AMIODARONE and DESETHYLAMIODARONE IN PLASMA BY UV FAST CODE Z33610
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1 AMIODARONE and DESETHYLAMIODARONE IN PLASMA BY UV FAST CODE Z6 INTRODUCTION Amiodarone is an antiarrhythmic agent used for various types of tachyarrhythmias, both ventricular and supraventricular arrhythmias. Amiodarone is fat-soluble, and tends to concentrate in tissues including fat, muscle, liver, lungs and skin. This confers a high volume of distribution ( liters in a 7 kg adult) and a long half-life. Due to the long half-life of Amiodarone, oral loading typically takes days to weeks. Amiodarone shows beta blocker-like and calcium channel blocker-like actions on the SA and AV nodes, increases the refractory period via sodium -and potassium channel effects, and slows intra-cardiac conduction of the cardiac action potential, via sodium-channel effects. Amiodarone is extensively metabolised in the liver, and can affect the metabolism of numerous other drugs. The major metabolite of Amiodarone is Desethylamiodarone (DEA), which also has antiarrhythmic properties. The metabolism of Amiodarone is inhibited by grapefruit juice, leading to elevated serum levels of Amiodarone. Amiodarone potentiates the action of warfarin. Individuals taking both of these medications should have their warfarin dose halved and their anticoagulant status (measured as prothrombin time (PT) and international normalized ratio (INR)) measured more frequently. Amiodarone inhibits the action of the cytochrome P isozyme family. This reduces the clearance of many drugs, including cyclosporine, digoxin, flecainide, procainamide, quinidine, sildenafil, simvastatin, theophylline and warfarin. Unlike most other drugs, which are excreted via the urine, excretion is primarily hepatic and biliary with almost no elimination via the renal route and it is not dialyzable. Elimination half-life average of 8 days for Amiodarone and 6 days for the active metabolite, Desethylamiodarone (DEA). There is - % transfer of Amiodarone and DEA in the placenta as well as presence in breast milk. Accumulation of Amiodarone and DEA occurs in adipose tissue and highly perfused organs, therefore, if an individual was taking Amiodarone on a chronic basis, if it is stopped it will remain in the system for weeks to months. EUREKA srl LAB DIVISION VAT N 7 info@eurekaone.com Head Quarter: Via Enrico Fermi 6 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 Amiodarone and Desethylamiodarone in plasma by UV - FAST March
2 TECHNICAL FEATURES Principle of the Method: Amiodarone and Desethylamiodarone are separated treating the serum/plasma with a suitable precipitant. µl are directly injected into HPLC. Recovery : % Sensitivity : <, mg/l Dynamic Range of the Method :,, mg/l Therapeutic Range in Plasma: Amiodarone Desethylamiodarone,, mg/l,, mg/l Components of the kit : All the components are ready-to-use and stable years at -8 C. The Calibrator is stable 8 months at 8 C. Reagent A Deproteinization Sol. with Internal Standard, x ml Reagent B Test Solution, x ml Reagent C Plasma Calibrator lyophil., x ml See Warnings Reagent M Mobile Phase, x ml Minimum Instrumental equipment required: Optional Equipment: Isocratic HPLC System with loop of µl. Spectrophotometric Detector UV/VIS λ= nm Chromatograms Recorder Autosampler Operational Computer Blood Collection Procedure: Collect ml of blood in a suitable test tube without gel and without anticoagulant. Centrifuge at rpm for min. Plasma samples should be stored at C for no longer than weeks..
3 PREANALYTICAL PROCEDURE Test Solution Preparation Pipette in a tube : 9 µl of H O HPLC grade µl of Reagent B Test Solution INJECTION : 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. ANALYTICAL PROCEDURE Important : Don t use this solution to calibrate STEP : Samples Preparation Pipette in a tube with cap: Calibrator Samples Calibrator µl Samples µl Reagent A Deproteinization Sol. µl µl Warning: deproteinize directly on Vortex for at least seconds STEP : Centrifuge at. rpm for minutes STEP : Take µl of clear surnatant and add µl of HCl M (not included) N.B.: at this step, the sample is stable days at -8 C INJECTION : Inject µl of this solution in the chromatographic system. Rev. N Amiodarone and Desethylamiodarone in plasma by UV - FAST March
4 REAGENT B : TEST SOLUTION AMIODARONE / DESETHYLAMIODARONE FAST - Warnings Amiodarone Desethylamiodarone About 6,9 mg/l About 7,6 mg/l REAGENT C : PLASMA CALIBRATOR LYOPHIL. Lot. N Amiodarone Desethylamiodarone Use and Reconstitution: Calibrators are used for calibration of the HPLC system. This lyophilised calibrator has to be prepared like a patient sample. Add exactly. ml HPLC water to the vial and mix for min. When all material is dissolved, the solution is ready to use. Stability: 8 months if stored at -8 C. After reconstitution the stability of the analytes is at least hours when stored at + C, at least hours at + C and at least month at - C. Store in a dark place. Don t use after expiry date. Packaging : x ml Warning: The calibrator derives from human plasma, so it could be potentially infected. It must be handled with care. mg/l mg/l SPECTROPHOTOMETRIC DETECTOR PARAMETERS λ GAIN INTEGRATION TIME nm, AUFS sec. HPLC COLUMN PROTECTION To save the analytical column Reverse Phase PURSUIT XRs C8 x,6 mm, µ, the use of Metasaver Precolumn Filter. um ( x pcs), cod. ZA6 is obligatory. HPLC COLUMN CONDITIONING COLUMN CLEANING HPLC PARAMETERS Install a new analytical C 8 Reversed-Phase column (PURSUIT XRs C8, x,6 mm, µ). Disconnect the detector and filter ml of H O: Methanol or Acetonitrile ( : 8 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. It is NOT possible to make analysis at recycling phase. If room temperature is > C store the Mobile Phase at -8 C between an analytical session and another. Disconnect the detector. Flux ml of H O and discharge. Flux a solution made of H O : Methanol or Acetonitrile ( : 8 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. LOOP µl FLOW, ml/min. PRESSURE About bar OPERATIONAL COMPUTER PARAMETERS IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE ACCESSORIES AND CONSUMABLES
5 CODE DESCRIPTION PACKAGING Z6 Calibrator in plasma for Amiodarone/Desethylamiodarone x ml Z7 Control in plasma for Amiodarone/Desethylamiodarone, Level x ml Z8 Control in plasma for Amiodarone/Desethylamiodarone, Level x ml Z9 Control in plasma for Amiodarone/Desethylamiodarone, Levels and x x ml ZA6C6 Pursuit XRs C8 ( x,6mm - um) Complete System PK ZA6R6 Pursuit XRs C8 ( x,6mm - um), Repl. PK ZA6CG ChromSep Guard Pursuit XRs C8 ( x,6mm - um), Repl. x PK ZA6 Metasaver Precolumn Filter. um x PK S97U Clear glass vials with reduced volume from, ml to ul + caps x PK
6
7 AMIODARONE and DESETHYLAMIODARONE IN PLASMA BY UV FAST ( Reference Chromatograms ) mv 9 test.data mv 9 C.DATA DESETILAMIODARONE 7 6 DESETILAMIODARONE AMIODARONE 7 6 IS AMIODARONE RT[min] - RT[min],,,,,, 6 6, 7,,,,,, 6 6, 7 Fig. Test Solution R.T..6 Desethylamiodarone R.T..7 Amiodarone Fig. Plasma Calibrator lyophil. R.T..9 Internal standard R.T..7 Desethylamiodarone,9 mg/l R.T..8 Amiodarone,6 mg/l 7
8 AMIODARONE and DESETHYLAMIODARONE IN PLASMA BY UV FAST ( Reference Chromatograms ) mv 9 C.DATA mv 9 MIX.DATA IS DESETILAMIODARONE AMIODARONE 7 6 IS DESETILAMIODARONE AMIODARONE ,,,,,, 6 RT[min] 6, 7 -,,,,,, 6 RT[min] 6, 7 Fig. Plasma Control lyophil. level R.T..8 Internal Standard R.T..7 Desethylamiodarone.78 mg/l R.T..98 Amiodarone.76 mg/l Fig. Enriched Plasma sample R.T..78 Internal Standard R.T..6 Desethylamiodarone.8 mg/l R.T..9 Amiodarone.8 mg/l 8
AMIODARONE and DESETHYLAMIODARONE IN PLASMA BY UV FAST CODE Z33610
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