Catecholamines Urine LC-MS/MS Analysis Kit User Manual
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1 Page 1 / 13 Catecholamines Urine LC-MS/MS Analysis Kit User Manual ZV
2 Page 2 / 13 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... 13
3 Page 3 / INTENDED USE Quantitative LC-MS/MS analysis kit for Epinephrine, Norepinephrine and Dopamine in urine. 2. SUMMARY AND EXPLANATION Catecholamines are hormones produced by the adrenal glands, which are found on top of the kidneys. They are released into the blood during times of physical or emotional stress. The major catecholamines are dopamine, norepinephrine, and epinephrine. The measurements of catecholamines are important for determination of pheochromocytomas in symptomatic patients like patients with persistent hypertension. It is also used in order to help monitor for recurrence when a pheochromocytoma has been discovered and removed. Zivak Catecholamines LC- MS/MS Analysis Kit was developed for accurate analysis of Epinephrine, Norepinephrine and Dopamine in urine samples. Analysis gives results in 16 minutes. 3. TEST PRINCIPLE Catecholamines are extracted from human urine samples by using an acidic extraction and deproteinization steps. After the extraction, these analytes derivatized via reagents. Then derivatizated catecholamines analyzed by LC-coupled tandem mass spectrometry. 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.
4 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 This analysis kit can be shipped at room temperature. Standards and controls, Reagent 1, 2, 3 and 4 should be stored at 2-8 C. Standards and controls (after reconstitution) should be stored at - 20 C. All other components of the kit should be stored at between room temperature. Reagent 2, 3 and 4 are volatile, please make sure that the lids of reagent bottles are closed tightly after use. All components are guaranteed until expiry date when stored at recommended temperatures and used as described in these instructions. 6. MATERIALS SUPPLIED ZV Volume Symbol Component ZV R1-20 1x 30ml R1 Reagent 1, Contains deuterated internal standard ZV R2-20 1x 50ml R2 Reagent 2, Contains base ZV R3-20 1x 50ml R3 Reagent 3, Contains base and derivatization reagent ZV R4-20 1x 50ml R4 Reagent 4, Contains organic extraction solvent ZV MA-20 2x 0,8 L MA Mobile Phase A, Contains organic solvent ZV MB-20 1x 0,6 L MB Mobile Phase B, Contains organic solvent ZV WB-20 1x 1 L WB Washing Solution ZV-3031-KK-20 1x 1pc User Guide
5 Page 5 / MATERIALS REQUIRED BUT NOT SUPPLIED Materials listed in the table below are required and should be ordered separately. ZV Volume Symbol Component ZV S1-20 1x 8ml CALIBRATOR LEVEL 1 Lyophilized Urine Calibrator Level 1 ZV K1-20 1x 8ml CONTROL LEVEL 1 Lyophilized Urine Control Level 1 CONTROL ZV K2-20 1x 8ml LEVEL 2 Lyophilized Urine Control Level 2 ZV C x 1 pcs Zivak Catecholamines Urine LC-MS/MS Analytical column ZS-2ML x100pcs 2 ml glass autosampler vial ZS x100pcs 2 ml glass autosampler vial caps preslit µl pipette Pipette tips Vortex mixer 2.0 ml glass sample preparation tube (for Manual Sample Preparation) Centrifuge Bidistilled or deionized water 0,5 ml conical autosampler vial or vial insert (for Manual Sample Preparation) 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.
6 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 have a significant effect on the test results. Urine samples must be kept frozen or acidified with appropriate acid solution before the analysis. (6N HCl, %50 Acetic acid, Na2CO3 (crystals), Boric acid (crystals)). If the urine sample is not clear, it has to 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 Calibrators: Add 8 ml of 0,2M HCl to lyophilised urine calibrator and dissolve. After reconstitution, Urine calibrator must be aliquoted into 120 µl volumes and stored at - 20 C. Preparation of Control Level 1: Add 8 ml of 0,2M HCl to urine control level I and dissolve. After preparation, control level I must be aliquoted into 120 µl volumes and stored at -20 C.
7 Page 7 / 13 Preparation of Control Level 2: Add 8 ml of 0,2M HCl to urine control level II and dissolve. After preparation, control level II must be aliquoted into 120 µl volumes and stored at -20 C. 11. TEST PROCEDURE Sample Pre-treatment (Automated): Take 100 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. Sample Pre-treatment (Manual): Take 100 L of patient sample (or calibrators and controls) into the sample preparation tube. Add 100 L of Reagent 1 and vortex for 10 seconds. Add 200 L of Reagent 2 and vortex for 10 seconds. Add 200 L of Reagent 3 and vortex for 10 seconds. Wait for 3 minutes and vortex again for 10 seconds. Wait for 1 minute. Add 200 L of Reagent 4 and vortex for 10 seconds. After mixing wait for a minute. Centrifuge at x rpm for 2 minutes. Take 200 L the upper phase into the autosampler vial.
8 Page 8 / 13 Inject 10 L to the LC-MS/MS system. Note: The prepared sample is stable at 2-8 C for 24 hours. Vortex mixing time should be equal for all samples. 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 2-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 standard have to be confirmed by a reference method. IS: Internal Standard
9 Page 9 / 13 Calculation for 24 hours urine: Concentration of the Sample (ug/l) x Volume of 24h Urine (L) = ug analyte / 24 hours Calculation for spot urine: [ (Concentration of the Sample (ug/l) ] [ (Concentration of Creatinine g/l) ] = ug analyte creatinine g *IS: Internal Standard 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 EPINEPHRINE <1 year: <2.6 mcg/24 hours 1 year: <3.6 mcg/24 hours 2-3 years: <6.1 mcg/24 hours 4-9 years: mcg/24 hours years: mcg/24 hours > or =16 years: <21 mcg/24 hours
10 Page 10 / 13 NOREPINEPHRINE <1 year: <11 mcg/24 hours 1 year: 1-17 mcg/24 hours 2-3 years: 4-29 mcg/24 hours 4-6 years: 8-45 mcg/24 hours 7-9 years: mcg/24 hours > or =10 years: mcg/24 hours DOPAMINE <1 year: <86 mcg/24 hours 1 year: mcg/24 hours 2-3 years: mcg/24 hours > or =4 years: mcg/24 hours It is recommended that each laboratory establishes its own range of normal values. 16. LC-MS/MS PARAMETERS Device Column LC-MS/MS Analysis System Zivak Catecholamines Urine LC-MS/MS Analytical column Injection volume 10 µl Gradient min 100 % A 08:30 min 10 % A 11:00 min 10 % A 11:01 min 100 % A 16:00 min 100 % A
11 Page 11 / 13 Flow 0,35 ml/min Ionization Mode: ESI positive CID Gas 2,4 mtorr API Nebulizing Gas 50 psi Scan Time 1,0 sec SIM Width 1,5 amu Drying Gas 35 psi Drying Gas Temp. 350 C Needle 6000V Shield 600V Capillary 20V Detector 1700 V 17. MS SCAN PARAMETERS RT Capillary CE Analyte MH+ (m/z) MS/MS No (min) (ev) (ev) 1 Adrenaline 7,1 (+)424,2 166,0 20V 24V 2 Adrenaline-IS 7,1 (+)427,2 169,0 20V 24V 3 Noradrenaline 6,3 (+)410,2 238,0 20V 12V 4 Noradrenaline -IS 6,3 (+)416,2 244,0 20V 12V 5 Dopamine 7,2 (+)429,3 412,2 20V 8V 6 Dopamine -IS 7,2 (+)433,2 416,2 20V 8V 18. ANALYTICAL PERFORMANCE No Analyte LOD (µg/l) LOQ (µg/l) Accuracy (%) Precision (%CV) 1 Adrenaline Noradrenaline Dopamine Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical substances tested.
12 Page 12 / SAMPLE CHROMATOGRAM
13 20. AUTOMATED SAMPLE PREPARATION METHOD PARAMETERS FOR ZIVAK MULTITASKER From 1 - Reagent addition (Parallel) Reagent tray 1;2;3 (Vial 0 mm 2 - Reagent addition (Parallel) Reagent tray 4;5;6 (Vial 0 mm 3- Reagent addition (Parallel) Reagent tray 4;5;6 (Vial 0 mm 4- Reagent addition (Parallel) Reagent tray 4;5;6 (Vial 0 mm 3 - Vortex 4 - Injection (Serial) Analysis tray Analysis tray 1 (Vial 1 (Vial 0 mm 110 µl 220 µl 220 µl 220 µl 10 µl 1.5 mm Page 13 / 13 To No liquid detection Analysis tray 1 (Vial 16 mm No liquid detection Analysis tray 1 (Vial 16 mm No liquid detection Analysis tray 1 (Vial 16 mm No liquid detection Analysis tray 1 (Vial 16 mm 100 µl 200 µl 200 µl 200 µl Liquid detection - active Washing Wash station A Wash station A Wash station A Wash station A Pump 2 Pump 2 Pump 2 Pump 2 Wash station A Loop Pump µl 1000 µl 1000 µl 1000 µl 1000 µl 250 µl Pump 5 repeat 2 repeat 2 repeat 2 repeat 1 repeat 1 repeat Pump 2 Pump 2 Pump 2 Pump 2 Pump 1 Syringes Air segment Vortex- Centrifuge Parameters ms (speedwait) ms (speedwait) ms (speed-wait) ms (speed-wait) ms (speed-wait) ms (speed-wait) ms (speed-wait) ms (speed-wait) First First First First First 100 µl 200 µl 200 µl 200 µl 10 µl 2 - Reagent addition (Parallel) Reagent tray 4;5;6 (Vial 0 mm (speed - wait) (speedwait)
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