METHADONE and in urine by GC-MS Code GC48010 Method of Confirmation by GC-MS INTRODUCTION is a synthetic opioid used as a drug of choice in the detoxification of heroin (and / or opiates). is metabolized in the body in 2-ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine (), metabolite that is found along with the parent drug in urine. The screening methods generally measure, through an antigen-antibody reaction, methadone. Because these methods can give false positive results for the presence of foreign substances that may interfere with the antigen-antibody reaction is always required for confirmation analysis. So if the analysis of a screening is positive (cut-off> or = 300 ng / ml) it is necessary an analysis of confirmation with chromatographic separation technique is that liquid gas, both coupled with a detector in mass spectrometry. The following method is used to determine the and the after solid phase extraction and subsequent determination in gas chromatography - mass spectrometry (Gas Chromatography Mass Spectrometry, GC-MS). EUREKA srl LAB DIVISION 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 This product fulfills all the requirements of Directive 98/79/EC on in vitro diagnostic medical devices (IVD). The declaration of conformity is available upon request. Release N 006 and in urine by GC/MS March 2012 1
TECHNICAL FEATURES Principle of the Method : This method allows to determinate the and its main metabolite,, after solid-liquid extraction and reading by GC/MS. Recovery : about 98 % Sensitivity : Dynamic Range of the Method : about 20 ng/ml 20 10 ng/ml Cut-off : 100 ng/ml 100 ng/ml Ref. Provvedimento Gazzetta Ufficiale 236 del 18-09-2008 Components of the kit : All the components are ready-to-use. Stability: 3 years at 2 8 C. Reagent A Conditioning Solution N 1, 1 x 50 ml Reagent B Conditioning Solution N 2, 1 x 50 ml Reagent C Buffer Solution, 1 x 50 ml Reagent D Wash Solution, 1 x 50 ml Reagent E Eluting Solution, 1 x 100 ml Reagent F Diluting Solution, 1 x 10 ml Reagent G Test Solution, 1 x 0,5 ml Reagent H Test Solution, 1 x 0,5 ml Reagent K Internal Standard Solution, 1 x 300 ul Reagent I1 Urine Calibrator lyophil Level 1, 2 x 5 ml Reagent I2 Urine Calibrator lyophil Level 2, 2 x 5 ml Reagent I3 Urine Calibrator lyophil Level 3, 2 x 5 ml Reagent I4 Urine Calibrator lyophil Level 4, 2 x 5 ml Reagent I5 Urine Calibrator lyophil Level 5, 2 x 5 ml Reagent J1 Urine Calibrator lyophil Level 1, 2 x 5 ml Reagent J2 Urine Calibrator lyophil Level 2, 2 x 5 ml Reagent J3 Urine Calibrator lyophil Level 3, 2 x 5 ml Reagent J4 Urine Calibrator lyophil Level 4, 2 x 5 ml Reagent J5 Urine Calibrator lyophil Level 5, 2 x 5 ml To dilute 1:10 with H 2O before use Reagent M Derivatization Solution, 5 x 1 ml (5 ampullas of 1 ml) 2
Clean-up Columns, 50 pcs Minimum Instrumental equipment required: Optional Equipment: GC-MS Instrument Operational Computer Autosampler Urine Collection Procedure: Take 10 ml of urine. If stored at - 20 C, the sample is stable about 3 months. Release N 006 and in urine by GC/MS March 2012 3
PREANALYTICAL PROCEDURE STEP 1 : Pipette in a glass tube with teflon cap: 400 µl of Reagent F Diluting Solution 50 µl of Reagent G Test Solution 50 µl of Reagent H Test Solution Vortex for 20 sec. STEP 2 : Dry the eluate by the help of an helium or nitrogen flow (at this step the work temperature must be < 40 C). STEP 3 : Put in 100 µl of Reagent M Derivatization Solution STEP 4: Incubate at 70 C for 30 minutes Vortex for 20 sec. Cooling at Room Temperature STEP 5 : Transfer the whole sample in vials goblet form NOTE : Before starting the analytical session it would be better to inject 1 µl of this solution in GC to identify the time of retention of and, that must be similar to that shown in Fig. 1. If the Test has given positive result, proceed with the analytical session. If it wouldn t be so, verify the functionality of the analytical system. ANALYTICAL PROCEDURE STEP 1 : Clean-up Columns Conditioning Condition clean up columns by fluxing 1 ml of Reagent A Conditioning Solution N 1 Discharge the percolated liquid Condition clean up columns by fluxing 1 ml of Reagent B Conditioning Solution N 2 Discharge the percolated liquid STEP 2: Dilute Reagent K Internal Standard Solution 1:10 with Water HPLC grade N.B.: this dilution is prepared fresh for each run Release N 006 and in urine by GC/MS March 2012 4
STEP 3 : Samples Preparation Make a Calibration Curve on 5 points : Sample Sample 1000 µl Reagent I1 Calibrator Level 1 Reagent J1 Calibrator Level 1 Reagent I2 Calibrator Level 2 Reagent J2 Calibrator Level 2 Reagent I3 Calibrator Level 3 Reagent J3 Calibrator Level 3 Reagent I4 Calibrator Level 4 Reagent J4 Calibrator Level 4 Reagent I5 Calibrator Level 5 Reagent J5 Calibrator Level 5 Calibrator + Level 1 Calibrator Methadon e + Level 2 Calibrator + Level 3 Calibrator + Level 3 Calibrator + Level 5 Control + Control Control Reagent K Internal Std Solution (diluted 1:10 with H 2 O) 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl Reagent C Buffer Solution 1000 µl 1000 µl 1000 µl 1000 µl 1000 µl 1000 µl 1000 µl Verify that the ph is between 6,5 and 7,5 Eventually correct it N.B: The values of concentration of the and calibrators mixed together are the HALF to that shown in the table of warnings!!!! Vortex for 10 sec. STEP 4 : Dispense all the samples, previously prepared, into clean up columns. Discharge the percolated liquid, if needed a vacuum system could be used for percolation, at speed of 1 ml / min ( drop by drop ). Release N 006 and in urine by GC/MS March 2012 5
STEP 5 : Clean-up Column Wash: Wash with 1 ml of Reagent D Wash Solution Discharge the percolated liquid and dry with a light flux of air STEP 6 : Eluting strictly in glass tubes with pyrex caps Elute with 2 ml of Reagent E Eluting Solution, at speedy of 1 ml / min. Collect the eluate and mix at Vortex If there is a turbidity or a precipitate in the eluate, centrifuge at 4000 rpm for 2 min. STEP 7 : Dry the eluate by the help of an helium or nitrogen flow (at this step the work temperature must be < 40 C). STEP 8 : Put in 100 µl of Reagent M Derivatization Solution STEP 9: Incubate at 70 C for 30 minutes Vortex for 20 sec. Cooling at Room Temperature STEP 10 : Transfer the whole sample in vials goblet form N.B.: at this step, the sample is stable 1 day at 2-8 C INJECTION : Inject 1 µl of the solution in the GC Release N 006 and in urine by GC/MS March 2012 6
REAGENT G TEST SOLUTION METHADONE METHADONE and in urine - Warnings ~ 50 ng/ml REAGENT H TEST SOLUTION ~ 10 ng/ml REAGENT I1-I2-I3-I4-I5 URINE CALIBRATORS LYOPHIL. METHADONE Lot. METHADONE Use and Reconstitution: Urine Calibrators are used for calibration of the GC system. Add exactly 5 ml of H 2 O of degree HPLC and agitate gently for 20 min. until the whole material is not dissolved. Before sampling mix up gently to assure homogeneity. Conservation and stability: Calibrators are stable 36 months if stored at 2 8 C. After reconstitution 7 days if stored at 2 8 C and 1 month at 20 C. Do not use after the expiry date. Packaging : 5 x 2 x 5 ml Warning: this calibrator in human matrix must be handled with care and must be considered as potentially infective. N.B.: Once reconstituted, the calibrators should be stored separately. Level 1 Level 2 Level 3 Level 4 Level 5 ng / ml REAGENT J1-J2-J3-J4-J5 URINE CALIBRATORS LYOPHIL. Lot. Use and Reconstitution: Urine Calibrators are used for calibration of the GC system. Add exactly 5 ml of H 2 O of degree HPLC and agitate gently for 20 min. until the whole material is not dissolved. Before sampling mix up gently to assure homogeneity. Conservation and stability: Calibrators are stable 36 months if stored at 2 8 C. After reconstitution 7 days if stored at 2 8 C and 1 month at 20 C. Do not use after the expiry date. Packaging : 5 x 2 x 5 ml Warning: this calibrator in human matrix must be handled with care and must be considered as potentially infective. N.B.: Once reconstituted, the calibrators should be stored separately. Level 1 Level 2 Level 3 Level 4 Level 5 ng / ml SET OF GAS-CROMATOGRAPH : VF-5ms Column 30 m x 0,25 mm, 0,25 µm (conditioned) Injector Temperature: 280 C Split Report Start: ON 20 0 min: OFF 1 min: ON 100 3 min: ON 20 Oven Column Temperature 50 C x 1 minute + 20 C/min till up 180 C x 0 minute + 10 C/min till up 300 C x 1.5 minute (run 21 minutes) Column Flow: 1 ml/min 7
SET OF MASSA DETECTOR : Range of Massa 80 600 Temperature of Massa: 180 C; Temperature of Transfer Line: 270 C; Temperature of Manifold: 80 C Filament on: 6 minutes Operative Method in Electronic Impact (EI) CONDITIONING OF VF-5 MS COLUMN and SYRINGE Before use wash the syringe with ACETONE. Follow the manufacturer prescription. Do not condition column/s if connected to the Massa Detector. At the beginning of each run to calibrate the masses and RF. CLEANING OF THE COLUMN Disconnect the detector. Keep the column at the highest temperature for the recommended time (see the manufacturer s instructions) with a speed of 30 C/min. ACCESSORIES AND CONSUMABLES CODICE DESCRIZIONE CONFEZIONE CC48016 Calibrator in urine for 2 x 5 x 5 ml CC48116 Calibrator in urine for 2 x 5 x 5 ml CC43017 Multiparametric Control for Drugs of Abuse Level 1 6 x 5 ml CC43018 Multiparametric Control for Drugs of Abuse Level 2 6 x 5 ml CC43019 Multiparametric Control for Drugs of Abuse Levels 1 and 2 2 x 6 x 5 ml CC48017 Control in urine for, 6-MAM and Cocaine Level 1 6 x 5 ml CC48018 Control in urine for, 6-MAM and Cocaine Level 2 6 x 5 ml CC48019 Control in urine for, 6-MAM and Cocaine Levels 1 and 2 2 x 6 x 5 ml Z394983500 Standard glass vial of 2 ml with screw cap 1 x 144 Pk S392611593 Insert 100 ul for vials of 2 ml 1 x 100 Pk 8
and in urine (Reference Chromatograms and Spectrums) 1.00 36 E 105-1_11-26-2010.SMS Ions: 277.0+262.0+276.0 Filtered 100% BP 276.0 (999=100%) 35 in EUREKA 276.0 999 277.0 939 13.218 m in 1.00 37 E 210-1_11-26-2010.SMS Ions: 277.0+262.0+276.0 Filtered 75% 13.227 m in 50% 220.0 458 262.0 451 1.00 13.238 m in 40 E 420-2_11-26-2010.SMS Ions: 277.0+262.0+276.0 Filtered 25% 115.0 271 105.0 182 165.0 198 127.0 83 153.0 58 200.0 178 179.0 118 190.0 72 221.0 93 235.0 151 247.0 103 278.0 162 0% 11 12 13 14 15 16 17 18 minutes 100 200 300 400 500 600 m/z Fig. 1 Urine Samples R.T. 13.7 105-210-420 ng/ml Fig. 2 Massa Spectrum of MOLECULAR IONS: 277-262-276 9
and in urine (Reference Chromatograms and Spectrums) 1.00 METADONE 25 M115-1_11-26-2010.SMS Ions: 165.0+115.0+294.0 Filtered 100% BP 165.0 (999=100%) 34 in EUREKA 165.0 999 METADONE METADONE 14.140 m in 1.00 METADONE 26 M224-1_11-26-2010.SMS Ions: 165.0+115.0+294.0 Filtered 75% 115.0 709 178.0 709 223.0 647 1.00 METADONE 14.136 min METADONE 14.136 min METADONE 27 M440-1_11-26-2010.SMS Ions: 165.0+115.0+294.0 Filtered 12 13 14 15 16 17 minutes 50% 25% 0% 105.0 392 117.0 345 102.0 227 103.0 144 104.0 65 129.0 221 139.0 140 152.0 332 150.0 55 166.0 174 179.0 333 167.0 107 195.0 173 193.0 128 190.0 62 224.0 142 294.0 421 295.0 130 100 200 300 400 500 600 m/z Fig. 3 Urine Samples R.T. 14.6 115-224-440 ng/ml Fig. 4 Massa Spectrum MOLECULAR IONS: 115-165-294 10
and in urine (Reference Chromatograms and Spectrums) 2.0 1.5 1.0 Nalorphine, o,o'-bis(trimethylsilyl)- 36 E 105-1_11-26-2010.SMS Ions: 455.0+440.0+414.0 Filtered 200.0 ng/m l N alorphine, o,o'-bis(trim e 17.988 m in 100% BP 455.0 (999=100%) 161816 in MAINLIB 73.0 949 Si O Nalorphine, o,o'-bis(trimethylsilyl)- C25H39NO3Si2, MW457 455.0 999 0.5 0.0 2.0 1.5 1.0 Nalorphine, o,o'-bis(trimethylsilyl)- 37 E 210-1_11-26-2010.SMS Ions: 455.0+440.0+414.0 Filtered 200.0 ng/m l Nalorphine, o,o'-bis 17.996 m in 75% 50% Si O O N 414.0 524 0.5 440.0 407 0.0 2.0 1.5 1.0 0.5 Nalorphine, o,o'-bis(trimethylsilyl)- 40 E 420-2_11-26-2010.SMS Ions: 455.0+440.0+414.0 Filtered 200.0 ng/ml Nalorphine, o,o'-bis(trim 17.992 min 25% 45.0 101 75.0 131 74.0 85 122.0 53 179.0 63 222.0 181 193.0 111 218.0 97 229.0 53 260.0 308 266.0 84 324.0 324 313.0 259 325.0 92 350.0 162 372.0 112 382.0 64 415.0 183 416.0 69 441.0 150 454.0 105 442.0 56 0.0 0% 13 14 15 16 17 18 19 20 minutes 100 200 300 400 500 m/z Fig. 5 Urine Samples with Internal Standard R.T. 18.8 Internal Standard Fig. 6 Massa Spectrum of Internal Standard MOLECULAR IONS: 455-440-414 11