HIPPURIC ACID, o-m-p-methylhippuric ACIDS and CREATININE IN URINE by UV CODE Z07510

Transcription

1 HIPPURIC ACID, o-m-p-methylhippuric ACIDS and CREATININE IN URINE by UV CODE Z07510 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 006 Hippuric/o-m-p Methylhippuric Acids 1 and Creatinine in urine by UV March 2010

2 Creatinine is a breakdown product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body (depending on muscle mass). Creatinine is mainly filtered by the kidney, though a small amount is actively secreted. There is little-to-no tubular reabsorption of creatinine. If the filtering of the kidney is deficient, blood levels rise. As a result, creatinine blood levels may be used to calculate creatinine clearance, which reflects the glomerular filtration rate (GFR). The GFR is clinically important because it is a measurement of renal function. However, in cases of severe renal dysfunction, the creatinine clearance rate will be overestimated because the active secretion of creatinine will account for a larger fraction of the total creatinine cleared. A higher than normal creatinine and blood urea nitrogen (BUN) may be indicative of dehydration when BUN-to-creatinine ratio is abnormal, with BUN levels rising higher than creatinine levels. Creatinine can give men abnormal amounts of bloating and gas. Men tend to have higher levels of creatinine because they have more skeletal muscle than women. Vegetarians tend to have lower creationine levels, because vegetables contain no creatine. The typical reference ranges are 0.5 to 1.0 mg/dl for women and 0.7 to 1.2 mg/dl for men. While a baseline serum creatinine of 2.0 mg/dl may indicate normal kidney function in a male body builder, a serum creatinine of 0.7 mg/dl can indicate significant renal disease in a frail old woman. More important than absolute creatinine level is the trend of serum creatinine levels over time. 2

3 TECHNICAL FEATURES Principle of the Method: Hippuric Acid, o-m-p Methylhippuric Acids and Creatinine 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 Acceptable values in urine of not exposed subjects: Hippuric Acid : < 1500 mg / l o-m-p Methylhipp. Total Acid : < 1 mg / l Creatinine about 1000 mg / l Acceptable values in urine after work shift: Hippuric Acid : < 1500 mg / l o-m-p Methylhipp. Total Acid : < 1600 mg / l Dynamic range of Method: Ref: G Ital Med Lav Erg 2002;24:3 Suppl. 0, mg/l Components of the kit : All the components are ready-to-use. Stability: 3 years at room temperature ( 20 C). Reagent A Diluting Solution, 1 x 200 ml Reagent B Stabilization Solution, 1 x 50 ml Reagent C Chemical Standard/ Test Sol. Hippuric Acids, 1 x 10 ml Reagent D Chemical Standard/ Test Sol. Creatinine, 1 x 10 ml Reagent M Mobile Phase, 4 x 500 ml See Warnings Store at 2-8 C See Warnings Store at 2-8 C Minimum Instrumental equipment required: Isocratic HPLC System with loop of 50µl Spectrophotometric Detector UV/VIS λ=236 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 3

4 PREANALYTICAL PROCEDURE Test Solution Preparation Pipette in a tube : 990 µl of Water HPLC grade 10 µl of Reagent C Test Solution Hippuric or 10 µl of Reagent D Test Solution Creatinine Vortex for 10 sec. 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 Standard Samples Water HPLC grade 0,02 ml Reagent C Chemical Std Hippuric Acids 0,02 ml Reagent D Chemical Std Creatinine 0,02 ml Samples 0,02 ml Reagent A Diluting Solution 1,8 ml 1,8 ml 1,8 ml Reagent B Stabilization Solution 0,5 ml 0,48 ml 0,5 ml Vortex for 10 sec. STEP 2 : Centrifuge at rpm for 5 minutes. N.B.: at this step, the sample is stable 3 days at 2-8 C INJECTION : Inject 50 µl of this solution in the chromatographic system. Rev. N 006 Hippuric/o-m-p Methylhippuric Acids and Creatinine in urine by UV March

5 HIPPURIC ACID, o-m-p- METHYLH IPPURIC ACIDS and CREATININE IN URINE - Warnings REAGENT C : CHEMICAL STANDARD HIPPURIC ACIDS/ TEST SOLUTION HIPPURIC ACID o-methylhippuric ACID m-methylhippuric ACID p-methylhippuric ACID mg/l mg/l 500 mg/l 500 mg/l REAGENT D : CHEMICAL STANDARD CREATININE/ TEST SOLUTION CREATININE mg/l SPECTROPHOTOMETRIC DETECTOR PARAMETERS λ GAIN INTEGRATION TIME 236 nm 0,01 AUFS 10 sec. 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. It ISN T possible to make analysis at recycling phase. 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 FLOW 1,2 ml/min. PRESSURE About 80 bar INTEGRATOR HP 3394 / 3395 / 3396 PARAMETERS ATTENUATION 8 OPERATIONAL COMPUTER PARAMETERS IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE 5

6 ACCESSORIES AND CONSUMABLES CODE DESCRIPTION PACKAGING Z07520 Chemical Standard for 4 parameters + Creatinine in urine 2 x 5 ml Z06016 Multiparametric Calibrator for 6 parameters in urine 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 Genesis C 18 (150 x 4,6mm -4 um) Analytical Column 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 6

7 HIPPURIC ACID/ o-m-p- METHYL HIPPURIC ACIDS and CREATININE IN URINE (Reference Chromatograms) Fig. 3: Test Solution Fig. 4: Enriched Urine Samples R.T Hippuric Acid 1000 mg/l R.T Hippuric Acid 240,00 mg/l R.T o-methylhippuric Acids 1000 mg/l R.T o-methylhippuric Acids 83,30 mg/l R.T Creatinine 1000 mg/l R.T Creatinine 833,00 mg/l R.T m-p-methylhippuric Acids 1000 mg/l R.T m-p-methylhippuric Acids 79,50 mg/l 7

8 HIPPURIC ACID/ o-m-p- METHYL HIPPURIC ACIDS and CREATININE IN URINE (Reference Chromatograms) Fig. 5: Urine Control lyophil. Level 1 Fig. 6: Urine Control lyophil. Level 2 R.T Hippuric Acid 678,60 mg/l R.T Hippuric Acid 1044,00 mg/l R.T o-methylhippuric Acid 33,30 mg/l R.T o-methylhippuric Acid 100,00 mg/l R.T Creatinine 583,00 mg/l R.T Creatinine 450,00 mg/l R.T m-p-methylhippuric Acid 68,00 mg/l R.T m-p-methylhippuric Acid 182,00 mg/l 8