Jennifer P. Pascali, PhD dtolabs, Resana (TV) Agilent Technologies Users Meeting, 20 May 2014
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Outline Introduction Hair analysis & drug incorporation Alcohol -Ethylglucuronide Materials & method Sample preparation Instrumentation Results Method validation & application
1. Introduction
Hair analysis What does it mean? 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. The toxicologist takes it as a method for retrospective detection of illegal and therapeutic drug exposure.
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 Alcohol is the most widely consumed psychoactive substance and is becoming a problematic addiction issue in millions of people worldwide. 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.
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 Decontamination Fragmentation Add of IS Hydrolysis 1-3 cm hair segments samples were: - decontaminated (CH 2 Cl 2 + CH 3 OH) - 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 Derivatization Injection 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 MSMS. finally injected into the GC-
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) TIC IS 266 73 NO INTERFERENCES WERE OBSERVED at RT 5.3 ETG 261 73 ETG 261 143 Figure: blank hair sample added of IS. TIC IS ETG Figure: spiked hair sample at the concentration of 30 pg/mg.
Results 2: Linearity Fig. Seven points calibration curve 2.5-100 pg/mg Linear, no weight R 2 = 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
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