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transcript
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