PKU (Phenylketonuria) Serum LC-MS/MS Analysis Kit User Manual
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1 Page 1 / 24 PKU (Phenylketonuria) Serum LC-MS/MS Analysis Kit User Manual ZV C
2 Page 2 / 24 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... 11
3 Page 3 / INTENDED USE LC-MS/MS analysis kit for quantitative determination of phenylketonuria in human serum samples. 2. SUMMARY AND EXPLANATION Phenylketonuria (PKU) is an autosomal recessive genetic disorder characterized by a deficiency in the enzyme phenylalanine hydroxylase (PAH). This enzyme is necessary to metabolize the amino acid phenylalanine to the amino acid tyrosine. When PAH is deficient, phenylalanine accumulates on the brain tissue and is converted into phenyl pyruvate (also known as phenyl ketone), which is detected in the urine. PAH is found on chromosome number 12. If left untreated, this condition can cause problems with brain development, leading to progressive mental retardation and seizures. However, PKU can be controlled by a special diet with low in phenylalanine and high in tyrosine can be a very effective treatment. There is no cure. The disorder is only detectable after irreversible damage is done hence early diagnosis is crucial. Most babies in developed countries are screened for PKU soon after birth. PKU is usually detected using the HPLC test, but some clinics still use the Guthrie test, as a part of national biochemical screening programs. ZIVAK PKU LC-MS/MS kit was developed for quantitative detection of Phenylketonuria in human serum, serum and dried blood samples. LC-MS/MS is an effective tool for the study of metabolic disorders (e.g. PKU). LC-MS/MS analysis gives results in 2 minutes with minimal sample preparation. Main methods and procedures that have been selected are based on EN ISO TEST PRINCIPLE Phenylalanine, tyrosine and tryptophan are eluted from human samples using an acidic extraction solution. After the extraction, these amino acids are derivatized via reagents. Then derivatized amino acids are separated on a reversed phase HPLC column with an isocratic elution and analyzed by 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.
4 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. It s recommended to work in a fume hood for your safety as Reagent 2, 3 and 4 are volatile and please avoid inhaling. Page 4 / STORAGE AND STABILITY This analysis kit can be shipped at room temperature. After arrival, calibrator and controls, Reagent 1, 2, 3, and 4 can be stored at 2-8 C. MP, WB can be stored at room temp. Reagent 2, 3 and 4 are volatile, please make sure that the lids of reagents 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 Order No. Volume Symbol Component ZV R x 20 ml R1 Reagent 1, Contains deuterated internal standards ZV R x 40 ml R2 Reagent 2, Contains acid ZV R x 5 ml R3 Reagent 3, Contains derivatisation reagent ZV R x 40 ml R4 Reagent 4, Contains dilution reagent ZV MP-10 1 x 0,5 L MP Mobile Phase, Contains organic solvent
5 Page 5 / 24 ZV WB-10 1 x 0,5 L WB Washing solution, Contains organic solvent KK ZV-3003-KK 10 1 x 1 pc User Guide 7. MATERIALS REQUIRED BUT NOT SUPPLIED Order No. Volume Symbol Component ZV S x 3 ml Calibrator Level 1 Lyophilized Serum Calibrator Level 1 ZV K x 3 ml Control Level 1 Lyophilized Serum Control Level 1 ZV K x 3 ml Control Level 2 Lyophilized Serum Control Level 2 ZV C pcs Zivak PKU Serum LC-MS/MS Analytical Column µl pipette µl pipette Pipette tips Vortex mixer Capped preparation tubes Centrifuge Bidistilled or deionised water 1.8 ml conical autosampler vial or vial insert
6 Page 6 / 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 has a significant effect on the test results. Any result with an elevated concentration has to be indicated as presumptive positive and has to be confirmed with further sampling and testing. A false negative result of this assay cannot be excluded with absolute certainty. Any anamnestic or clinical hint of Phenylketonuria has to lead to a repeated measurement. A direct influence of applied drugs to the patients on the phenylalanine results cannot be excluded. Therefore, it is highly recommended to review in these cases even a normal phenylalanine value with a confirmation test if there are anamnestic or clinical signs of a phenylketonuria in a patient. 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.
7 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 Calibrators: Page 7 / 24 Add 3 ml of deionised water to serum calibrator and dissolve. After preparation, calibrator must be aliquoted into 75 µl volumes and stored at -20 C. Preparation of Zivak Control Level I: Add 3 ml of deionised water to serum control level I and dissolve. After preparation, control level I must be aliquoted into 75 µl volumes and stored at -20 C. Preparation of Zivak Control Level II: Add 3 ml of deionised water to serum control level II and dissolve. After preparation, control level II must be aliquoted into 75 µl volumes and stored at -20 C. 11. TEST PROCEDURE Sample Pre-treatment (Manual) Take 50 L of serum calibrator into a 2 ml capped preparation. Add 100 L of R1. Mix by vortex for 10 seconds. Add 200 L of R2. Mix by vortex for 10 seconds. Add 25 µl of R3. Mix by vortex for 10 seconds. Wait for 1 minute. Mix by vortex for 10 seconds. Add 800 µl of R4. Mix by vortex for 10 seconds.
8 Page 8 / 24 Centrifuge at x rpm for 5 minutes. Take 500 µl upper liquid into autosampler vial. Inject 5 L to the LC-MS/MS system. Note: The prepared sample is stable at 2-4 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)
9 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. Page 9 / 24 *IS: Internal Standard 14. INTERPRETATION OF RESULTS Various societies for neonatal screening recommend different Cut-Off values for repetition of the measurement and the application of confirmatory assays. Depending on the application of samples of different populations of newborns 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 Phe/Tyr molar ratio must be 2.5 to confirm the diagnosis of hyperphenylalaninemia. It is recommended that each laboratory establishes its own range of normal values. 16. LC-MS/MS PARAMETERS Device Column Zivak Tandem Gold LC-MS/MS System Zivak PKU Serum LC-MS/MS Analytical Column Injection Volume 5 µl Pump Program Flow 0:00 min 100 % B 2:00 min 100 % B 0.30 ml/min Ionization Mode CID Gas ESI positive 2.4 mtorr
10 Page 10 / 24 API Nebulizing Gas Scan Time SIM Width Drying Gas 55 psi sec 1.2 amu 35 psi Drying Gas Temp. 350 C Needle Shield Capillary Detector 5000V 600V 50V 1700 V 17. MS SCAN PARAMETERS No Analyte RT (min) MH+ (m/z) MS/ MS Capillary (ev) 1 Phenylalanine (PHE) V 15V 2 Phenylalanine-IS (PHE-IS) V 15V 3 Tryptophan (TRP) V 15V 4 Tryptophan-IS (TRP-IS) V 15V 5 Tyrosine (TYR) V 15V CE (ev) 6 Tyrosine-IS (TYR-IS) V 15V 18. ANALYTICAL PERFORMANCE No Analyte Intra-Assay LOD LOQ Accuracy Precision (µmol/l) (µmol/l) (%) (%CV) Inter-Assay Precision (%CV) Linearity (R 2 ) 1 Phenylalanine (PHE) Tryptophan (TRP) Tyrosine (TYR) Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical substances tested.
11 Page 11 / SAMPLE CHROMATOGRAM
12 Page 12 / 24 PKU (Phenylketonuria) Dried Blood Spots LC-MS/MS Analysis Kit User Manual ZV C
13 Page 13 / 24 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... 23
14 Page 14 / INTENDED USE LC-MS/MS analysis kit for quantitative determination of phenylketonuria in human dried blood spot samples. 2. SUMMARY AND EXPLANATION Phenylketonuria (PKU) is an autosomal recessive genetic disorder characterized by a deficiency in the enzyme phenylalanine hydroxylase (PAH). This enzyme is necessary to metabolize the amino acid phenylalanine to the amino acid tyrosine. When PAH is deficient, phenylalanine accumulates on the brain tissue and is converted into phenyl pyruvate (also known as phenyl ketone), which is detected in the urine. PAH is found on chromosome number 12. If left untreated, this condition can cause problems with brain development, leading to progressive mental retardation and seizures. However, PKU can be controlled by a special diet with low in phenylalanine and high in tyrosine can be a very effective treatment. There is no cure. The disorder is only detectable after irreversible damage is done hence early diagnosis is crucial. Most babies in developed countries are screened for PKU soon after birth. PKU is usually detected using the HPLC test, but some clinics still use the Guthrie test, as a part of national biochemical screening programs. ZIVAK PKU LC-MS/MS kit was developed for quantitative detection of Phenylketonuria in human serum, serum and dried blood samples. LC-MS/MS is an effective tool for the study of metabolic disorders (e.g. PKU). LC-MS/MS analysis gives results in 2 minutes with minimal sample preparation. Main methods and procedures that have been selected are based on EN ISO TEST PRINCIPLE Phenylalanine, tyrosine and tryptophan are eluted from human samples using an acidic extraction solution. After the extraction, these amino acids are derivatized via reagents. Then derivatized amino acids are separated on a reversed phase HPLC column with an isocratic elution and analyzed by Tandem Mass spectrometry. 4. WARNING AND PRECAUTIONS For in-vitro diagnostic use only. For professional use only. Read the instructions carefully, before you start.
15 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. All calibrators and controls of this kit which contain human blood or dried blood was tested and found negative for HIV 1/2 and HCV antibodies, HbsAg, HIV 1/2 and HCV genome. Nevertheless, the blood controls should be considered as potentially infectious and treated with appropriate care. Page 15 / STORAGE AND STABILITY This analysis kit can be shipped at room temperature. After arrival, calibrator and controls, Reagent 1, 2, 3, and 4 can be stored at 2-8 C. MP, WB can be stored at room temp. Reagent 2, 3 and 4 are volatile, please make sure that the lids of reagents 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 Order No. Volume Symbol Component ZV R x 20 ml R1 Reagent 1, Contains deuterated internal standards ZV R x 40 ml R2 Reagent 2, Contains acid ZV R x 5 ml R3 Reagent 3, Contains derivatisation reagent ZV R x 40 ml R4 Reagent 4, Contains dilution reagent
16 Page 16 / 24 ZV MP-15 1 x 0,5 L MP Mobile Phase, Contains organic solvent ZV WB-15 1 x 0,5 L WB Washing solution, Contains organic solvent KK ZV-3003-KK 10 1 x 1 pc User Guide 7. MATERIALS REQUIRED BUT NOT SUPPLIED Order No. Volume Symbol Component ZV S x 1 spot Calibrator Level 1 Dried Blood Spot Calibrator Level 1 ZV K x 1 spot Control Level 1 Dried Blood Spot Control Level 1 ZV K x 1 spot Control Level 2 Dried Blood Spot Control Level 2 ZV C pcs Zivak PKU Dried Blood Spots LC-MS/MS Analytical Column µl pipette µl pipette Pipette tips Vortex mixer Capped preparation tubes Centrifuge Bidistilled or deionised water 1.8 ml conical autosampler vial or vial insert
17 Page 17 / 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 has a significant effect on the test results. Please always follow the universal safety precautions for accurate and safe DBS sampling given below. Universal Safety Precautions for DBS sampling: Treat all blood samples as though they are infectious Wash hands, wear gloves and apron/lab coat Take precaution to avoid needle injury Dispose of contaminated sharps and waste appropriately Clearly label each card with appropriate identification number. It is unacceptable to submit a blood card for testing that has not been properly labelled. Apply gentle pressure to the heel and allow a large drop of free flowing blood to collect at the puncture site. Working quickly, hold the filter paper by the edges and touch the filter paper gently against the large drop of blood and in one step allow a sufficient quantity of blood to soak through and completely fill or saturate a circle. A completed saturated spot will contain approx.100 µl of blood.
18 Repeat, until you have collected enough blood to fill at least 3 circles on the blood collection card. It is critical that entire circle be uniformly saturated with blood. If collecting spots using a pipette, collect 100 µl of blood and gently apply to filter paper. Page 18 / 24 Any result with an elevated concentration has to be indicated as presumptive positive and has to be confirmed with further sampling and testing. A false negative result of this assay cannot be excluded with absolute certainty. Any anamnestic or clinical hint of Phenylketonuria has to lead to a repeated measurement. A direct influence of applied drugs to the patients on the phenylalanine results cannot be excluded. Therefore, it is highly recommended to review in these cases even a normal phenylalanine value with a confirmation test if there are anamnestic or clinical signs of a phenylketonuria in a patient. 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. 11. TEST PROCEDURE Sample Pre-treatment (Manual) Take dried blood spot sample in 3.5 mm diameter by a puncher into a 2 ml capped preparation tube. Add 200 µl of R1. Mix by vortex for 10 seconds. Mix by vortex for 5 sec. Incubate for 30 minutes at room temperature.
19 Page 19 / 24 Add 200 L of R2. Mix by vortex for 10 seconds. Add 25 µl of R3. Mix by vortex for 10 seconds. Wait for 2 minute. Mix by vortex for 10 seconds. Add 800 µl of R4. Mix by vortex for 10 seconds. Centrifuge at x rpm for 2 minutes. Take 500 µl upper liquid into autosampler vial. Inject 5 L to the LC-MS/MS system. Note: The prepared sample is stable at 2-4 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.
20 Page 20 / 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 14. INTERPRETATION OF RESULTS Various societies for neonatal screening recommend different Cut-Off values for repetition of the measurement and the application of confirmatory assays. Depending on the application of samples of different populations of newborns 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 Phe/Tyr molar ratio must be 2.5 to confirm the diagnosis of hyperphenylalaninemia. It is recommended that each laboratory establishes its own range of normal values.
21 Page 21 / LC-MS/MS PARAMETERS Device Column Zivak Tandem Gold LC-MS/MS System Zivak PKU Dried Blood Spots LC-MS/MS Analytical Column Injection Volume 5 µl Pump Program Flow 0:00 min 100 % B 2:00 min 100 % B 0.30 ml/min Ionization Mode CID Gas API Nebulizing Gas Scan Time SIM Width Drying Gas ESI positive 2.4 mtorr 55 psi sec 1.2 amu 35 psi Drying Gas Temp. 350 C Needle Shield Capillary Detector 5000V 600V 50V 1700 V 17. MS SCAN PARAMETERS No Analyte RT (min) MH+ (m/z) MS/ MS Capillary (ev) 1 Phenylalanine (PHE) V 15V 2 Phenylalanine-IS (PHE-IS) V 15V 3 Tryptophan (TRP) V 15V 4 Tryptophan-IS (TRP-IS) V 15V 5 Tyrosine (TYR) V 15V CE (ev) 6 Tyrosine-IS (TYR-IS) V 15V
22 Page 22 / ANALYTICAL PERFORMANCE No Analyte Intra-Assay LOD LOQ Accuracy Precision (µmol/l) (µmol/l) (%) (%CV) Inter-Assay Precision (%CV) Linearity (R 2 ) 1 Phenylalanine (PHE) Tryptophan (TRP) Tyrosine (TYR) Analytical Specificity (Cross Reactivity): No cross-reactivity was found with the typical substances tested.
23 Page 23 / SAMPLE CHROMATOGRAM
24 Page 24 / 24
PKU (Phenylketonuria) Serum HPLC Analysis Kit User Manual
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