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

Transcription

1 Page 1 / 18 Psychoactive Drugs Urine LC-MS/MS Analysis Kit User Manual ZV C

2 Page 2 / 18 Table of Contents 1. INTENDED USE SUMMARY AND EXPLANATION TEST PRINCIPLE WARNING AND PRECAUTIONS STORAGE AND STABILITY MATERIALS SUPPLIED MATERIALS REQUIRED BUT NOT SUPPLIED PROCEDURE NOTES LIMITATIONS OF THE PROCEDURE PRE-TEST SET-UP INSTRUCTIONS TEST PROCEDURE QUALITY CONTROL CALCULATION OF RESULTS INTERPRETATION OF RESULTS EXPECTED VALUES LC-MS/MS PARAMETERS MS SCAN PARAMETERS ANALYTICAL PERFORMANCE SAMPLE CHROMATOGRAM AUTOMATED SAMPLE PREPARATION METHOD PARAMETERS FOR ZIVAK MULTITASKER... 18

3 Page 3 / 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 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.

4 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 / 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 R x 37 ml R1 Reagent 1, Contains base ZV R2-20 2x 45 ml R2 Reagent 2, Contains internal standards ZV MA-20 1x 0,5 L MA Mobile Phase A, Contains organic solvent and acid ZV MB-20 1x 0,5 L MB Mobile Phase B, Contains organic solvent and acid

5 Page 5 / 18 ZV WB-10 2x 1 L WB Washing solution, Contains organic solvent SU ZV SU-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 S x 1 ml S1 Standard 1, in organic solvent ZV C pcs Zivak Psychoactive Drugs Urine LC-MSMS Analytical Column µl pipette 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.

6 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 / 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.

7 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 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.

8 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

9 Page 9 / 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.

10 Page 10 / 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

11 Page 11 / MS SCAN PARAMETERS MH+ MS/ Capillar CE IS to Use No Analyte (m/z) MS y (ev) (ev) 1 Amphetamine Amphetamine D6 2 Amphetamine Amphetamine D6 3 Methamphetamine Methamphetamine D5 4 Methamphetamine Methamphetamine D5 5 MDA MDA D5 6 MDA MDA D5 7 MDMA MDMA D5 8 MDMA MDMA D5 9 MDEA MDEA D6 10 MDEA MDEA D6 11 Diazepam Diazepam D5 12 Diazepam Diazepam D5 13 Morphine Morphine D6 14 Morphine Morphine D6 15 Codeine Codeine D6 16 Codeine Codeine D6 17 Alprazolam Alprazolam D5 18 Alprazolam Alprazolam D5 19 Cocaine Cocaine D3 20 Cocaine Cocaine D3 21 Flunitrazepam Flunitrazepam D7 22 Flunitrazepam Flunitrazepam D7 23 Delta-9-THC Delta-9-THC D3 24 Delta-9-THC Delta-9-THC D3 25 Clonazepam Clonazepam D4 26 Clonazepam Clonazepam D Acetylmorphine Acetylmorphine D Acetylmorphine Acetylmorphine D6 29 Heroin Heroin D9 30 Heroin Heroin D9 MH+ (m/z) MS/MS Capillary (ev) CE No Analyte (ev) 31 Amphetamine D Methamphetamine D MDA D MDMA D

12 Page 12 / MDEA D Diazepam D Morphine D Codeine D Cocaine D Alprazolam D Delta-9-THC D Clonazepam D Flunitrazepam D Acetylmorphine D Heroin D ANALYTICAL PERFORMANCE No Analyte Linearity Recovery (R 2 ; (%) µg/l) Limit of Detection (µg/l) Limit of Quantitation (µg/l) Intra-Assay Precision (%CV) Inter-Assay Precision (%CV) 1 Amphetamine Methamphetamine MDA MDMA MDEA Diazepam Morphine Codeine Cocaine Alprazolam Delta-9-THC Clonazepam Flunitrazepam acetylmorphine Heroin Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical substances tested.

13 Page 13 / SAMPLE CHROMATOGRAM

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18 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 From ,0 mm 270 µl 320 µl - 20 µl No Detection No Detection - Use Detection Analysis Tray Analysis Tray - - To µl 300 µl - - Wash Station A Wash Station A - Wash Station A Washing Reagent Syringe Reagent Syringe - Reagent Syringe x 1 - I4 / O x x 1 - Injection Valve I4 / O1 I4 / O1 - Reagent Syringe x 1 - I4 / O1 Reagent Syringe Reagent Syringe - Injection Syringe Syringes / I / I / O / O Waste Before Air Segment Vortex- Centrifuge Parameters µl µl - - Wash Station A Wash Station A µl At First minute RPM minute Vortex Purge Before Run Syringe Pump Wash Place Flush Volume Repeat Time Reagent Syringe Wash Station A I6 / O1 1 Batch 100