BASIC DRUGS OF ABUSE in hair by GC-MS Code GC49010 Confirm method in GC-MS INTRODUCTION

BASIC DRUGS F ABUSE in hair by GC-MS Code GC49010 (Morphine, Codeine, Dihydrocodeine, 6-MAM, Ethylmorphine, Cocaine, BEG, Cocaethylene, Amphetamine, Methamphetamine, MDA, MDMA, MDE, MBDB, Methadone, EDDP, Buprenorphine) Confirm method in GC-MS INTRDUCTIN In the early 80 's, began a series of studies on the possibility of determining substances of abuse in keratinous array and then use hair analysis as a means of finding the previous use of these substances. nce embedded in the hair, by the systemic circulation and by the contribution of sweat and sebum, substances of abuse remain impounded in the keratinous array in a stable way over time as a function of their lipophilic, molecular weight, the pka and the steric encumbrance. The analysis of substances of abuse in keratinous array is an ideal addendum to the analysis of blood or urine as it provides information regarding a time earlier than that covered by other biological matrices and refers to a broader temporal space (one or more months) as a function of the length of the hair. Considering an average growth of 1 cm per month, you can learn about the history of the past by analyzing consumption segmented hair per cm or fractions of cm. It is also possible to highlight external contamination by washing solvents review comparing them with the results obtained by digested hair. The search for substances of abuse in hair can be used to test a use, abuse and misuse continued over time to determine the intensity and its history and then provide analytical data with medico-legal value. For this reason the determination in the hair may be required in the following cases: deaths related to the use of substances of abuse, assessment of unsuitability driving, criminal liability, reliance custody of infants, prenatal exposure to substances of abuse and finally as confirmation of a sporadic or consumption of a dependency in workers with jobs at risk. Immunochemical tests can be used as initial methods to analyse in a short time a large number of samples in a standardized, efficient and economic way and exclude samples that are negative, i.e. samples that did not contain the substance or class of substances, or those in which the concentration is below a threshold value (cut-off). 1

Confirmation analyses are defined as those that serve to verify false positive due to the nonspecificity of screening tests. In the case of keratinous array, because there are no real screening tests we cannot talk about real "confirmatory analysis". The "Society of Hair Testing" (SHT) recommends the use of methodologies coupled to chromatographic separative disclosure in mass spectrometry for the determination of substances of abuse in keratinous matrix medical-legal purposes and/or clinical trials. Taking into account the guidelines of SHT, this method allows to determine all basic substances of abuse (6-MAM, Morphine, Codeine, Ethylmorphine, Dihydrocodeine, Cocaine, BEG, Cocaethylene, Amphetamine, Methamphetamine, MDA, MDE, MDMA, MBDB, Buprenorphine, Methadone, EDDP) after extraction of molecules from keratinous matrix and solid phase extraction (SPE) column of the analytes of interest (to eliminate the presence of complex matrix interferences) and determination in Gas chromatography/mass spectrometry (GC/MS). EUREKA srl LAB DIVISIN VAT N 01547310423 E-mail:info@eurekaone.com www.eurekaone.com Head Quarter: Via Enrico Fermi 25 60033 Chiaravalle (AN) ITALY Tel. +39 071 7450790 Fax + 39 071 7496579 Release N 003 Abuse Drugs in hair by GC/MS January 2013 2

TECHNICAL FEATURES Principle of the method : This method determines Morphine, Codeine, Dihydrocodeine, 6-Monoacetylmorphine, Ethylmorphine, Cocaine, Beg, Cocaethylene, Methadone, Amphetamine, methamphetamine, MDMA, MDA, MDE, MBDB, Buprenorphine, EDDP after hydrolysis of the keratin matrix, SPE extraction and GC-MS in reading. The method was validated according to the criteria established at the international level by the FDA and by the ICH. ANALYTE Intra-Day Accuracy Inter-Day-Precision Inter-Day-Accuracy (Error %) (CV%) (Error %) Calibrator Calibrator Calibrator Low Medium High Low Medium High Low Medium High AMPHETAMINE 5,51 7,82 3,51 28,1 19,07 15,87 20,61 21,29 10,78 METHAMPHETAMINE 7,41 4,94 2,37 13,26 19,07 11,96 19,51 12,64 7,49 3,4-MDMA 22,27 11,79 4,96 16,5 15,46 9,39 26,33 16,42 10,88 3,4- MDA 5,17 6,2 4,13 21,63 14,83 8,13 28,55 13,16 12,76 3,4-MDE 18,07 5,54 1,88 28,05 5,81 11,44 28,59 4,58 6,93 BENZILECGNINE 11,48 47,4 46,97 14,21 10,74 8,91 11,46 14,3 16,74 CCAETHYLENE 25,64 3,52 6,49 28,23 7,2 8,4 26,84 6,5 6,77 CCAINE 22,48 6,5 1,84 16,46 11,51 3,73 17,57 7,88 3,07 MRPHINE 14,43 35,8 39,38 23,91 16,64 10,07 24,09 22,75 17,53 6-MAM 26,35 16,83 13,05 31,37 13,68 11,88 34,85 12,48 10,54 ETHYLMRPHINE 26,39 10,24 4,06 27,36 10,14 8,97 29,36 8,04 7,47 CDEINE 24,95 8,36 8,55 24,16 12,51 9,92 26,25 15,74 7,76 DIHYDRCDEINE 14,45 3,98 9,75 27,26 11,42 10,57 25,75 6,57 9,06 METHADNE 7,05 8,78 2,91 10,52 10,67 9,17 9,72 10,04 7,34 EDDP 24,52 27,91 7,26 17,7 14,79 20,63 21,07 27,32 12,33 BUPRENRPHINE 36,18 19,84 29,62 51,99 48,11 25,87 22,41 18,4 13,91 The table below shows for each analyte concentrations corresponding to the calibrators whose values Low, Medium and High cited above refer. ANALYTE Concentration (ng/mg) Calibrator Low Medium High AMPHETAMINE 0,5 5 20 METHAMPHETAMINE 0,5 5 20 3,4-MDMA 0,5 5 20 3,4- MDA 0,5 5 20 3,4-MDE 0,5 5 20 BENZILECGNINE 0,5 5 20 CCAETHYLENE 0,5 5 20 CCAINE 0,5 5 50 MRPHINE 0,5 5 15 6-MAM 0,5 5 20 ETHYLMRPHINE 0,5 5 15 CDEINE 0,5 5 15 DIHYDRCDEINE 0,5 5 15 METHADNE 0,5 5 20 EDDP 0,5 2 10 BUPRENRPHINE 0,5 5 20 3

ANALYTE Correlation Coefficent R2 ± SD LD (ng/mg) LQ (ng/mg) AMPHETAMINE 0,9990 ± 0,0011 0,07 0,1 METHAMPHETAMINE 0,9990 ± 0,0011 0,07 0,1 3,4-MDMA 0,9992 ± 0,0009 0,07 0,1 3,4- MDA 0,9997 ± 0,0002 0,08 0,1 3,4-MDE 0,9985 ± 0,0029 0,07 0,1 BENZILECGNINE 0,9997 ± 0,0006 0,05 0,1 CCAETHYLENE 0,9999 ± 0,0002 0,07 0,1 CCAINE 1,0000 ± 0,0000 0,05 0,1 MRPHINE 0,9933 ± 0,0052 0,05 0,1 6-MAM 0,9985 ± 0,0021 0,08 0,1 ETHYLMRPHINE 0,9969 ± 0,0023 0,05 0,1 CDEINE 0,9892 ± 0,0113 0,07 0,1 DIHYDRCDEINE 0,9976 ± 0,0037 0,05 0,1 METHADNE 0,9999 ± 0,0001 0,06 0,1 EDDP 0,9973 ± 0,0021 0,08 0,1 BUPRENRPHINE 0,9982 ± 0,0024 0,02 0,04 ANALYTE Recovery (%) Calibrator Low Medium High AMPHETAMINE 70,43 104,19 90,16 METHAMPHETAMINE 111,89 98,25 85,11 3,4-MDMA 82,65 86,06 87 3,4- MDA 82,58 90,88 81,08 3,4-MDE 78,14 81,69 104,69 BENZILECGNINE 90,24 74,37 77,02 CCAETHYLENE 65,96 76,91 84,56 CCAINE 90,36 85,2 93,16 MRPHINE 92,86 98,03 91,56 6-MAM 42,81 33,31 30,73 ETHYLMRPHINE 85,55 74,03 77,4 CDEINE 56,28 59,9 50,71 DIHYDRCDEINE 78,26 86,84 92,34 METHADNE 78,08 68,71 83,87 EDDP 74,26 53,41 51,63 BUPRENRPHINE 89,56 95,14 88,64 Cut-off : piates, Amphetamines, Cocaine, Methadone: BEG, Buprenorphine: 0.2 ng/mg 0.05 ng/mg Rif. Gazzetta Ufficiale n 236 8 ott 2008 4

Components of the kit (50 test) : Reagent A Diluting Solution, 2 x 10 ml All the reagents are ready to use and stable 3 years at 2 8 C. Reagent B Buffer Solution, 1 x 100 ml Reagent C Conditioning Solution 1 and Wash 3, 1 x 250 ml Reagent D Conditioning Solution 2, 1 x 100 ml Reagent E Eluting Solution, 1 x 100 ml Reagent F Wash Solution 1, 1 x 100 ml Reagent G Wash Solution 2, 1 x 100 ml Reagent H Derivatization Solution 1 for Amphetamines, 1 x 5 ml Reagent I Derivatization Solution 2 for Amphetamines, 1 x 5 ml Reagent L Test Solution Amphetamines, 1 x 1 ml Reagent N Test Solution piates, Methadone, Cocaine, Buprenorphine and metabolites, 1 x 0,5 ml Reagent M Derivatization Solution, 3 x 1 ml (3 ampullas of 1 ml) Reagent Internal Standard Solution for Basic Drugs, 1 x 2 ml Reagent P Internal Standard Solution for Amphetamines, 1 x 2 ml Reagent Q Estraent Solution, 1 x 50 ml See Warnings. For the sampling of Reagent M, the cap sealing requires the use of 100 ul Hamilton syringe Cod. Z81000 See Warnings. For the sampling of Reagent M, the cap sealing requires the use of 50 ul Hamilton syringe Cod. Z80500 See Warnings See Warnings See Warnings. For the sampling of Reagent M, the cap sealing requires the use of 100 ul Hamilton syringe Cod. Z81000 See Warnings See Warnings Reagent R Hair Wash Solution, 1 x 250 ml Reagent S - Blank Hair, 1 x 1500 mg Reagent T1 Chemical Standard Solution 1, 1 x 400 µl Reagent T2 Chemical Standard Solution 2, 1 x 400 µl Reagent T3 Chemical Standard Solution 3, 1 x 400 µl Reagent U Chemical Standard Solution for MBDB, 1 x 5 ml Clean-up Columns, 50 pcs Not included in the kit. Saleable product apart, code Z43120 Minimum Instrumental equipment required: ptional Equipment: Hair collection procedure: Hair collection procedure : GC-MS Instrument perational Computer Autosampler Recommended length: 5 cm, starting from the scalp. Is cut a strand, not torn (the bulb has no purpose at the end of these investigations) in the region of the vertex back of the head, of at least 200 mg which is divided into 2 aliquots of similar weight (A and B) of each of which is fixed to the proximal end. These aliquots are placed in separate containers nontransparent bearing caps airtight and sealed with tape irremovable and stored at room temperature ambiente.il cutting the hair must be performed with clean scissors with a small amount of alcohol and dry well. And it is necessary to cut 200 mg of hair from the pubic region; the hairs thus collected is divided into two aliquots (A and B). 5

PREANALITICAL PRCEDURE for all substances of abuse of basic EXCEPT AMPHETAMINES: STEP 1 : Pipette in a glass tube with teflon cap : 50 µl of Reagent N Test Solution piates, Methadone, Cocaine, Buprenorphine and metabolites STEP 2 : Evaporate to dryness with the aid of a stream of helium or nitrogen (in this step the temperature must be < 40 C ) STEP 3 : Add 70 µl of Reagent M - Derivatization Solution using the appropriate syringe Hamilton of 100 µl (code Z81000) STEP 4 : Incubate at 70 C for 20 minutes Seal the tube with the cap Vortex for 20 sec. Cooling at Room Temperature pen glass tube STEP 5 : Transferring the sample vials with reduction volume autosampler NTE: Before starting the analytical session is advisable to inject 1 ml of this freshly prepared solution in the gas chromatograph to identify the retention time and mass spectrum of all basic substances that must be similar to those shown in Figures below. If the test was successful, proceed to assay. If not, check the functionality of the analytical system. PREANALITICAL PRCEDURE for AMPHETAMINES: STEP 1 : Pipette in a glass tube with teflon cap : 100 µl of Reagent L Test Solution Amphetamines STEP 2 : Evaporate to dryness with the aid of a stream of helium or nitrogen (in this step the temperature must be < 40 C) STEP 3 : Add 100 µl of Reagent H Derivatization Solution 1 for Amphetamines using the appropriate syringe Hamilton of 100 µl (code Z81000) Seal the tube with the cap Vortex for 20 sec. STEP 4: Incubate at 80 C for 5 minutes Cooling at Room Temperature pen glass tube Release N 003 Abuse Drugs in hair by GC/MS January 2013 6

STEP 5 : Add 30 µl of Reagent I Derivatization Solution 2 for Amphetamines using the appropriate syringe Hamilton of 50 µl (code Z80500) STEP 6: Incubate at 80 C for 5 minutes Seal the tube with the cap Vortex for 20 sec. Cooling at Room Temperature pen glass tube STEP 7 : Transferring the sample vials with reduction volume autosampler NTE: Before starting the analytical session is advisable to inject 1 ml of this freshly prepared solution in the gas chromatograph to identify the retention time and mass spectrum of all basic substances that must be similar to those shown in Figures below. If the test was successful, proceed to assay. If not, check the functionality of the analytical system. N.B.: it is recommended to use different syringes for derivatization reagents H / I and for Reagent M. Release N 003 Abuse Drugs in hair by GC/MS January 2013 7

ANALYTICAL PRCEDURE : A) PREPARATIN of unknown samples STEP 1 : Wash hair intact inside a glass tube of 10 ml with 2 ml of Reagent R Hair Wash Solution Seal the tube with the cap Vortex for 1 minute Centrifuge at 3.500 rpm for 5 minutes pen glass tube STEP 2 : Evaporate to dryness the solvent with the aid of a light stream of helium or nitrogen paying attention to remain in the hair / fur on the bottom of the tube. Alternatively remove with a clean forceps with Acetone (not supplied in the kit), the hair / fur from the tube and place on paper towels until completely dry. In this last case the blotter paper should be changed for each sample. Repeat steps 1 and 2 for two consecutive STEP 3 : Cut your hair, washed and dried, in segments of 1-2 cm with scissors cleaned with Acetone (not supplied in the kit) STEP 4 : Weigh an aliquot of sample, between 20 and 50 mg depending on the availability of the sample, in a Pyrex glass tube with screw cap clean and previously named. Release N 003 Abuse Drugs in hair by GC/MS January 2013 8

B) PREPARATIN of CALIBRATRS N 5 LEVELS Preparation of the Standard Chemical for enrichment after: Prepare 6 glass tubes, call them 1 A, 1 B, 2 A, 2 B, 3 A, 3 B and dilute each Standard Chemical (T1-T2-T3) with Reagent A Diluting Solution as reported in tab. below: Tubes 1 A 1 B 2 A 2 B 3 A 3 B Reagent T1 Chemical Std. Solution 1 Reagent T2 Chemical Std. Solution 2 Reagent T3 Chemical Std. Solution 3 Reagent A Diluting Sol. 30 µl // // 120 µl 10 µl // // 490 µl // 30 µl // 120 µl // 1 µl // 490 µl // // 30 µl 120 µl // // 1 µl 490 µl Close the tubes Vortex for 1 minute Prepare 5 glass tubes of 10 ml called C0-C1-C2-C3-C4. Add at each tube (C0-C1-C2-C3-C4) the standard chemical just prepared (1A, 1B, 2A, 2B, 3A, 3B) referring to the table below: Calibrator C0 Calibrator C1 Calibrator C2 Calibrator C3 Calibrator C4 50 µl of Reagent A Diluting Sol. 25 µl of 1 B 50 µl of 1 B 25 µl of 1 A 50 µl of1 A 50 µl of Reagente A Diluting Sol. 25 µl of 2 B 50 µl of 2 B 25 µl of 2 A 50 µl of 2 A 50 µl of Reagente A Diluting Sol. 10 µl of 3 B 25 µl of 3 B 50 µl of 3 B 25 µl of 3 A Evaporate to dryness with the aid of a stream of helium or nitrogen (in this step the temperature must be < 40 C). Add to each tube of the calibrators C0-C1-C2-C3-CA just brought to dryness, 50 mg of Reagent S Blank Hair (supplied in kit). Release N 003 Abuse Drugs in hair by GC/MS January 2013 9

At this point Calibrators of 5 points are ready and the concentrations to be used for the calibration curve will be: Analyte Calibrator C0 Calibrator C1 Calibrator C2 Calibrator C3 Calibrator C4 Buprenorphine Morphine, Codeine, Dihydrocodeine, 6- MAM, Ethylmorphine, Cocaine, Beg, Cocaethylene, Amphetamine, Methamphetamine, MDA, MDE, MDMA, Methadone, EDDP 0 ng/mg 0.2 ng/mg 0.5 ng/mg 1 ng/mg 5 ng/mg 0 ng/mg 0.5 ng/mg 1 ng/mg 5 ng/mg 10 ng/mg Release N 003 Abuse Drugs in hair by GC/MS January 2013 10

C) PREPARATIN F INTERNAL STANDARDS Reagent - Internal standard Sol for basic substances and Reagent P - Internal Standard Sol for Amphetamines supplied in the kit should be diluted 1:10 with water before each use, in the necessary amount for the entire assay. ES. for an analytic session with 20 unknown samples and 5 calibrators are required 625 µl of each Standard Internal dilution (diluted Reagent and diluted Reagent P). Therefore you fetch 62.5 µl of Reagent - Internal standard Sol for basic substances and 62.5 µl of Reagent P - Internal standard Sol for Amphetamine and are diluted respectively with 562.5 µl of H2 in two glass tubes. Vortex for at least 10 sec. The solutions are now ready for use. The Reagent - Internal standard Sol for basic substances and the Reagent P - Internal standard Sol for Amphetamine undiluted and not used are closed again tightly and kept to the residual quantity in the freezer at -20 C. Release N 003 Abuse Drugs in hair by GC/MS January 2013 11

D) DIGESTIN F KERATINIC MATRIX STEP 5 : In each tube previously prepared (phase A and phase B) dispense: Reagent diluted - (Internal Std Sol. for BASIC Substances diluted as shown in STEP C) Reagent P diluted - (Internal Std Sol. for Amphetamines diluted as shown in STEP C) Reagent Q - Estraent Sol. Calibrator C0 (prepared in Step B) Calibrator C1 (prepared in Step B) Calibrator C2 (prepared in Step B) Calibrator C3 (prepared in Step B) Calibrator C4 (prepared in Step B) Controls Samples (prepared in Step A) 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 25 ul 1000 µl 1000 µl 1000 µl 1000 µl 1000 µl 1000 µl 1000 µl Seal the tubes with the cap - without invert gently mix the vial for 10 seconds STEP 6 : Incubate at 45 C for 18 hours Cooling at Room Temperature pen the glass tube STEP 7 : Add at each tube 2 ml of Reagent B Buffer Sol. Check that the ph is 7.8 + 0.2 with indicator strips (dip the tip of a pasteur pipette or a pipette in the tube; use a different pipette for each tube). In any case that the ph is not correct, you can make corrections with about 170 µl of Potassium hydroxide 0.1 M and about 30 µl of Potassium hydroxide 2 M mixing and re-inspecting the ph after each addition. Adjust if necessary by adding additional µl of Potassium hydroxide 0.1 M. Seal the tubes and mix for 1 minute Centifuge at 3.500 rpm for 5 minutes Release N 003 Abuse Drugs in hair by GC/MS January 2013 12

E) EXTRACTIN F ANALYTES STEP 8 : Column SPE Conditioning Prepare a number of columns corresponding to the sum of the calibrators, controls and unknown samples Add 1,5 ml of Reagent C Conditioning Solution 1 and Wash 3 Pass in the column and discard the eluate without leading to dry the column Add 2 ml of Reagent D Conditioning Solution 2 Pass in the column and discard the eluate without leading to dry the column STEP 9 : Add all the supernatant of the calibrators, controls and unknown samples, prepared in steps 5-7, in columns packed and download at a flow rate of 1 ml / minute (drop by drop) also with the aid of a moderate vacuum. STEP 10 : Column SPE Washing Wash with 2 ml of Reagent F Wash Solution 1 Pass in column and discard the eluate Wash with 2 ml of Reagent G Wash Pass in column and discard the eluate Wash with 2 ml of Reagent C Conditioning Solution 1 and Wash 3 Pass in column and discard the eluate bringing to dryness under a gentle stream of air Release N 003 Abuse Drugs in hair by GC/MS January 2013 13

F) ELUTIN and DERIVATIZATIN F ANALYTES Elute strictly in Pyrex glass tubes with cap THERE ARE 3 CASES: 1) In the case where the sample was positive at screening only for the class of Amphetamine: Elute with 1 ml of Reagent E Eluting Solution Evaporate to dryness with a gentle stream of air Collect the eluate of Pyrex tubes with screw cap Evaporate to dryness with the aid of a stream of helium or nitrogen (in this step the temperature must be < 40 C) Derivatization: Add at each tube 100 µl of Reagent H Derivatization Solution 1 for Amphetamines Amphetamines using the appropriate syringe Hamilton of 100 µl (code Z81000) Seal the tubes with the cap Incubate at 80 C x 5 min Cooling at Room Temperature pen the glass tube Add 30 µl of Reagent I Derivatization Solution 2 for Amphetamines using the appropriate syringe Hamilton of 50 µl (code Z80500) Seal the tubes with the cap Incubate at 80 C x 5 min Cooling at Room Temperature pen the glass tube Injection: Transfer the derivatized in the vial with reducing volume and inject 1 µl in the gas chromatograph. 2) In the case where the sample was positive at screening only for the class of piates, Methadone, Cocaine, Buprenorphine: Elute with 1 ml of Reagent E Eluting Solution Evaporate to dryness with a gentle stream of air Collect the eluate of Pyrex tubes with screw cap Evaporate to dryness with the aid of a stream of helium or nitrogen (in this step the temperature must be < 40 C) Derivatization: Add at each tube 70 µl of Reagent M Derivatization Solution using the appropriate syringe Hamilton of 100 µl (code Z81000) Seal the tubes with the cap Release N 003 Abuse Drugs in hair by GC/MS January 2013 14

Incubate at 70 C x 20 min Cooling at Room Temperature pen the glass tube Injection: Transfer the derivatized in the vial with reducing volume and inject 1 µl in the gas chromatograph. 3) In the case where the sample was positive at screening for both the class of Amphetamines and for the other classes of substances of abuse: Elute with 1 ml of Reagent E Eluting Solution Evaporate to dryness with a gentle stream of air Collect the eluate of glass tubes and vortex for 10 seconds, then split into two aliquots (500 µl + 500 µl) in two pyrex tubes with screw cap clean. Evaporate to dryness with the aid of a stream of helium or nitrogen (in this step the temperature must be < 40 C) Derivatization rate amphetamines: Add, in each tube dedicated at rate for Amphetamine, 50 µl of Reagent H Derivatization Solution 1 for Amphetamines using the appropriate syringe Hamilton of 100 µl (code Z81000) Incubate at 80 C x 5 min Seal the tubes with the cap Cooling at Room Temperature pen the glass tube Add 15 µl of Reagent I Derivatization Solution 2 for Amphetamines using the appropriate syringe Hamilton of 50 µl (code Z80500) Incubate at 80 C x 5 min Seal the tubes with the cap Cooling at Room Temperature pen the glass tube Injection: Transfer the derivatized in the vial with reducing volume and inject 1 µl in the gas chromatograph. Derivatization rate other classes of Abuse Drugs: Add, in each tube dedicated at rate for Basic Drugs, 35 µl of Reagent M Derivatization Solution using the appropriate syringe Hamilton of 100 µl (code Z81000) Incubate at 70 C x 20 min Seal the tubes with the cap Cooling at Room Temperature pen the glass tube Release N 003 Abuse Drugs in hair by GC/MS January 2013 15

Injection: Transfer the derivatized in the vial with reducing volume and inject 1 µl in the gas chromatograph. NB: in this way the analytical sensitivity will be increased compared to that obtained in steps 1 and 2 of point F. GENERAL CNSIDERATINS about QUANTITIVE CALCULATIN F UNKNWN SAMPLES The calculation of the unknown samples is performed as a function of the equation obtained for the calibration curve where the abscissa axis shows the concentration in ng/mg and along the ordinate the ratio between the areas of the analyte/internal standard. To calculate the concentration in ng/mg hair in the case of unknown samples, please note that once you have the value of concentration on the line, it should be multiplied by the mg of hair that has built the calibration curve (50 mg) and divided by the actual weight of the sample under examination (eg. 20 mg if it is weighed amount of this hair). Release N 003 Abuse Drugs in hair by GC/MS January 2013 16

BASIC DRUGS F ABUSE in hair - Warnings REAGENT N: TEST SLUTIN PIATES, METHADNE, CCAINE, BUPRENRPHINE AND METABLITES Morphine Codeine 6-Monoacethylmorphine Ethylmorphine Dihydrocodeine Methadone BEG Cocaethylene Cocaine Buprenorphine EDDP Storage: nce you open the vial, withdrawn the necessary volume, close it with the reagent used and stored at -20 C. About 10.000 ng/ml REAGENT L: TEST SLUTIN AMPHETAMINES Amphetamine Methamphetamine MDA MDMA MDE Storage: nce you open the vial, withdrawn the necessary volume, close it with the reagent used and stored at -20 C. About 10.000 ng/ml REAGENT T1: CHEMICAL STANDARD SLUTIN 1 Amphetamine Methamphetamine MDA MDMA MDE Cocaine Cocaethylene Morphine Ethylmorphine Codeine Dihydrocodeine Methadone Storage: nce you open the vial, withdrawn the necessary volume, close it with the reagent used and stored at -20 C. 100.000 ng/ml REAGENT T2: CHEMICAL STANDARD SLUTIN 2 Benzoilecgonine 6-Monoacethylmorphine EDDP Storage: nce you open the vial, withdrawn the necessary volume, close it with the reagent used and stored at -20 C. 100.000 ng/ml REAGENT T3: CHEMICAL STANDARD SLUTIN 3 Buprenorphine Storage: nce you open the vial, withdrawn the necessary volume, close it with the reagent used and stored at -20 C. 100.000 ng/ml 17

REAGENT H: DERIVATIZATIN SLUTIN 1 FR AMPHETAMINES nce opened ampoule transfer all the content, 1 ml, in a glass vial. Close cap tightly and store at 2-8 C. REAGENT I: DERIVATIZATIN SLUTIN 2 FR AMPHETAMINES nce opened ampoule transfer all the content, 1 ml, in a glass vial. Close cap tightly and store at 2-8 C. REAGENT M: DERIVATIZATIN SLUTIN nce opened ampoule transfer all the content, 1 ml, in a glass vial. Close cap tightly and store at 2-8 C. REAGENT : INTERNAL STANDARD SLUTIN FR BASIC DRUGS Nalorphine Storage: at 20 C 100.000 ng/ml REAGENT P: INTERNAL STANDARD SLUTIN FR AMPHETAMINES MDPA Storage: at 20 C 100.000 ng/ml DETECTR DI MASSA CNDITINS (4000) : (these conditions may be varied depending on the instrument used) Massa Range 60 530 m/z Massa Temperature: 180 C; Transfer Line Temperature: 270 C; Manifold Temperature: 80 C Filament on: 5 minutes GAS-CHRMATGRAPH CNDITINS : (these conditions may be varied depending on the instrument used) Column VF-5ms or Rxi-5Sil MS 30 m x 0,25 mm, 0,25 µm (after conditioning according to the indications provided by the manufacturer) Injector Temperature 280 C Spli Ratio Inizio: N split 20 0 min: FF 1 min: N split 100 3 min: N split 20 NB: The split ratio can be varied depending on the instrument used Heat Schedule: 120 C x 3,25 minutes 120-250 C at 20 C/min 250 C x 1 min 250-300 C at 40 C/min 300 x 3 minutes (run 15 minutes) Helium Gas Flow 1 ml/min CLUMN VF-5 MS or Rxi-5Sil MS CNDITINING Follow the manufacturer's instructions. CLUMN VF-5 MS or Rxi-5Sil MS CLEANING If the analytical performance suffer significant changes (e.g. Low sensitivity or presence of interfering in chromatograms), you must make the cleaning of the column as reported in accordance with the indications of the manufacturer. 18

AUTSAMPLER WASH Wash the syringe before each injection with ACETNE. PERATINAL CMPUTER PARAMETERS ACCRDING T THE SPECIFICATIN F ACCUNTING SFTWARE ACCESSRES AND CNSUMABLES CDE DESCRIPTIN PACKAGING GC49016 Calibrator in hair for Abuse Drugs 1 CF Z43120 Chemical Standard for MBDB 2 x 5 ml GC49019 Control in hair for Abuse Drugs Levels 1 and 2 1 CF S29057U Standard Glass Tubes of 2 ml with screw caps 1 x 100 Pk S24717 Insert 100 ul for vials of 2 ml 1 x 100 Pk Z1636/26 Pyrex Tubes of 10 ml with SWL caps 1 x 40 Pk SCP8944 VF-5MS Column (30 m x 0,25 mm - 0,25 µm) 1 Pk S13623 Rxi-5Sil MS Column (30 m x 0,25 mm 0,25 um) 1 Pk Z81000 Hamilton Syringe of 100 ul 1 Pk Z80500 Hamilton Syringe of 50 ul 1 Pk 19

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) MCou t 60:50 0 03 Tpp-Amf-Coc-Metad5200+ISnalo-MDPA1000M- 100MS 30MBT SMS TIC Filt d 6 MD E 9.4 TM 18 S min TFA 5 4 3 AM PH ET AMI NE TM6.0 S 59 TFA min ME TA MP HE TA MIN E TM6.9 S 72 TFA min 2 MD A 8.4 TM 68 S min TFA MD MA TM9.1 S 87 TFA min MD PA TM9.7 S 58 TFA min 1 6 7 8 9 10 11 12 minut Fig. 1 : Test Solution Basic Abuse Drugs Fig. 2 : Test Solution Amphetamine R.T. 10.8 EDDP R.T. 6.0 Amphetamine R.T. 11.4 Methadone R.T. 7.0 Methamphetamine R.T. 11.8 Cocaine R.T. 8.4 MDA R.T. 12.2 Benzoilecgonine R.T. 9.2 MDMA R.T. 12.2 Cocaethylene R.T. 9.4 MDE R.T. 12.8 Dihydrocodeine R.T. 9.7 Internal Standard for Amphetamines R.T. 13.2 Codeine R.T. 13.3 Ethylmorphine R.T. 13.4 Morphine R.T. 13.8 6-Monoacethylmorphine R.T. 14.0 Internal Standard for Basic Drugs 20

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) kcounts Ionization ff 390:530 Buprenorfina.SMS Ions: 450.0+482.0+506.0 Filtered 10 BP 276.0 (999=10) 146823 in MAINLIB Pyrrolidine, 1,5-dimethyl-3,3-diphenyl-2-ethylidene- CAS No. 30223-73-5, C20H23N, MW277 276.0 999 125 100 BUPRENRFINA-TMS 13.393 min 75% N 277.0 845 75 5 262.0 484 50 25 25% 42.0 76 56.0 122 69.0 91 99.0 109 105.0 265 115.0 124 129.0 68 165.0 76 178.0 191.0 83 81 220.0 233 200.0 180 205.0 135 221.0 77 235.0 83 248.0 69 263.0 96 278.0 222 0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 minutes 50 100 150 200 250 m/z Fig. 3 : Test Solution Buprenorphine Fig. 4 : Massa Spectrum of EDDP R.T. 13.3 Buprenorphine MLECULAR INS: 276-277-262 21

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 10. BP 72.0 (999=10) 31682 in MAINLIB 57.0 72.0 125 999 Methadone(reduced) CAS No. 545-90-4, C21H29N, MW311 10 BP 82.0 (999=10) 40834 in MAINLIB 82.0 999 Cocaine CAS No. 50-36-2, C17H21N4, MW 303 N 7.5% 73.0 69 N 75% 182.0 716 H 5. 42.0 49 56.0 48 71.0 48 H H 43.0 41 70.0 41 5 2.5% 0. 44.0 25 45.0 6 55.0 31 58.0 11 69.0 16 77.0 10 91.0 18 97.0 6 115.0 13 129.0 9 165.0 17 178.0 12 179.0 7 25% 42.0 234 51.0 121 55.0 78 83.0 367 81.0 125 105.0 296 96.0 249 97.0 111 122.0 88 198.0 183.0 95 83 272.0 69 303.0 172 50 100 150 200 m/z 0 50 100 150 200 250 300 m/z Fig. 5 : Massa Specrum of Methadone Fig. 6 : Massa Spectrum of Cocaine MLECULAR INS: 72-165-115 MLECULAR INS: 82-182-94 22

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) BP 82.0 (999=10) 40851 in MAINLIB 82.0 999 Benzoylecgonine-TMS C19H27N4Si, MW361 6 BP 373.0 (999=10) 159067 in MAINLIB Dihydrocodeine TMS derivative C21H31N3Si, MW373 373.0 999 7 6 N Si 5 N 5 240.0 514 4 Si 4 3 2 1 42.0 121 83.0 379 73.0 185 94.0 231 81.0 108 75.0 65 105.0 297 96.0 182 108.0 51 122.0 83 361.0 218 241.0 99 346.0 239.0 73 45 3 2 1 73.0 179 59.0 73 75.0 38 129.0 39 146.0 179 178.0 90 199.0 36 226.0 47 236.0 216 242.0 75 244.0 47 268.0 38 282.0 137 283.0 78 315.0 127 316.0 69 358.0 70 374.0 278 372.0 136 375.0 70 0 100 200 300 400 500 m/z 50 100 150 200 250 300 350 m/z Fig. 7 : Massa Spectrum of Benzoilecgonine Fig. 8 : Massa Spectrum of Dihydrocodeine MLECULAR INS: 82-240-361 MLECULAR INS: 373-146-236 23

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 4 BP 73.0 (999=10) 34871 in MAINLIB 178.0 196.0 411 391 Morphinan-6-ol, 7,8-didehydro-4,5-epoxy-3-methoxy-17-methyl-6-(trimethylsilyl)- CAS No. 74367-14-9, C21H29N3Si, MW371 371.0 761 10 BP 73.0 (999=10) 17 in EUREKA 73.0 999 ETILMRFINA-TMS N 385.0 852 3 75% 146.0 696 2 1 179.0 177 176.0 37 188.0 111 197.0 87 189.0 51 195.0 32 207.0 44 212.0 24 229.0 187 225.0 22 234.0 135 235.0 58 242.0 33 254.0 27 266.0 28 282.0 63 280.0 32 299.0 41 313.0 93 314.0 51 312.0 21 343.0 65 Si 356.0 85 357.0 30 372.0 226 370.0 109 373.0 63 5 25% 74.0 91 115.0 159 107.0 82 192.0 519 196.0 395 193.0 340 152.0 123 179.0 147.0 90 60 234.0 540 243.0 392 235.0 150 214.0 253.0 105 106 228.0 58 280.0 154 296.0 78 328.0 204 341.0 88 357.0 267 386.0 294 384.0 137 370.0 75 429.0 63 503.0 96 200 250 300 350 m/z 100 200 300 400 500 600 m/z Fig. 9 : Massa Spectrum of Codeine Fig. 10 : Massa Spectrum of Ethymorphine MLECULAR INS: 371-234-343 MLECULAR INS: 385-146-73 24

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) BP 73.0 (999=10) 34417 in MAINLIB 73.0 146.0 999 493 Morphinan, 7,8-didehydro-4,5-epoxy-17-methyl-3,6-bis[(trimethylsilyl)oxy]-, (5. CAS No. 55449-66-6, C23H35N3Si2, MW429 236.0 586 10 BP 399.0 (999=10) 9 in EUREKA 399.0 999 6-MAM-TMS 4 196.0 409 Si 234.0 327 429.0 326 75% 341.0 770 3 220.0 260 Si 2 1 75.0 132 59.0 110 74.0 84 70.0 52 94.0 73 96.0 31 129.0 42 152.0 42 165.0 68 173.0 27 203.0 89 217.0 195.0 47 N 216.0 125 235.0 87 238.0 64 243.0 40 63 222.0 41 266.0 75 269.0 40 287.0 197 288.0 60 324.0 135 325.0 42 372.0 50 401.0 110 402.0 38 414.0 156 430.0 124 428.0 54 5 25% 81.0 95 162.0 145 204.0 359 205.0 83 288.0 445 283.0 136 273.0 91 325.0 215 326.0 56 357.0 62 400.0 374 401.0 86 100 200 300 400 m/z 100 200 300 400 m/z Fig. 11 : Massa Spectrum of Morphine Fig. 12 : Massa Spectrum of 6-Monoacethylmorphine MLECULAR INS: 429-414-234 MLECULAR INS: 399-340-287 25

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 10 BP 455.0 (999=10) 161816 in MAINLIB Nalorphine, o,o'-bis(trimethylsilyl)- C25H39N3Si2, MW457 455.0 999 10 BP 118.0 (999=10) 41 in EUREKA 118.0 999 AMPHETAMINE TMS TFA Si 140.0 779 75% N 75% 91.0 679 5 Si 414.0 524 5 440.0 407 456.0 387 25% 179.0 63 193.0 111 222.0 181 218.0 97 234.0 66 260.0 308 266.0 281.0 84 81 324.0 324 313.0 259 325.0 92 350.0 162 371.0 136 366.0 53 382.0 64 415.0 183 416.0 69 441.0 150 442.0 56 457.0 143 25% 65.0 214 63.0 64 92.0 171 89.0 65 117.0 306 119.0 129 115.0 82 232.0 72 200 250 300 350 400 450 m/z 50 100 150 200 250 300 350 m/z Fig. 13 : Massa Spectrum of Internal Standard for Basic Drugs Fig. 14 : Massa Spectrum of Amphetamine MLECULAR INS: 455-440-414 MLECULAR INS: 91-118-140 26

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 10 BP 154.0 (999=10) 42 in EUREKA 154.0 999 METAMPHETAMINE TMS TFA 10 BP 135.0 (999=10) 43 in EUREKA 135.0 999 MDA TMS TFA 75% 75% 110.0 640 5 5 162.0 507 91.0 362 118.0 421 25% 25% 77.0 231 56.0 56 69.0 148 117.0 111 119.0 68 155.0 60 246.0 73 51.0 64 69.0 132 78.0 73 105.0 77 136.0 105 163.0 81 275.0 96 50 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z Fig. 15 : Massa Spectrum of Methamfetamine Fig. 16 : Massa Spectrum of MDA MLECULAR INS: 118-110-154 MLECULAR INS: 135-162-275 27

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 10 BP 162.0 (999=10) 45 in EUREKA 162.0 999 MDMA TMS TFA 10 BP 168.0 (999=10) 46 in EUREKA 168.0 999 MDE TMS TFA 154.0 850 162.0 901 75% 75% 135.0 670 110.0 603 5 5 135.0 429 25% 51.0 95 69.0 182 77.0 294 78.0 113 105.0 83 136.0 81 163.0 157 25% 51.0 57 69.0 100 77.0 212 104.0 90 124.0 79 163.0 242 169.0 65 50 100 150 200 250 300 m/z 50 100 150 200 250 300 350 m/z Fig. 17 : Massa Spectrum of MDMA Fig. 18 : Massa Spectrum of MDE MLECULAR INS: 110-135-154-162 MLECULAR INS: 135-140-162-168 28

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 10 BP 140.0 (999=10) 48 in EUREKA 140.0 999 162.0 994 MDPA TMS TFA 10 BP 196.0 (999=10) 122244 in MAINLIB 8-Azabicyclo[3.2.1]octane-2-carboxylic acid, 3-(benzoyloxy)-8-methyl-, ethyl es CAS No. 529-38-4, C18H23N4, MW317 196.0 999 N 75% 182.0 774 75% 82.0 730 5 135.0 442 5 25% 51.0 71 77.0 301 78.0 144 105.0 115 103.0 72 131.0 75 163.0 178 183.0 61 25% 83.0 262 96.0 200 81.0 82 94.0 333 97.0 94 122.0 94 166.0 65 197.0 123 212.0 136 272.0 167 317.0 110 50 100 150 200 250 300 m/z 50 100 150 200 250 300 m/z Fig. 19 : Massa Specrum of Internal Standard for Amphetamines Fig. 20 : Massa Spectrum of Cocaethylene MLECULAR INS: 135-140-162-163-182 MLECULAR INS: 82-196-94 29

DRUGS F ABUSE IN HAIR (Reference Chromatograms/Spectra) 10 Spectrum1A BP: 450.6 (139272=10), buprenorfina.sms 450.6 139272 13.394 min, Scan: 1635, 390:530, Ion: 939 us, RIC: 518000, EBC 75% 5 482.4 57842 25% 451.5 43296 506.4 39598 408.6 10265 424.6 10178 438.7 11753 452.4 12788 483.4 21688 492.4 23408 493.4 8211 507.3 17251 524.3 11462 400 425 450 475 500 525 m/z Fig. 21 : Massa Spectrum of Buprenorphine MLECULAR INS: 450-482-506 30