HYDROXYPROLINE IN URINE by UV/VIS Code Z Chemical characteristics Hydroxyproline or (S,R)--Hydroxy-L-pyrrolidine--carboxylic acid, is an aminoacid with an hydroxyl group on the pyrrolidine ring, in position. Its M.W. is,. Biochemistry Hydroxyproline is present almost exclusively in collagen, where represents % of the total amount of amino acids. It is formed from proline by a post-translational modification by an enzymatic hydroxylation reaction. Collagen, main component of the bone tissue has a protein structure composed by glycin, proline, hydroxyproline and hydroxylysine. It contains virtually the total content of hydroxyproline and hydroxylysine in the body. Urinary hydroxyproline is an important marker of bone resorption. Its urinary excretion is an index for the rate of bone resorption. It can increase in osteoporosis, Paget s disease, skeletal metastases, hyperparathyroidism, acute osteomyelitis, osteomalacia. A large body of evidence highlighted a close relationship between hydroxyproline and direct measurements of bone resorption (bone mineral density, calcium kinetics). In postmenopausal osteoporosis more than % of patients has high urinary hydroxyproline values. Therefore, it is possible to conclude that urinary hydroxyproline is an important marker of bone resorption. EUREKA srl LAB DIVISION VAT N E-mail:info@eurekaone.com www.eurekaone.com Head Quarter: Via Enrico Fermi Chiaravalle (AN) ITALY Tel. +9 9 Fax + 9 99 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 Hydroxyproline in urine by UV/VIS August
TECHNICAL FEATURES Principle of the Method: Hydroxyproline is derivatized and injected into the HPLC after acid hydrolysis at C for h. Recovery: % Sensitivity: Linearity: Normal h urinary values : Components of the kit : Reagent A Alkaline Solution, x ml, mg/l, mg/l,, mg/ h All the components are ready-to-use, except for Reagent D. Stability: years at - C. Reagent B Buffer Solution, x ml Reagent C Internal Standard Solution, x ml Reagent D Derivatization Solution N, x, ml Reagent E Derivatization Solution N, x ml To reconstitute with ml of Acetonitrile (not included in the kit) Reagent H Test Solution, x ml Reagent I Urine Calibrator Liophil, x ml See Warnings Reagent W Acidifying Solution, x ml Reagent M Mobile Phase, x ml Minimum Instrumental equipment required: Isocratic HPLC System with loop of µl UV/VIS Detector λ = 9 nm Chromatograms Recorder Optional Equipment: Autosampler Operational Computer h Urine Collection Procedure: h urine must be collected into a container with ml of HCl M per liter of urine. After collection, ml of urine should be delivered to the lab with the indication of the total diuresis. Delayed analyses require sample freezing at C or less. Stability of samples: until months. Warning for the collection procedure : Patients must avoid the intake of foods containing collagen (meat and derivatives, fish, cakes and all the products containing gelatin) at least h before sample collection.
PREANALYTICAL PROCEDURE Before proceeding with analysis check the analytical system with Test Solution without Internal Standard. Follow this procedure : Pipette in a tube of, ml with cap: 9 µl of Reagent B Buffer Solution µl of Reagent H Test Solution µl of Reagent E Derivatization Solution N (verify that ph is ± 9.) Close the tube, Vortex for sec INJECTION : Incubate at Room Temperature for minutes 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. You can use again this prepared Solution as a Test for the following analytical session, only if you make some aliquotes of it and store them at C ( stable about month ). Rev. N Hydroxyproline in urine by UV/VIS August
ANALYTICAL PROCEDURE STEP : Acid hydrolysis of samples Pipette in a labeled hydrolysis tube (with tefloned cap) µl of calibrator, control and patient sample of urine and add µl of N HCl (not included in the kit). Close the tubes and place them at C for h. STEP : Preparation of samples for derivatization Cooling. The hydrolyzate may be quoted and stored at - C. Stable about month. Pipette in a plastic ml tube: µl of hydrolysed urine µl of Reagent A - Alkaline Solution Warnings: Presence of CO bubbles µl of KOH concentrate % (not included in the kit) µl of Reagent B Buffer Solution µl of Reagent C Internal Standard Vortex for seconds Check that the solution has a ph at 9. STEP : Derivatization Transfer in a, ml glass vial with cap : µl of the final solution µl of Reagent D Derivatization Solution N (after reconstitution with ml of Acetonitrile) Add: µl of Reagent E Derivatization Solution N Close the conical test tube Vortex for sec. Wait minute Incubate at Room Temperature for minutes Vortex for seconds Incubate at Room Temperature for minutes Add : µl of Reagent W - Acidifying Solution STEP : Centrifuge at rpm for min. Vortex for seconds The solution is stable over hours if stored in a closed tube In a dark place and at room temperature INJECTION : Inject µl of the solution into the chromatographic system. Rev. N Hydroxyproline in urine by UV/VIS August
REAGENT H : TEST SOLUTION URINARY HYDROXYPROLINE Warnings HYDROXYPROLINE About mg/l REAGENT I : URINE CALIBRATOR LYOPHIL HYDROXYPROLINE 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 H O HPLC grade 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 Room Temperature, 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. mg/l PARAMETERS FOR THE SPECTROPHOTOMETRIC DETECTOR - VARIAN λ 9 nm GAIN Low or Medium INTEGRATION TIME or 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 Install a new analytical C Reversed-Phase column (GENESIS, mm x mm, µ). Disconnect the detector and flux ml of H O : Methanol or Acetonitrile ( : v/v ) solution and subsequently ml of H O HPLC grade, 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. It s 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. COLUMN CLEANING Disconnect the detector. Flux ml of water and discharge. Flux a solution of H O : Methanol or Acetonitrile ( : v/v ) for minutes. Discharge. A new use of the column needs a flow of ml of H O before a new conditioning with the mobile phase. HPLC PARAMETERS LOOP µl Flow, ml / min Pressure About bar INTEGRATOR HP 9 / 9 / 9 PARAMETERS ATTENUATION or OPERATIONAL COMPUTER PARAMETERS IN CONFORMITY WITH THE SPECIFICATION OF COMPUTER SOFTWARE
ACCESSORIES AND CONSUMABLES CODE DESCRIPTION PACKAGING Z Urine Calibrator lyophil. for Hydroxyproline x ml Z Urine Control lyophil. for Biogenic Amines, Level x ml Z Urine Control lyophil. for Biogenic Amines, Level x ml Z9 Urine Control lyophil. for Biogenic Amines, Levels and x x ml ZFM9E Analytical Column Genesis C ( x,mm - u) PK ZA Metasaver Precolumn Filter. um x PK S9U Clear glass vials with reduced volume from, ml to ul + caps x PK
HYDROXYPROLINE IN URINE by UV ( Reference Chromatograms ) mv 9 TEST.DATA [MIB - Channel ] mv 9 CAL.DATA 9 9 OH-prolina Std Int 9 RT [min] - 9 RT [min] Fig. : Test Solution R.T.. Hydroxyproline Fig. : Urine Calibrator lyophil R.T.. Hydroxyproline mg/l R.T.. Internal Standard
HYDROXYPROLINE IN URINE BY UV ( Reference Chromatograms ) mv 9 C.DATA mv 9 C+.DATA OH-prolina 9 Std Int 9 Std Int OH-prolina - RT [min] - RT [min] 9 9 Fig. : Urine Control lyophil Level R.T.. Hydroxyproline 9 mg/l R.T.. Internal Standard Fig. : Urine Sample not pathological R.T.. Hydroxyproline R.T.. Internal Standard