Amino Acid Biological Fluids LC-MS/MS Analysis Kit (Plasma & Serum & Urine & Cerebrospinal Fluid) ZV-3002-0200-10 200 2-8 °C User Manual D. No: ZV-3002-KK-10_Rev13 Table Of Contents Page 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: ..................................................................................................................................................... 5 7. MATERIALS REQUIRED BUT NOT SUPPLIED: ......................................................................................................... 6 8. PROCEDURE NOTES: .......................................................................................................................................................... 7 9. LIMITATIONS OF THE PROCEDURE: ........................................................................................................................... 7 10. PRE-TEST SET-UP INSTRUCTIONS: ............................................................................................................................. 7 11. PRE-TEST SET-UP INSTRUCTIONS: ............................................................................................................................. 9 Sample Pre-treatment (Automated): .................................................................................................................. 9 Sample Pre-treatment (Manual): ......................................................................................................................... 9 Sample Pre-treatment (Automated) for Homocysteine, Cysteine ....................................................... 10 Sample Pre-treatment (Manual) for Homocysteine, Cysteine .............................................................. 10 12. QUALITY CONTROL: ......................................................................................................................................................... 11 13. CALCULATION OF RESULTS:......................................................................................................................................... 11 14. INTERPRETATION OF RESULTS: ................................................................................................................................ 12 15. EXPECTED VALUES: .......................................................................................................................................................... 13 16. LC-MS/MS PARAMETERS: .............................................................................................................................................. 14 17. MS SCAN PARAMETERS: ................................................................................................................................................. 15 18. ANALYTICAL PERFORMANCE ...................................................................................................................................... 17 19. SAMPLE CHROMATOGRAM: .......................................................................................................................................... 18 20. AUTOMATED SAMPLE PREPARATION METHOD PARAMETERS FOR ZİVAK MULTITASKER .......... 27 D. No: ZV-3002-KK-10_Rev13 2 1. INTENDED USE: Quantitative LC-MS/MS analysis kit for Amino Acids in biological fluids (Plasma & Serum & Urine & Cerebrospinal Fluid). 2. SUMMARY AND EXPLANATION: Amino acids are the basic structural units of the proteins. Quantitative analysis of the amino acids in biological fluids is one of the most important parts of the clinical diagnosis of patients with inborn errors of amino acid metabolism or monitoring the healthy people. Amino acid analysis in urine is important for diagnosis of disorders which are related with renal amino acid transport. Quantitative analysis of amino acids in plasma is preferred over urine for the diagnosis of aminoacidopathies which cause to accumulation or the deficiency of amino acids in biological fluids. Quantitative analysis of the amino acids in biological fluids also has clinical importance for determining some neurological and endocrinal disorders. LC-MS/MS is an influential device for the study of disorders related with amino acids. LC-MS/MS analysis gives results for 47 free amino acids in 20 minutes. Zivak Quantitative Free Amino Acids LC-MS/MS Analysis Kit was developed for quantitative detection of free amino acids in human serum, plasma, urine and cerebrospinal fluid LC-MS/MS is an influential device for the study of metabolic disorders and LC-MS/MS analysis gives results in 20 minutes with minimal sample preparation. Main methods and procedures that have been selected are based on EN ISO 13485 and 98/79/EC. 3. TEST PRINCIPLE: Free amino acids are extracted from human samples by using an acidic extraction and deproteinization steps. After the extraction, these amino acids derivatised via reagents. Then derivatisated amino acids are separated on a reversed phase HPLC column with a gradient elution and analysed by Tandem Mass spectrometry. 4. WARNING AND PRECAUTIONS: • For in-vitro diagnostic use only. • For professional use only. D. No: ZV-3002-KK-10_Rev13 3 • 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 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. • It’s recommended to work in a fume hood for your safety as Reagent 2, 3 and 4 are volatile and please avoid inhaling. 5. STORAGE AND STABILITY: • This analysis kit can be shipped at room temperature. • After arrival, standards and controls should be stored at 2-8 °C. • Reagent 1, 2 ,3 ,4 should be stored at 2-8 °C. • All other components of the kit should be stored at between room temp. • 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. D. No: ZV-3002-KK-10_Rev13 4 6. MATERIALS SUPPLIED: Order No. Volume Symbol Component Reagent 1, R1 ZV-3002-02R1-10 1 x 30 ml Contains deuterated internal standards R2 Reagent 2, ZV-3002-02R2-10 1 x 50 ml Contains base R3 Reagent 3, ZV-3002-02R3-10 1x 50 ml Contains derivatisation reagent R4 Reagent 4, ZV-3002-02R4-10 1 x 50 ml Contains organic modifier RH Reagent H, ZV-3002-02RH-10 1 x 30 ml Protective Reagent for HCYS MA Mobile Phase A, ZV-3002-02MA-10 1 x 1 L Contains organic solvent MB Mobile Phase B, ZV-3002-02MB-10 1 x 1 L Contains organic solvent WB Washing solution, ZV-3002-02WB-10 1 x 1 L Contains organic solvent ZS-2ML-8-1001 2x100pcs 2 ml glass autosampler vial for automated Sample Preparation ZS-9004-0100-00 2x100pcs 2 ml glass autosampler vial caps pre- slit for automated Sample Preparation KK ZV-3002-KK-10 1x1 pc User Guide D. No: ZV-3002-KK-10_Rev13 5 7. MATERIALS REQUIRED BUT NOT SUPPLIED: Calibrator Level 1 ZV-3002-02S1-10 1 x 3 ml Lyophilized Plasma Calibrator Level-1 Calibrator ZV-3002-02S2-10 1 x 3 ml Level 2 Lyophilized Plasma Calibrator Level-2 Calibrator ZV-3002-02S3-10 1 x 3 ml Level 3 Lyophilized Plasma Calibrator Level-3 Control Level 1 ZV-3002-02K1-10 1 x 3 ml Lyophilized Plasma Control Level 1 Control Level 2 ZV-3002-02K2-10 1 x 3 ml Lyophilized Plasma Control Level 2 Control Level 3 ZV-3002-02K2-10 1 x 3 ml Lyophilized Plasma Control Level 3 Calibrator SGLevel 1 Sulfur Group Lyophilized Plasma ZV-3002-02SGS1-10 1 x 3 ml Calibrator Level 1 Calibrator ZV-3002-02SGS2-10 1 x 3 ml SGLevel 2 Sulfur Group Lyophilized Plasma Calibrator Level 2 Control ZV-3002-02SGK1-10 1 x 3 ml SGLevel 1 Sulfur Group Lyophilized Plasma Control Level 1 • Zivak Amino Acid Biological Fluids LC-MS/MS Analytical Column • 20-200 µL pipette • Pipette tips • 2.0 mL glass sample preparation tube (for Manual Sample Preparation) • Vortex mixer • Centrifuge • Bidistilled or deionised water • 0.5 ml conical autosampler vial or vial insert (for Manual Sample Preparation) D. No: ZV-3002-KK-10_Rev13 6 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. 9. LIMITATIONS OF THE PROCEDURE: Specimen collection and storage have a significant effect on the test results. Plasma 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. D. No: ZV-3002-KK-10_Rev13 7 Preparation of Zivak Calibrators: Add 3 ml of deionised water to lyophilised plasma calibrators and controls and dissolve. After reconstitution, plasma calibrator must be aliquoted into 150 µl volumes and stored at -20°C. Preparation of Zivak lyophilized Sulfur Group Calibrators & Control Add 3 ml of deionised water to lyophilised plasma calibrators and control and dissolve. After reconstitution, plasma calibrator must be aliquoted into 150 µl volumes and stored at -20°C. Note: Please use zivak lyophilized sulfur group calibrators, control for Homocysteine, cysteine, cystine, homocystine. Zivak lyophilized sulfur group calibrators contain homocysteine, cysteine, cystine, homocystine. Use zivak lyophilized plasma calibrators, controls for other 43 aminoacids. Reagent-H for only homocysteine, cysteine. Don’t use Reagent-H if analysis cystine, homocystine and other 43 aminoacids. Reagent-H is protective reagent for Cysteine, Homocysteine. Please make sure it is completely resolution before using, if there are particules please resolution. D. No: ZV-3002-KK-10_Rev13 8 11. PRE-TEST SET-UP INSTRUCTIONS: 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 2 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 5.000 x rpm for 3 minutes. Take 100 L the upper phase into the autosampler vial. Inject 3 L to the LC-MS/MS system. Note: The prepared sample is stable at 2-8 °C for 24 hours. D. No: ZV-3002-KK-10_Rev13 9 Sample Pre-treatment (Automated) for Homocysteine, Cysteine 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. (Please use Aminoacids HCYS automated method) Note: The prepared sample is stable at 2-8°C for 24 hours. Sample Pre-treatment (Manual) for Homocysteine, Cysteine Take 100 L of patient sample (or calibrators and controls) into the sample preparation tube. Add 100 L of Reagent H and vortex for 10 seconds. Wait for 1 minute and vortex again for 10 seconds. 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 2 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 5.000 x rpm for 3 minutes. Take 100 L the upper phase into the autosampler vial. Inject 3 L to the LC-MS/MS system. Note: The prepared sample is stable at 2-8 °C for 24 hours. D. No: ZV-3002-KK-10_Rev13 10