Psychoactive Drugs Urine LC-MS/MS Analysis Kit User Manual

Page 1 / 18 Psychoactive Drugs Urine LC-MS/MS Analysis Kit User Manual ZV-2001-0200-20 200 2-8 C

Page 2 / 18 Table of Contents 1. INTENDED USE... 3 2. SUMMARY AND EXPLANATION... 3 3. TEST PRINCIPLE... 3 4. WARNING AND PRECAUTIONS... 3 5. STORAGE AND STABILITY... 4 6. MATERIALS SUPPLIED... 4 7. MATERIALS REQUIRED BUT NOT SUPPLIED... 5 8. PROCEDURE NOTES... 5 9. LIMITATIONS OF THE PROCEDURE... 6 10. PRE-TEST SET-UP INSTRUCTIONS... 6 11. TEST PROCEDURE... 7 12. QUALITY CONTROL... 8 13. CALCULATION OF RESULTS... 8 14. INTERPRETATION OF RESULTS... 9 15. EXPECTED VALUES... 9 16. LC-MS/MS PARAMETERS... 10 17. MS SCAN PARAMETERS... 11 18. ANALYTICAL PERFORMANCE... 12 19. SAMPLE CHROMATOGRAM... 13 20. AUTOMATED SAMPLE PREPARATION METHOD PARAMETERS FOR ZIVAK MULTITASKER... 18

Page 3 / 18 1. INTENDED USE Quantitative LC-MS/MS analysis kit for Psychoactive Drugs in urine samples. 2. SUMMARY AND EXPLANATION Psychoactive drugs are chemical substances that affect the brain functions, behaviors and consciousness. These drugs can be used therapeutically to treat both physical and psychological disorders. Monitoring the concentrations of these drugs is very important issue for clinicians if they used for therapy. The analytic methods must be highly sensitive and selective for accurate and precise quantification of these drugs for therapeutic monitoring. Detection and quantification of these drugs can be performed using UV or fluorescence spectrophotometers, immunoassays and radio-receptor assays. Mass spectrometers coupled with chromatographic systems such as LC- MS, LC-MS/MS, GC-MS are more suitable for this purpose through their sensitivity and specifity. ZinMass Psychoactive Drugs LC-MS/MS Analysis Kit is developed for quantitative detection of 15 selected psychoactive drugs from different drug groups in urine samples with a minimum sample preparation in 7 minutes. Main methods and procedures that have been selected are based on EN ISO 13485 and 98/79/EC. 3. TEST PRINCIPLE The 15 selected psychoactive drugs including amphetamine, methamphetamine, MDA, MDMA, MDEA, diazepam, morphine, codeine, cocaine, alprozolam, flunitrazepam, delta-9-thc, clonazepam, 6-MAM and heroin are extracted from urine samples by using an organic extraction mixture. After the extraction, these analytes separated with a gradient elution on Zivak PD HPLC column and measured by Tandem Mass spectrometry. Accuracy and reliability of the analysis could be checked by the deuterated internal standards for each analyte which are supplied with the kit. Confirmation and quantification ions for all analytes and internal standards determined in ESI(+) ionization mode. 4. WARNING AND PRECAUTIONS For in-vitro diagnostic use only. For professional use only.

Read the instructions carefully, before you start. In case of damage of the kit package, please contact Zivak or your supplier. Do not use expired kits and components. Please check the batch no and expiry date before start. Protective gloves and goggles should be worn. Please take any necessary precautions to prevent infection with blood borne pathogens while working with biological fluids. Appropriate bio-safety precautions and disposal of bio-hazardous wastes should be followed. Please check the labels on reagent bottles. Reagents of this kit contain hazardous material may cause eye and skin irritations. Page 4 / 18 5. STORAGE AND STABILITY Please check the labels on reagent bottles. Reagents of this kit contain hazardous material may cause eye and skin irritations. This analysis kit can be shipped at room temperature. After arrival, reagent 2 and standard 1 must be stored at -20 C. Synthetic Urine must be stored at 2-8 C All other components must be stored at room temperature. Please make sure that you keep reagent 1 at room temperature otherwise it will become unusable. All components are guaranteed until expiry date when stored at recommended temperatures and used as described in these instructions. 6. MATERIALS SUPPLIED Order No Volume Symbol Content ZV-2001-02R1-20 2 x 37 ml R1 Reagent 1, Contains base ZV-2001-02R2-20 2x 45 ml R2 Reagent 2, Contains internal standards ZV-2001-02MA-20 1x 0,5 L MA Mobile Phase A, Contains organic solvent and acid ZV-2001-02MB-20 1x 0,5 L MB Mobile Phase B, Contains organic solvent and acid

Page 5 / 18 ZV-2001-02WB-10 2x 1 L WB Washing solution, Contains organic solvent SU ZV-2001-02SU-10 1x 10ml Synthetic Urine KK ZV-2001-KK-20 1x 1 pc User Guide 7. MATERIALS REQUIRED BUT NOT SUPPLIED Order No Volume Symbol Content ZV-2001-02S1-20 1 x 1 ml S1 Standard 1, in organic solvent ZV-2001-02C1-20 1 pcs Zivak Psychoactive Drugs Urine LC-MSMS Analytical Column 20-200 µl pipette 100-1000 µl pipette Pipette tips Vortex mixer 2.0 ml sample preparation tube Centrifuge 0.5 ml autosampler vial and vial caps 8. PROCEDURE NOTES Any inappropriate handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pre-treatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time.

Leave aside all reagents and specimens to reach room temperature (18-25 C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each reagent, standard or specimen. Do not interchange the vial caps. Always keep vials closed when not been used. Do not re-use wells/tubes or reagents. Incubation time affects results. All tubes or wells should be handled in the same order and time sequences. Page 6 / 18 9. LIMITATIONS OF THE PROCEDURE Specimen collection and storage has a significant effect on the test results. Urine samples from earlier days must be kept frozen before the analysis. If the urine sample is not clear, it must be centrifuged before sample preparation. 10. PRE-TEST SET-UP INSTRUCTIONS Set-up the Instrument: Purge the HPLC pumps with a high flow rate of mobile phase(s). This should be done by pumping the mobile phase(s) through the system for 2 minutes at a flow rate of 4.0 ml/min. Switch off the pump and connect the column in flow direction. Activate the method and allow mobile phase(s) to flow through the column for 15 minutes. Make sure the bottle of mobile phase bottle is closed well, otherwise components of the mobile phase could evaporate; this alters the retention times. Preparation of Zivak Calibrator Level I: Add 1900 µl synthetic urine into a preparation tube. Add 100 L of Standard and vortex for 10 seconds. After preparation, calibrator must be aliquoted into 350 µl volumes and stored at -20 C. Preparation of Zivak Calibrator Level II: Add 1800 µl synthetic urine into a preparation tube. Add 200 L of Standard and vortex for 10 seconds. After preparation, calibrator must be aliquoted into 350 µl volumes and stored at -20 C.

Page 7 / 18 Preparation of Zivak Control Level I: Add 1850 µl synthetic urine into a preparation tube. Add 150 L of Standard and vortex for 10 seconds. After preparation, control must be aliquoted into 350 µl volumes and stored at -20 C. 11. TEST PROCEDURE Sample Pre-treatment (Manual) Take 300 L of urine sample (or calibrator) into a preparation tube (centrifuge first if the sample is blurry) Add 250 L of Reagent 1 and vortex for 10 seconds. Add 300 L of Reagent 2 and vortex for 10 seconds. Centrifuge at 5.000 x rpm for 3 minutes. Take 200 L of upper phase into the vials. Take 200 L of upper phase into the vials. Inject 20 L to the LC-MS/MS system. Note: The prepared sample is stable at 2-4 C for 24 hours.

Page 8 / 18 Sample Pre-treatment (Automated): Take 300 L of patient sample (or calibrators and controls) into the Zivak Multitasker Sample Preparation vials Make sure that reagents and wash solution are connected to the reagent pump and purged properly. Place the vials on the MULTITASKER system tray. Note: The prepared sample is stable at 2-8 C for 24 hours. 12. QUALITY CONTROL The test results are only valid if the test has been performed by following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All standards and kit controls must be found within the acceptable ranges as stated on the QC (Quality Control) Certificate. If the criteria are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. In case of any deviation, the following technical issues should be proven: Expiry dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. It is recommended to participate at appropriate quality assessment trials. 13. CALCULATION OF RESULTS The obtained area of the standard is plotted against their concentration. The standard curve is calculated by a linear regression or a weighted linear regression function. Using computer programs, the curve is best described by a 1-point linear regression fit with linear axes. For the calculation of the regression curve, apply each signal of the standards. (One obvious outlier of duplicates might be omitted and the more plausible single value might be used) The concentration of the samples can be read directly from the regression function. Sample signals above the highest control have to be confirmed by a reference method. *IS: Internal Standard

Page 9 / 18 14. INTERPRETATION OF RESULTS Various societies recommend different Cut-Off values for repetition of the measurement and the application of confirmatory assays. Depending on the application of patient samples from different populations, it is highly recommended that each laboratory establishes its own range of normal values and that this distribution of values is coordinated with the recommendations of the responsible society of this geographic region. The results themselves should not be the only reason for any therapeutic consequences. They have to be correlated to other clinical observations and diagnostic tests. 15. EXPECTED VALUES Psychoactive drug concentration <50 ng/ml It is recommended that each laboratory establishes its own range of normal values.

Page 10 / 18 16. LC-MS/MS PARAMETERS Device Column Zivak Tandem Gold LC-MS/MS System Zivak Psychoactive Drugs Urine LC-MS/MS Analytical Column Injection Volume 20 µl Pump Program Flow 00:00 min 5 %B 01:00 min 5 %B 03:00 min 95 %B 04:30 min 95 %B 04:31 min 5 %B 07:00 min 5 %B 0.25 ml/min Ionization Mode CID Gas API Nebulizing Gas Scan Time SIM Width Drying Gas vesi positive 2.4mTorr 55 psi 0,725 sec 1.0 amu 35 psi Drying Gas Temp. 350 C Vortex Gas 25psi Vortex Gas Temp. 250 C Needle Shield Capillary Detector 5000V 600V 50V 1600 V

Page 11 / 18 17. MS SCAN PARAMETERS MH+ MS/ Capillar CE IS to Use No Analyte (m/z) MS y (ev) (ev) 1 Amphetamine-1 136 91 50 16 Amphetamine D6 2 Amphetamine-2 136 119 50 8 Amphetamine D6 3 Methamphetamine-1 150 91 50 18 Methamphetamine D5 4 Methamphetamine-2 150 119 50 10 Methamphetamine D5 5 MDA-1 180 135 50 15 MDA D5 6 MDA-2 180 163 50 8 MDA D5 7 MDMA-1 194 135 50 18 MDMA D5 8 MDMA-2 194 163 50 10 MDMA D5 9 MDEA-1 208 135 50 20 MDEA D6 10 MDEA-2 208 163 50 12 MDEA D6 11 Diazepam-1 285 154 50 24 Diazepam D5 12 Diazepam-2 285 194 50 28 Diazepam D5 13 Morphine-1 286 165 50 36 Morphine D6 14 Morphine-2 286 201 50 24 Morphine D6 15 Codeine-1 300 165 50 38 Codeine D6 16 Codeine-2 300 215 50 24 Codeine D6 17 Alprazolam-1 309 205 50 40 Alprazolam D5 18 Alprazolam-2 309 281 50 24 Alprazolam D5 19 Cocaine-1 304 150 50 22 Cocaine D3 20 Cocaine-2 304 182 50 16 Cocaine D3 21 Flunitrazepam-1 314 239 50 32 Flunitrazepam D7 22 Flunitrazepam-2 314 268 50 24 Flunitrazepam D7 23 Delta-9-THC-1 315 123 50 30 Delta-9-THC D3 24 Delta-9-THC-2 315 193 50 20 Delta-9-THC D3 25 Clonazepam-1 316 214 50 34 Clonazepam D4 26 Clonazepam-2 316 270 50 24 Clonazepam D4 27 6-Acetylmorphine-1 328 165 50 34 6-Acetylmorphine D6 28 6-Acetylmorphine-2 328 211 50 24 6-Acetylmorphine D6 29 Heroin-1 370 165 50 40 Heroin D9 30 Heroin-2 370 268 50 26 Heroin D9 MH+ (m/z) MS/MS Capillary (ev) CE No Analyte (ev) 31 Amphetamine D6 142 125 50 8 32 Methamphetamine D5 155 121 50 10 33 MDA D5 185 138 50 16 34 MDMA D5 199 135 50 18

Page 12 / 18 35 MDEA D6 214 166 50 12 36 Diazepam D5 290 198 50 28 37 Morphine D6 292 165 50 36 38 Codeine D6 306 218 50 24 39 Cocaine D3 307 185 50 16 40 Alprazolam D5 314 210 50 38 41 Delta-9-THC D3 318 123 50 30 42 Clonazepam D4 320 274 50 24 43 Flunitrazepam D7 321 275 50 24 44 6-Acetylmorphine D6 334 165 50 34 45 Heroin D9 379 272 50 26 18. ANALYTICAL PERFORMANCE No Analyte Linearity Recovery (R 2 ; 0-100 (%) µg/l) Limit of Detection (µg/l) Limit of Quantitation (µg/l) Intra-Assay Precision (%CV) Inter-Assay Precision (%CV) 1 Amphetamine 0.9994 99.24 0.27 0.90 2.6 2.9 2 Methamphetamine 0.9998 100.88 0.31 1.02 4.8 5.3 3 MDA 0.9976 95.32 0.08 0.26 2.6 2.9 4 MDMA 0.9991 98.54 0.12 0.39 2.9 3.3 5 MDEA 0.9998 98.74 0.07 0.23 3.4 3.7 6 Diazepam 0.9999 95.86 0.10 0.33 2.4 2.6 7 Morphine 0.9999 100.48 0.08 0.26 6.7 6.9 8 Codeine 0.9969 92.10 0.31 1.02 4.4 4.7 9 Cocaine 0.9989 96.84 0.09 0.30 2.8 3.1 10 Alprazolam 0.9996 96.96 0.09 0.30 2.8 3.2 11 Delta-9-THC 0.9998 98.82 0.50 1.65 4.1 4.5 12 Clonazepam 0.9998 97.42 0.16 0.53 2.9 3.3 13 Flunitrazepam 0.9996 98.76 0.13 0.43 4.4 4.7 14 6-acetylmorphine 0.9986 99.12 0.08 0.26 2.6 2.8 15 Heroin 0.9987 98.10 0.12 0.40 6.3 6.7 Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical substances tested.

Page 13 / 18 19. SAMPLE CHROMATOGRAM

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20. AUTOMATED SAMPLE PREPARATION METHOD PARAMETERS FOR ZIVAK MULTITASKER Page 18 / 18 Reagent Adding (R1) Reagent Adding (R2) Centrifuge Injection Reagent Tray Reagent Tray Analysis Tray Analysis Tray 1 1 1 1 From 0 0 0 1,0 mm 270 µl 320 µl - 20 µl No Detection No Detection - Use Detection Analysis Tray Analysis Tray - - To 1 1 - - 15 18 - - 250 µl 300 µl - - Wash Station A Wash Station A - Wash Station A Washing Reagent Syringe Reagent Syringe - Reagent Syringe - 1000 x 1 - I4 / O1 8000 x 1 8000 x 1 - Injection Valve I4 / O1 I4 / O1 - Reagent Syringe - 500 x 1 - I4 / O1 Reagent Syringe Reagent Syringe - Injection Syringe Syringes 13-1000 / I3 13-1000 / I2-22 - 1000 14-1000 / O1 14-1000 / O1-24 - 1000 Waste Before Air Segment Vortex- Centrifuge Parameters 10000 µl 10000 µl - - Wash Station A Wash Station A - - - - - 20 µl - - - At First - - 3 minute - - - 5000 RPM - - - 1 minute Vortex - - - - - Purge Before Run Syringe Pump Wash Place Flush Volume Repeat Time Reagent Syringe Wash Station A 10000 - I6 / O1 1 Batch 100