaro Head of Forensic Unit

Dept. of Public Health and Community Medicine Univ. of Verona, Italy

Contacts:

Jennifer Pascali, PhD

dtoLABS-Resana (TV)

j.pascali@dtolabs.eu

www.dtolabs.eu

dtoLABS – Web site

dtoLABS

Via Fratta, 25

31023 Resana (TV)

Tel: +39 0423 717666

Fax +39 0423 717667

info@dtolabs.eu

www.dtolabs.eu