Hair analyses Hair analysis beyond workplace testing Detlef Thieme IDAS Kreischa IRM + FTC Munich. anabolic (steroids, clenbuterol). kinetic (segmentation and profiling) 3. genetic (amitriptyline vs.nortriptyline Ia: Fatal steroid abuse Identification of stanozolol in hair Bodybuilder 996: Administration of.5g/month (endogenous prod.-5mg/day) Cardiac infarction Heart weight ~ 55 g Exact administration data available 5 4 3 mg Apr-8 androlone Decanoate Testosterone (4 Esters) Metandienone Metenolone Enantate Stanozolol 47.335 Stanozolol (8 pg/mg hair) 6.8E6 5: CH3 3.4E6.E : 3: 4: 5: 6: 7: 8: 56.365 545.345 3'--Stanozolol (6 pg/mg hair).9e5 5:49 3' CH3 9.7E4.E : 3: 4: 5: 6: 7: 8: date Mai-. Steroid-precursor C R Identification of testosterone esters H 46.747 458.36 49.36 54.384 Testosterone esters. Active Anabolic Steroids Androstendiol CH 3 Std. Mixture (ng) Prop () Iso () Dec (4) PhP () 5.4E5 4.3E5 3.3E5.E5.E5 Testosterone Stanozolol.E : : 3: 4: 5: 6: 7: 3. Inaktive Metabolites C Suspicious Hair Sample () (.4) (5) (.8).7E5.4E5.E5 6.8E4 H H Phase : Androsterone H Phase : Androsterone glucuronid : : 3: 4: 5: 6: 7: 3.4E4.E
Ib: Stallion licensing and hair testing 35 3 5 5 5 Clenbuterol Profiles in Hair (after 5 therapeutic doses) H Cl Cl C CH H C(CH3)3 5 d d 35 d 4 d 9 d d 5 d 36 d 5 5 5 3 35 Mane Hair Segment [cm] androlone in horse hair Stallion Licensing androlone hair concentration in untreated stallions 3.5..5..5. 3. 4. 5. >5 Concentration Concentration [pg/mg] 3.5.5.5 5 5 5 3 Age [a] androlone concentration in mane hair after transdermal treatment 8 6 4 day 84: expected maximum 6 4 8 6 3 Physiology of Skin, Hair and Sweat Eccrine Sweat Glands: High Amount Water, Electrolytes Skin Surface Thermoregulation Apocrine Glands: Small Amount ily Liquid Hair Follicle Bacterial Degradation dorants, Pheromones
Bioavailability I Transdermal Application of 9-orandrost-4-ene-diol Bioavailability II ral Application of 9-orandrost-4-ene-diol Blood conc [ng/ml]. 8. 4. MB: orandrosterone AS: androlone PC: orandrostendiol Blood conc [ng/ml] 5 5 5 Phase-MB: orandrosterone-glucuronide. 3 time [h] 3 time [h] androlone in horse hair Stallion Licensing Relevance in doping control?..5..5. 3. 4. 5. >5 4 3 4 Doping according to WADA: the detection of a prohibited substance or metabolites or markers of that substance in the athlete s specimen, the use of prohibited substances or methods, possession or trafficking prohibited substances or Concentration Conclusions Anabolics may be identified in hair Parent compound > metabolite Information about applied compound (e.g. differentiation of steroid esters) refusing without compelling justification to submit a sample. 3
. Illegal administration of clozapine Hair profile of clozapine 3 cm segments of a hair strand at least 5 victims duration > year date of termination was relevant analyses started 6 weeks later Clozapin [pg/mg] 5 5 Cl H 5 4 6 8 4 6 8 Single hair analysis, reproducibility Segments of.5 mm, taken -ov-3 Second hair collection. ov.3 vs. 3. Mai 4 Individual hair growth rate of.66 cm / month ± 7% 6 6 7d+X 4 conc [pg/mg] 4 3 4 5 6 7 8 Segment [o] 3 4 5 6 7 8 Segment [o] Miniaturization Segment length -5 cm -5 mm weight 5- mg -5 µg Solvent 5 ml 3 µl HPLC analytical micro LC.5e4 Intensity, cps Fast Chromatography.5 mm segment ~ 6 µg hair ~7 fg clozapine on column.64 clozapin ~3 pg/mg desmethyl-mb...3.5.7.9. 4
Hair Blank Desmethyl-metabolite 9 LD ~ 5 fg on column 7 6 Clozapin orclozapin 4 Intensity, cps Conc Clocapin [pg/mg] 5 4 3 8 6 4 Conc. orclozapin [a.u.]..3.5.7.9. 5 5 5 Hair Segment [count] Maximum (?) resolution mm (~ days per data point) 3 5 Conclusions Accurate long term profiles of hair concentrations may be obtained from analyses of single hairs Conc. [pg/mg] 5 5 5 5 5 3 Haar Segment [r] 3. Amitriptyline in hair of >3 children Illegal administration by a nurse Side effects: dizziness, somnolence, 8 unconsciousness, speech disorders, 6 diarrhoea, constipation 4 Large variations between children Investigation started 3-4 weeks after last administration [pg/cm] 6 4 Critical Timeframe Ami-T or-t 5 5 5
ortriptyline Profile (reproducibility) 9 8 7 Ami-T or-t hair conc. [pg/cm] 8 6 4 [pg/cm] 6 5 4 3 Critical Timeframe 5 5 5 5 5 3 Amitriptyline Biotransformation Amitriptyline - (E/Z)- Hydroxy-Amitriptyline CYP D6 78./7. Max..e4 cps. 5.9.e4 Amitriptyline 94./5. Max.. cps. -- Amitriptyline E 4.43 4.64 4.74 Z. 3 4 5 6 64./33. Max..e5 cps. 3 4 5 6 Max. cps. 8./5. 344. CYP C9 CYP D6 CYP C9.8e5 ortriptyline 5. 344 -- ortriptyline E 4.37 4.59 Z H. 3 4 5 6 3 4 5 6 ortriptyline - (E/Z)- Hydroxy-ortriptyline -T/T Variation of biotransformation (9 children).6.4...8.6 8 6 4 AT T E--T Z--T E--AT Z--AT 4 6 8.4. 4.... 3. 4. 5. 6. 7. 8. 9. T/AT 5 5 6
Conclusions 3 Quantitation ratios in hair are reliable and highly reproducible Hair concentrations reflect bioavailability better than urinary levels Metabolite ratios examined in hair are consistent markers for individual biotransformation 6 5 4 3 Limitation (uncertain incorporation via sweat) Citalopram in hairof a complient patient Hair Concentration Theory 5 5 5 Segment [cm] C F 7