trans,trans-muconic ACID IN URINE BY UV - CODE Z23010

Transcription

1 trans,trans-muconic ACID IN URINE BY UV - CODE Z BIOCHEMISTRY The measuring of trans,trans-muconic Acid is suggested as a biological and reliable marker for monitoring workers exposed to benzene. Benzene is an industrial chemical product which often appears in mineral oils following combustion processes. For this reason there is a high risk of environmental pollution. Its levels in the air can easily increase due to emissions from coal and oil burning, benzene leak and storage operations, exhaust fumes from cars, evaporation from gasoline service stations, and use of industrial solvents. Smoking is another source of benzene in air since tobacco contains high levels of benzene. Acute exposure to benzene can cause drowsiness, dizziness, rapid heart rate, headaches, tremors, confusion, unconsciousness and death. People who breathe benzene for long periods may have different harmful effects. In fact, it is haematoxic and has been identified as a carcinogen, can bring to anaemia, excessive bleeding and it can be harmful to the immune system and the reproductive organs. Regarding cancerogenesis, according to the American Department of Health and Human Services ( DHHS ) benzene is a human carcinogen. Long-term exposure to relatively high levels of benzene in the air can cause leukemia. For all these reasons monitoring and control of workers exposed to benzene is very important Benzene is primarily metabolised by a cythocrome P- ( CYP- ) to phenolic compounds. The range of metabolic conversion of benzene to t,t-ma is about % and dependent on the benzene exposure level. Once oxidized, the metabolites play a substantial role in benzene s toxicity by inducing either DNA alkylation or oxidative stress that lead to the above mentioned systemic disorders. EUREKA srl LAB DIVISION VAT N info@eurekaone.com Head Quarter: Via Enrico Fermi Chiaravalle (AN) ITALY Tel Fax This product fulfills all the requirements of Directive 9/9/EC on in vitro diagnostic medical devices (IVD). The declaration of conformity is available upon request. Rev. N T,t-Muconic Acid in urine by UV December

2 TECHNICAL FEATURES Principle of the Method: trans,trans-muconic Acid is diluted with a suitable diluent and after purification in a clean up column it is injected into the HPLC System. Recovery: > 9% Sensitivity: < µg /l CV% :,% Exposure Biological Index E.B.I. : Acceptable values in urine of not exposed subjects: Metabolic Interferences : t,t-ma µg / l This value is due to the Exposure Average of ppm of Benzene in the air <, mg/g creatinin (not smokers) Ref.: G Ital Med Lav Erg ;: Suppl Sorbic Acid : about, % of Sorbic Acid got with diet is converted in t,t-ma Components of the kit : All the components are ready-to-use and stable years at room temperature ( C ). The Urine Calibrator liophyl. is stable months at - C. Reagent A Dilution Solution, x ml Reagent B Conditioning Solution N, x ml Reagent C Conditioning Solution N, x ml Reagent D Conditioning Solution N, x ml Reagent E Wash Solution N, x ml Reagent F Wash Solution N, x ml Reagent G Eluting Solution, x ml Reagent H Test Solution, x ml Store at - C Reagent I Internal Standard Solution, x ml Store at - C Reagent U Urine Calibrator lyoph., x ml See Warnings Reagent M Mobile Phase, x ml Clean up Columns, pcs Minimum Instrumental equipment required: Isocratic HPLC System with loop of µl Spectrofotometric Detector UV/VIS λ= nm Chromatograms Recorder Optional Equipment: Autosampler Operational Computer Urine Collection Procedure: After work shift, collect ml of urine in a suitable test tube. Delayed analyses require sample freezing at C or less. Stable over weeks.

3 PREANALYTICAL PROCEDURE Test Solution at µg/l Preparation. Dispense in a tube : 9 µl of Water HPLC grade µl of Reagent H Test Solution µl of Reagent I Internal Standard Solution INJECTION : Vortex for seconds Inject µl of this solution in the chromatographic system. Verify that the Test Solution has retention time similar to fig.. If the Test is all right you can start with the analytical procedure; if not, check the functionality of the analytical system. Important : Don t use this solution to calibrate! ANALYTICAL PROCEDURE STEP : Calibrator, Samples and Controls preparation As soon as the urine samples are ready for processing check if the ph is >. Dispense in a tube : Blank Calibrator Sample Water HPLC grade µl Reagent U Urine Calibrator µl Sample µl Reagent A Diluting Solution µl µl µl Reagent I Internal Standard Sol. µl µl µl Vortex for seconds STEP : Prepare Clean up columns per each Sample, Calibrator and Controls. Condition clean up columns by fluxing, ml of Reagent B-Conditioning Sol. and don t let the columns be dry Condition clean up columns by fluxing, ml of Reagent C- Conditioning Sol. and don t let the columns be dry Condition clean up columns by fluxing, ml of Reagent D- Conditioning Sol. and don t let the columns be dry STEP : Sample loading Dispense, ml of sample, previously prepared, into clean up columns Discharge the percolated liquid, if needed a vacuum system could be used for percolation, at speed of ml/min ( drop by drop ). STEP : Washing Wash with ml of Reagent E - Wash Solution discharge the percolated liquid Wash with ml of Reagent F - Wash Solution discharge the percolated liquid Rev. N T,t-Muconic Acid in urine by UV December

4 STEP : Extraction Extract with ml of Reagent G - Eluting Solution Let dry with a light flux of air! Vortex for seconds N.B.: at this step, the sample is stable days at - C INJECTION : Inject µl of the eluate into the chromatographic system. Rev. N T,t-Muconic Acid in urine by UV December

5 REAGENT H : TEST SOLUTION trans,trans-muconic ACID IN URINE Warnings trans,trans-muconic ACID. µg / l REAGENT U : URINE CALIBRATOR LYOPHIL.- Lot N trans,trans-muconic ACID Use and Reconstitution: Urine Calibrators are used for calibration of the HPLC system. This lyophilised calibrator has to be prepared like a patient sample. Add exactly. ml of HPLC water to the vial and mix for min. When all material is dissolved, the solution is ready to use. Stability: months if stored at - C. After reconstitution the stability of the analytes is at least hours when stored at + C, at least days at + C and at least month at - C. Don t use after expiry date. Mix always before use. Packaging: x ml Warning: The calibrator derives from human urine, so it could be potentially infected. It must be handled with care., µg / l PARAMETERS FOR THE SPECTROFOTOMETRIC DETECTOR λ GAIN INTEGRATION TIME nm, AUFS sec HPLC COLUMN PROTECTION To save the analytical column Reverse Phase GENESIS, x mm, µ, the use of Metasaver Precolumn Filter. um ( x pcs.) cod. ZA is obligatory. HPLC COLUMN CONDITIONING COLUMN CLEANING Install a new analytical C Reversed-Phase column (GENESIS mm x, mm, µ). Disconnect the detector and filter ml of H O: Methanol ( : v/v ) solution and subsequently ml of H O per HPLC, set flow at, ml / min. Don t recycle the washing solutions. Filter the mobile phase with a vacuum system and a suitable filter of, µ. Condition the column with the mobile phase at a flow of, ml / min. and discharge the first ml. Condition further on the column for min. also at recycling phase. Finally inject the Chemical Standard and verify the quality of the HPLC run. It is NOT possible to make analysis at recycling phase. If room temperature is > C store the Mobile Phase at - C between an analytical session and another. Disconnect ESA detector. Flux ml of H O for min and discharge. Flux a solution made of H O : Methanol ( : v/v) for min and discharge. When you re-use the column, flux ml of H O before a new conditioning with the Mobile Phase. INJECTION NEEDLE WASHING HPLC PARAMETERS Wash with a solution of Water / Ethanol ( : v/v). LOOP µl Recommended Flow rate, ml / min Pressure About bar INTEGRATOR HP 9 / 9 / 9 PARAMETERS ATTENUATION OPERATIONAL COMPUTER PARAMETERS IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE

6 ACCESSORIES AND CONSUMABLES CODE DESCRIPTION PACKAGING Z Urine Calibrator lyophil. for t,t-muconic Acid x ml Z Urine multiparametric Control lyophil. for toxic organic compounds, Level x ml Z Urine multiparametric Control lyophil. for toxic organic compounds, Level x ml Z9 Urine multiparametric Control lyophil. for toxic organic compounds, Levels, x x ml ZFM9E Genesis C ( x,mm - u) Analytical Column Pk ZA Metasaver Precolumn Filter. um x PK S9U Standard glass vials of ml with screw cap x PK

7 t,t-muconic ACID (Reference Chromatograms) mv test fmvertua-isop.data mv.data TTM IS 9 - TTM IS - 9 RT[min] RT[min] 9 Fig. Test Solution Fig. Urine Calibrator lyophil. R.T.. t,t-muconic Acid R.T.. t,t-muconic Acid µg/l R.T.. Internal Standard R.T..9 Internal Standard

8 t,t-muconic ACID (Reference Chromatograms) mv 9.DATA mv camp.data IS IS TTM TTM - 9 RT[min] 9 9 RT[min] 9 Fig. Urine Control lyophil. Level Fig. Urine Sample R.T.. t,t-muconic Acid µg/l R.T.. t,t-muconic Acid, µg/l R.T.. Internal Standard R.T.. Internal Standard