FORMALDEHYDE IN URINE by UV Code Z65010 BIOCHEMISTRY The dose of urinary Formaldehyde is made after exposure to Formaldehyde and Methanol. The Formaldehyde is a pollutant so-called ubiquist, because it is practically everywhere, even in cities and along the very busy roads. And it is one of the main pollutants present in homes and offices, due not only to cigarette smoke, but also the slow release of furniture (it's present in the shavings in plywood and glue). We find also Formaldehyde in food (in Italy is admitted as a preservative by the initials E240), in cosmetics, in packaging, in the glue used for books and magazines. In the industrial field, the Formaldehyde is used in the manufacture of synthetic resins, glues, solvents, paints, preservatives, disinfectants and deodorants, soaps and detergents, cosmetics, textiles and also as an intermediate in the synthesis of drugs. The Methanol is used in the production of cellulose, synthetic resins, formaldehyde, acetic acid and fuel. It is metabolized in the liver and excreted in the urine as Formaldehyde, Formic acid and CO 2. Alcohol dehydrogenase is the first metabolic pathway that oxidizes the Methanol in Formaldehyde. The following oxidation of Formaldehyde to Formic Acid takes place through aldehyde dehydrogenase, extremely fast reaction, so that in some patients with poisoning from Methanol can not be determined the Formaldehyde either in tissues or in fluids. TOXICITY Since a long time toxic effects of Formaldehyde, already at low doses, are known. This is a strong irritant, and its presence is immediately felt from the characteristic smell and from eyes typical burning. The most worrying effect is its ability to induce mutations and cancer. IARC (International Agency for Research on Carcer), in the month of June 2004 classified formaldehyde in Group 1 of carcinogens, namely in certain carcinogenic to humans. This method allows the determination of the urinary Formaldehyde in HPLC. It 'a substance excreted by diffusion and therefore does not depend on the concentration of creatinine (see guidelines AIDII-Italian Association Of Industrial Hygienist). EUREKA srl LAB DIVISION VAT N 01547310423 E-mail:info@eurekaone.com www.eurekaone.com Head Quarter: Via Enrico Fermi 25 60033 Chiaravalle (AN) ITALY Tel. +39 071 7450790 Fax + 39 071 7496579 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. Release N 002 Formaldehyde in urine by UV March 2010 1
TECHNICAL FEATURES Principle of the Method: This method allows to determine the urinary concentrations of Formaldehyde after derivatization urine sample to 70 C for 15 minutes. After the sample is diluted and injected into the chromatograph HPLC. Recovery of Method: 100 % Sensitivity of Method: 0,15 mg/l Dynamic range of the method : 0,15 100 mg/l Reference Values : < 5,6 mg/l Components of the kit : All the reagents are ready-to-use and stable 3 years at room temperature (< 20 C). Reagent A - Buffer Solution 1, 1 x 10 ml Reagent B Buffer Solution 2, 1 x 10 ml Reagent C - Derivatization Solution, 1 x 10 ml Reagent D Test Solution/Chemical Standard, 1 x 10 ml Reagent M Mobile Phase, 3 x 500 ml See Warnings Store at 2-8 C Minimum Instrumental equipment required: Isocratic HPLC System with loop of 50 µl. Spectrophotometric Detector λ=385 nm Chromatograms Recorder Optional Equipment: Autosampler Operational Computer Urine Collection Procedure: After work shift, collect 10 ml of urine in a suitable test tube and store at 2-8 C for 2 days and at - 20 C until 3 months. 2
PREANALYTICAL PROCEDURE Test Solution at 36,5 mg/l preparation STEP 1 : Derivatization of Test Solution Test Reagent A Buffer Solution 1 100 µl Reagent D Test Solution 50 µl Reagent B Buffer Solution 2 100 µl Reagent C Derivatization Solution 100 µl Vortex for 10 seconds STEP 2 : Incubate at 70 C for 15 minutes Cooling STEP 3 : Add to derivatizated Test 500 µl of H 2 O HPLC grade INJECTION : Inject 50 µl of this solution in the chromatographic system. Verify that the Test Solution has retention time similar to fig. 1. 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 : Preparation of Chemical Standard Solution 3,65 mg/l Pipette in a tube: 900 µl of H 2 O HPLC grade 100 µl of Reagent D - Chemical Standard IMPORTANT: It is important to use standard solution just prepared STEP 2 : Derivatization of Blank (necessary as point 0 of calibration), of Samples, of Chemical Standard and of Controls Pipette in a tube as eppendorf by 1,5 ml with cap: Blank Standard Sample Control Reagent A Buffer Solution 1 100 µl 100 µl 100 µl 100 µl H 2 O HPLC grade 50 µl Chemical Standard 3,65 mg/l 50 µl Sample 50 µl Control 50 µl Reagent B Buffer Solution 2 100 µl 100 µl 100 µl 100 µl Reagent C Derivatization Solution 100 µl 100 µl 100 µl 100 µl Vortex for 10 seconds 3
Release N 002 Formaldehyde in urine by UV March 2010 STEP 3 : Incubate at 70 C for 15 minutes Cooling STEP 4 : Add to derivatizated Sample 500 µl of H 2 O HPLC grade. N.B.: at this step, the sample is stable 2-3 days at 2-8 C INJECTION : Inject 50 µl of the solution into the chromatographic system. 4
FORMALDEHYDE - Warnings Release N 002 Formaldehyde in urine by UV March 2010 REAGENT D : TEST SOLUTION/CHEMICAL STANDARD FORMALDEHYDE 36,5 mg/l SPECTROPHOTOMETRIC DETECTOR PARAMETERS λ GAIN INTEGRATION TIME 385 nm 0,002 AUFS 5 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 150 mm x 4,6 mm, 4 µ. Disconnect the detector and filter 30 ml of H 2 O: Methanol ( 20 : 80 v/v ) solution and subsequently 30 ml of H 2 O per HPLC, set flow at 1,2 ml / min. Don t recycle the washing solutions. Condition the column with the mobile phase at a flow of 1,2 ml / min. and discharge the first 30 ml. It is possible to make analysis at recycling phase; in which case you will see a progressive yellowing of mobile phase that, in general, does not disturb the linearity of the baseline. If room temperature is > 20 C store the Mobile Phase at 2-8 C between an analytical session and another. Disconnect ESA 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 minutes 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/minute PRESSURE about 100 bar INTEGRATOR HP 3394 / 3395 / 3396 PARAMETERS ATTENUATION 8 OPERATIONAL COMPUTER PARAMETERS IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE ACCESSORIES AND CONSUMABLES CODE DESCRIPTION PACKAGING Z65020 Chemical Standard for Formaldehyde 2 x 5 ml ZFM15960E Analytical Column Genesis C18 (150 x 4,6mm -4 um) 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
FORMALDEHYDE in URINE (Reference Chromatograms ) Fig. 1 Test Solution Fig. 2 Blank Fig. 3 Calibration Standard R.T. 10.39 Formaldehyde 36,5 mg/l R.T. 10.31 Formaldehyde R.T. 10.33 Formaldehyde 3,65 mg/l 6
FORMALDEHYDE in URINE (Reference Chromatograms) Fig. 4 Diluted Calibration Standard (Control) Fig. 5 Urine Sample enriched R.T. 10.29 Formaldehyde 0,365 mg/l R.T. 10.31 Formaldehyde 0,40 mg/l 7