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Microsoft word - z04310 levetiracetam rev.8.docLEVETIRACETAM IN PLASMA BY UV – CODE Z04310
Treatment of the epileptic patients requires a multidisciplinary medical knowledge, regarding i.e.
pharmacology, psychology and social science. In this contest the various antiepileptic drugs (AEDs) are used to decrease the frequency and/or severity of seizures in people with epilepsy. Monitoring of plasmatic concentrations of AEDs is important to improve patient’s theraphy. Our method allows to determine in the same HPLC run the sieric and/or plasmatic concentration of EUREKA srl – LAB DIVISION
VAT N° 01547310423
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.
Rev. N° 008
Lavetiracetam in plasma by UV
Principle of the Method :
Levetiracetam is separated treating the serum/plasma with a suitable precipitant. After addition of a
stabilization Reagent the solution is directly injected into HPLC.
Dynamic Range of the Method :
0,2 – 200 µg/ml
Therapeutic Range in Plasma :
Components of the kit :
All the components are ready-to-use. Stability: 3 years at 2–8 °C. The Calibrator is stable 24 months at 2–8°C.
Reagent A – Deproteinization Sol. with Internal Standard, 1 x 20 ml
Reagent B – Stabilization Solution, 1 x 20 ml
Reagent C – Test Solution, 1 x 5 ml
Reagent D – Plasma Calibrator lyophil., 1 x 1 ml
Reagent M – Mobile Phase, 2 x 500 ml
Minimum Instrumental equipment required:
Isocratic HPLC System with loop of 50 µl
Spectrophotometric Detector UV λ=205 nm
Whole Blood Collection Procedure:
Collect 3 ml of blood in a suitable test tube without gel and with anticoagulant Heparin. Centrifuge at 4000 rpm for 5 minutes. Separate the serum and store at – 20 °C. Stable 4 weeks. PREANALYTICAL PROCEDURE
Pipette in a tube :
• 50 µl of Reagent C - Test Solution
• 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!
STEP 1 : Samples Preparation
Pipette in a suitable tube with cap :
Water HPLC grade
Reagent D - Calibrator
Reagent A – Deproteinization with
Warning: deproteinize directly on Vortex for at least 10 seconds
STEP 2 : Centrifuge at 5000 rpm for 5 min.
STEP 3 : Pipette in a tube 200 µl of clear surnatant.
• Add 200 µl of Reagent B – Stabilization Sol.
Vortex for 10 seconds
N.B.: at this step, the sample is stable 3 days at 2-8 °C
• Inject 50 µl of this solution in the chromatographic system.
Rev. N° 008
Lavetiracetam in plasma by UV
LEVETIRACETAM - Warnings
REAGENT C: TEST SOLUTION
REAGENT D: PLASMA CALIBRATOR LYOPHIL.Lot.0708
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 1.0 ml HPLC water to the vial and mix for 15 min.
When all material is dissolved, the solution is ready to use.
Stability: 24 months if stored at 2-8 °C. After reconstitution the stability of
the analytes is at least 15 days at +4°C and at least 3 months at -20° C.
Don’t use after expiry date.
Packaging : 1 x 1 ml
Warning: The calibrator derives from human plasma, so it could be
potentially infected. It must be handled with care.
SPECTROPHOTOMETRIC DETECTOR PARAMETERS
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
Install a new analytical C18 Reversed-Phase column (GENESIS 150 mm x 4,6 mm, 4 µ). Disconnect the detector and filter 30 ml of H2O: Methanol (20 : 80 v/v) solution and subsequently 30 ml of H2O 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 is possible to make analysis at recycling phase, providing that you filter the same
mobile phase with a filter of 0,22 µ before any analytical run. 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 H2O and discharge. Flux a solution made of H2O : Methanol or Acetonitrile (20 : 80 v/v) for 30 min and discharge. When you re-use the column, flux 30 ml of H2O before a new conditioning with the Mobile Phase.
INTEGRATOR HP – 3394 / 3395 / 3396 PARAMETERS
OPERATIONAL COMPUTER PARAMETERS
IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE
ACCESSORIES AND CONSUMABLES
Control for Levetiracetam in plasma – Level 1 Control for Levetiracetam in plasma – Level 2 Control for Levetiracetam in plasma – Levels 1 and 2 ZFM15960E
Genesis C 18 (150 x 4,6mm -4 um) Analytical Column Clear glass vials with reduced volume from 1,5 ml to 15 ul + Fig. 1 : Test Solution
Fig. 2 : Plasma Calibrator lyophil.
Fig. 3 : Plasma Control lyophil. Level 2
Fig. 4 : Enriched Plasma Sample
New Spirit 4 Aussie Rescue Heartworm Treatment Protocol as of Fall 2011 Introduction: The drug, Immiticide, has been the treatment of choice for heartworm positive dogs. New Spirit has always opted for full treatment with Immiticide, even though it’s very expensive. In conjunction with a pre- and post treatment protocol, this was our preferred approach. and we had very good outcomes