CLOZAPINE AND NORCLOZAPINE IN SERUM AND/OR IN PLASMA BY UV CODE Z76010
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1 CLOZAPINE AND NORCLOZAPINE IN SERUM AND/OR IN PLASMA BY UV CODE Z76010 INTRODUCTION Clozapine (Leponex, Clozapine ) is an atypical neuroleptic drug class chemistry of dibenzodiazepines, widely used in the treatment of refractory schizophrenia. Unlike other neuroleptics, does not induce extrapyramidal side effects, despite its efficacy, its use is limited because it can potentially cause agranulocytosis. The oral absorption of Leponex is 90-95%, the literature shows that it is possible that plasma levels of clozapine and norclozapine are influenced by various factors such as sex, age, compliance, caffeine intake, smoking and drug interactions. Monitoring improves the use of this neuroleptic, since sufficiently high concentrations, increase the response and, furthermore, it seems that the side effects (seizures, sedation, hypersalivation, etc...) are related to concentrations supra therapeutic. It s recommended the regular monitoring of WBC count. The following method allows to determine in the same chromatographic run both clozapine that its main metabolite, the norclozapine or N-desmethyl clozapine, which has no therapeutic range of reference but an indication of how the patient metabolizes the drug. 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. Release N 006 Clozapine and Norclozapine in serum and/or in plasma by UV July
2 TECHNICAL FEATURES Principle of the Method : This method is used to determine plasma concentrations of Clozapine and its metabolite, Norclozapine.At an aliquot of plasma (250 µl) is added a derivatization solution and a coprecipitant and, after centrifugation, the supernatant is transferred into the tube and injected into HPLC. Recovery : 100% Sensitivity : 15 ng/ml Dynamic Range of the Method : ng/ml CV% inter serie : Clozapine : 2,8 % Norclozapine : 2,9 % CV% intra serie : Clozapine : 2,2 % Norclozapine : 2,4 % Normal Range : Clozapine : ng/ml Components of the kit : All the components are ready to use. Stability: 3 years at room temperature. Plasma Calibrator is stable 36 months at 2 8 C. Reagent A Deproteinization Solution with Internal Standard, 1 x 25 ml Reagent B Coprecipitant Solution, 1 x 5 ml Reagent C Test Solution, 1 x 5 ml Reagent D Plasma Calibrator, 1 x 2 ml See Warnings Reagent M Mobile Phase, 7 x 500 ml Minimum Instrumental equipment required: Isocratic HPLC System with loop of 50 µl Spectrophotometric Detector UV/VIS λ=254 nm Chromatograms Recorder Optional Equipment: Autosampler Operational Computer Blood Collection Procedure: In the morning, before the next dose. Plasma: tubes with lithium heparin or EDTA. Serum: tubes without gel. The sample must necessarily be stored at 1 week at 2 8 C and 2 months at - 20 C. 2
3 PREANALITYCAL PROCEDURE Pipette in a vial : 800 µl of H 2 O HPLC grade 100 µl of Reagent C - Test Solution 100 µl of Reagent A Deproteinization with Internal Stanadrd NOTE : 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!. ANALITYCAL PROCEDURE STEP 1 : Samples Preparation Dispense in a tube: Calibrator Sample Control Reagent D Plasma Calibrator 250 µl Sample 250 µl Control 250 µl Reagent A Deproteinization Sol. 100 µl 100 µl 100 µl Reagente B Coprecipitant Sol. 50 µl 50 µl 50 µl Warning: deproteinize directly on Vortex for at least 30 sec. STEP 2 : Centrifuge at rpm for 10 minutes. STEP 3 : Put all the surnatant in a clean tube. 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. Release N 006 Clozapine and Norclozapine in serum and/or in plasma by UV July
4 CLOZAPINE AND NORCLOZAPINE IN SERUM and/or IN PLASMA Warnings REAGENT C : TEST SOLUTION CLOZAPINE NORCLOZAPINE µg/l µg/l REAGENT D : PLASMA CALIBRATOR LIOPHYL. Lot. 003 CLOZAPINE NORCLOZAPINE Use and Reconstitution: Plasma Calibrators are used for calibration of the HPLC system. This lyophilised calibrator has to be prepared like a patient sample. Add exactly 2.0 ml HPLC water to the vial and mix for 15 min. When all material is dissolved, the solution is ready to use. Stability: 36 months if stored at 2-8 C. After reconstitution the stability of the analytes is at least 1 day at +4 C and at least 7 days at 20 C. Don t use after expiry date. Packaging : 1 x 2 ml Warning: The calibrator derives from human plasma, so it could be potentially infected. It must be handled with care. 538,4 µg/l 624,9 µg/l SPECTROPHOTOMETRIC DETECTOR PARAMETERS λ GAIN INTEGRATINO TIME 254 nm 0,001 AUFS 10 sec HPLC COLUMN PROTECTION To save the analytical column Reverse Phase PURSUIT XRs C18 50 x 4,6 mm, 3 µ, the use of Metasaver Precolumn Filter 0.5 um (1 x 10 pcs), cod. ZA6005 is obligatory. COLUMN CONDITIONING COLUMN CLEANING HPLC PARAMETERS Install a new analytical C 18 Reversed-Phase column PURSUIT XRs C18 (50 x 4,6 mm - 3 µm). 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. 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 further on the column for 30 min. also at recycling phase. Finally inject the Chemical Standard and verify the quality of the HPLC run. It s NOT possible to make analysis at recycling phase. Disconnect the detector. Flux 30 ml of H 2 O and discharge. LOOP 50 µl RECOMMENDED FLOW 1,0 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 4
5 ACCESSORIES AND CONSUMABLES CODE DESCRIPTION PACKAGING Z76016 Calibrator in plasma for Clozapine and Norclozapine 4 x 2 ml Z76017 Control in plasma for Clozapine and Norclozapine, Level 1 5 x 2 ml Z76018 Control in plasma for Clozapine and Norclozapine, Level 2 5 x 2 ml Z76019 Control in plasma for Clozapine and Norclozapine, Levels 1 and 2 2 x 5 x 2 ml ZFM15960E Genesis C18 (150 x 4,6mm -4 um) Analytical Column 1 PK ZA6005 Metasaver Precolumn Filter 0.5 um 1 x 10 PK S29307U Clear Glass Vials with reduced volume (goblet form) from 1,5 ml to 15 ul, with caps 1 x 100 PK 5
6 CLOZAPINE AND NORCLOZAPINE IN PLASMA ( Reference Chromatograms ) Fig. 1 : Test Solution Fig. 2 : Plasma Calibrator R.T Norclozapine R.T Norclozapine 497 µg/l R.T Internal Standard R.T Internal Standard R.T Clozapine R.T Clozapine 538 µg/l 6
7 CLOZAPINE AND NORCLOZAPINE IN PLASMA ( Reference Chromatograms ) Fig. 3 : Low Control Fig. 4 : High Control R.T Norclozapine 122 µg/l R.T Norclozapine 1034 µg/l R.T Internal Standard R.T Internal Standard R.T Clozapine 154 µg/l R.T Clozapine 1000 µg/l 7
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