HIPPURIC ACID and o-m-p- METHYLHIPPURIC ACIDS IN URINE BY UV CODE Z06010
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1 HIPPURIC ACID and o-m-p- METHYLHIPPURIC ACIDS IN URINE BY UV CODE Z06010 Biochemistry The determination of Hippuric Acid ( HPA ) in urine is important to determine the risk due to the exposition to Toluene. The Toluene is metabolised mostly in Benzoic Acid and subsequently combined with Glycine is eliminated by urine as Hippuric Acid (fig. 1). Fig. 1: Biotransformation of Toluene in vivo The determination of o-m-p Methylhippuric Acid ( m-mhpa, o-mhpa and p-mhpa ) is important to determine the risk due to the exposition to a mixture of o-m-p-xilene. The Xilene, which is present as a mixture of three isomers o-m-p, is metabolised in o-m-p-methyl-benzoic and after conjugation with Glycine are eliminated by urine as a mixture of o-m-p-methyl-hippuric Acids. (fig. 2). Fig. 2 Biotransformation of Xilene in vivo 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 98/79/EC on in vitro diagnostic medical devices (IVD). The declaration of conformity is available upon request. Rev. N 017 Hippuric and o-m-p-methylhippuric 1 Acids in urine by UV March 2010
2 TECHNICAL FEATURES Principle of the Method: Hippuric Acid and o-m-p Methylhippuric Acids are analysed after dilution and after the addition of a stabilization solution. This solution is then directly injected into HPLC System. Recovery: 100% Sensitivity: 0,2 µg/ml CV% : Hippuric Acid 3,14% o-m-p Methylippuric Total Acids 3,02% Acceptable values in urine of not exposed subjects: Hippuric Acid : < 1500 mg / l o-m-p Methylhipp. Total Acids : < 1 mg / l Acceptable values in urine after work shift: Hippuric Acid : < 1600 mg / l o-m-p Methylhipp. Total Acids : < 1500 mg / l Dynamic range of Method: Ref.: G Ital Med Lav Erg 2002;24:3 Suppl 0, µg/ml Components of the kit: Reagent A Dilution Solution, 1 x 200 ml All the components are ready-to-use. Stability: 3 years at room temperature ( 20 C). The Calibrator is stable 36 months at 2 8 C. Reagent B Stabilization Solution, 1 x 50 ml Reagent C Test Solution, 1 x 10 ml Store at 2-8 C Reagent D Urine Calibrator lyophil, 1 x 5 ml See Warnings Reagent M Mobile Phase, 2 x 500 ml Minimum Instrumental equipment required: Isocratic HPLC System with loop of 50µl Spectrophotometric Detector UV/VIS λ=207 nm Chromatograms Recorder Optional Equipment: Autosampler Operational Computer Urine Collection Procedure: Before and soon after work shift, collect 10 ml of urine in a suitable test tube. Pre-analytics phase: Centrifuge at 4000 rpm for 5 min. The sample is stable up to 4 weeks at 2-8 C or 6 months at 20 C. 2
3 PREANALYTICAL PROCEDURE Test Solution Preparation Pipette in a tube : 990 µl of Water HPLC grade 10 µl of Reagent C Test Solution Vortex for 10 seconds INJECTION : Inject 50 µl of this solution in the chromatographic system. Verify that the Test Solution has retention time similar to fig. 3. 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 1: Samples Preparation Pipette in suitable tubes for centrifugation in sequence : Blank Calibrator Sample Reagent A Dilution Solution 1,8 ml 1,8 ml 1,8 ml Reagent B Stabilization Solution 0,5 ml 0,5 ml 0,5 ml Water HPLC grade 0,02 ml Reagent D Urine Calibrator 0,02 ml Sample 0,02 ml Vortex for 10 seconds STEP 2: Centrifuge at rpm for 5 min. N.B.: at this step, the sample is stable 5 days at 2-8 C. INJECTION : Inject 50 µl of this solution in the chromatographic system. 3
4 Rev. N 017 Hippuric and o-m-p-methylhippuric Acids in urine by UV March 2010 HIPPURIC ACID and o-m-p-methylhippuric ACIDS IN URINE - Warnings REAGENT C : TEST SOLUTION Hippuric Acid o-methyl-hippuric Acid m-methyl-hippuric Acid p-methyl-hippuric Acid 1000 mg/l 1000 mg/l 500 mg/l 500 mg/l REAGENT D : URINE CALIBRATOR LYOPHIL. Lot. n 005 Hippuric Acid 1.699,4 mg/l o- Methyl-hippuric Acid 534,6 mg/l p- Methyl-hippuric Acid 519,1 mg/l m- Methyl-hippuric Acid 523,7 mg/l Intend 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 5 ml HPLC water to the vial and mix for 15 min. When all material is dissolved, the solution is ready to use. Mix always before use. Storage and stability: Originally closed and stored at 4 C, the lyophilised urine calibrator is stable for 36 months, but no longer than the expiration date printed on the label. After reconstitution the stability of the analytes is at least 12 hours when stored at +20 C, at least 7 days at +4 C and at least 1 month at -20 C. Packaging: 1 x 5 ml Caution : this calibrator was manufactured from human urine. Therefore the material has to be considered as potentially infectious and should be treated with appropriate care. SPECTROPHOTOMETRIC DETECTOR PARAMETERS λ GAIN INTEGRATION TIME 207 nm 0,01 AUFS 10 seconds HPLC COLUMN PROTECTION To save the analytical column Reverse Phase GENESIS 4,6 x 150 mm, 4 µ, the use of Metasaver Precolumn Filter 0.5 um (1 x 10 pcs.) cod. ZA6005 is obligatory. HPLC COLUMN CONDITIONING COLUMN CLEANING HPLC PARAMETERS Install a new analytical C 18 Reversed-Phase column (GENESIS 4,6 mm x 150 mm, 4 µ). Disconnect the detector and flux 30 ml of solution and subsequently 30 ml of HPLC water, set flow at 1,2 ml / min. Don t recycle the washing solutions. Filter the mobile phase with a vacuum system and a suitable filter of 0,22 µ. Condition the column with the mobile phase at a flow of 1,2 ml / min. and discharge the first 30 ml. Condition furtherly the column for 30 min. also at recycling phase. Finally inject the Chemical Standard and verify the quality of the HPLC run. It is possible to make analysis at recycling phase, providing that you filter the same mobile phase with a filter of 0,22 µ before any analytical run. If Room Tem. is > 20 C is better to preserve the Mobile Phase at 2 8 C among runs. Disconnect the detector. Flux 30 ml of H 2 O and discharge. Flux a solution made of H 2 O: Methanol or Acetonitrile ( 20 : 80 v/v ) for 30 min and discharge. When you re-use the column, flux 30 ml of H 2 O before a new conditioning with the mobile phase. LOOP 50 µl Recommended Flow rate 1,2 ml / min Pump Pressure About 80 bar INTEGRATOR HP PARAMETERS 3394 / 3395 /
5 ATTENUATION 8 OPERATIONAL COMPUTER PARAMETERS IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE ACCESSORIES AND CONSUMABLES CODE DESCRIPTION PACKAGING Z06016 Urine multiparametric Calibrator lyophil. for 6 parameters 4 x 5 ml Z38017 Urine multiparametric Control lyophil. for Toxic Organic Compounds, Level 1 5 x 10 ml Z38018 Urine multiparametric Control lyophil. for Toxic Organic Compounds, Level 2 5 x 10 ml Z38019 Urine multiparametric Control lyophil. for Toxic Organic Compounds, Levels 1,2 2 x 5 x 10 ml ZFM15960E Analytical Genesis C18 (150 x 4,6mm -4 u) 1 Pk ZA6005 Metasaver Precolumn Filter 0.5 um 1 x 10 PK S29057U Standard glass vials of 2 ml with screw cap 1 x 100 PK 5
6 HIPPURIC ACID and o-m-p-methylhippuric ACIDS IN URINE ( Reference Chromatograms ) Fig. 3 Test Solution R.T Hippuric Acid R.T o-methylhippuric Acid R.T p-methylhippuric Acid R.T m-methylhippuric Acid Fig. 4 Urine Calibrator lyophil. R.T Hippuric Acid 1360 mg/l R.T o-methylhippuric Acid 55 mg/l R.T p-methylhippuric Acid 56 mg/l R.T m-methylhippuric Acid 56 mg/l 6
7 HIPPURIC ACID and o-m-p-methylhippuric ACIDS IN URINE ( Reference Chromatograms ) Fig. 5 Urine Control lyophil. Level 1 R.T Hippuric Acid 696,8 mg/l R.T o-methylhippuric Acid 25,9 mg/l R.T p-methylhippuric Acid 30,2 mg/l R.T m-methylhippuric Acid 45,9 mg/l Fig. 6 Urine Control lyophil. Level 2 R.T Hippuric Acid 1311,4 mg/l R.T o-methylhippuric Acid 99,6 mg/l R.T p-methylhippuric Acid 80,7 mg/l R.T m-methylhippuric Acid 137,9 mg/l 7
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