LC05010 Antiepileptics Rev - eurekakit.com€¦ · Antiepileptics are separated treating the serum/plasma with a suitable precipitant. After addition of a stabilization Reagent the

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ANTIEPILEPTICS in plasma by LC-MS – Deuterated internal Std - Code LC05010 (Levetiracetam, Lamotrigine, Primidone, Oxcarbazepine, Carbamazepine Epoxide, 10,11-Dihydro-10-Hydroxy-Carbamazepine, Carbamazepine, Phelbamate, Desmethylsuximide, Rufinamide, Phenytoin, Methsuximide, Topiramate, Phenobarbital, Valproic Acid, Ethosuximide, Lacosamide, Zonisamide, Perampanel)

INTRODUCTION

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

the following drugs: Levetiracetam, Lamotrigine, Primidone, Oxcarbazepine, Carbamazepine

Epoxide, 10,11-Dihydro-10-Hydroxy-Carbamazepine, Carbamazepine, Phelbamate,

Desmethylsuximide, Rufinamide, Phenytoin, Methsuximide, Topiramate, Phenobarbital, Valproic

Acid, Ethosuximide, Lacosamide, Zonisamide, Perampanel.

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 Antiepileptics in plasma by LC/MS February 2017

EUREKA srl – LAB DIVISION VAT N° 01547310423 E-mail:[email protected] www.eurekaone.com

Head Quarter: Via Enrico Fermi 25 60033 Chiaravalle (AN) ITALY Tel. +39 071 7450790 Fax + 39 071 7496579

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TECHNICAL FEATURES Principle of the Method : Antiepileptics are separated treating the serum/plasma with a suitable precipitant. After addition of a stabilization Reagent the solution is directly injected into HPLC. Sensitivity: Levetiracetam 0,07 µg/ml

Lamotrigine 0,03 µg/ml

Primidone 0,07 µg/ml

Oxcarbazepine 0,03 µg/ml

Carbamazepine Epoxide 0,02 µg/ml 10,11-Dihydro-10-Hydroxy-Carbamazepine 0,07 µg/ml

Carbamazepine 0,01 µg/ml

Phelbamate 0,1 µg/ml

Rufinamide 0,02 mg/l

Desmethylsuximide 0,3 mg/l

Phenytoin 0,3 mg/l

Methsuximide 0,3 mg/l

Topiramate 0,1 mg/l

Phenobarbital 1,5 µg/ml

Valproic Acid 2,5 mg/l

Ethosuximide 5,0 µg/ml

Lacosamide 0,03 mg/l

Zonisamide Perampanel

0,07 µg/ml 0,001 µg/ml

Dynamic Range : Levetiracetam 0,2 – 100 µg/ml

Lamotrigine 0,1 – 30 µg/ml

Primidone 0,2 – 30 µg/ml

Oxcarbazepine 0,1 – 100 µg/ml

Carbamazepine Epoxide 0,5 – 100 µg/ml 10,11-Dihydro-10-Hydroxy-Carbamazepine 0,1 – 100 µg/ml

Carbamazepine 0,02 – 30 µg/ml

Phelbamate 0,3 – 150 µg/ml

Rufinamide 0,05 – 100 mg/l

Desmethylsuximide 1 – 100 mg/l

Phenytoin 0,8 – 50 mg/l

Methsuximide 1 – 100 mg/l

Topiramate 0,3 – 100 mg/l

Phenobarbital 5 – 100 µg/ml

Valproic Acid 10 – 200 mg/l

Ethosuximide 20 – 150 µg/ml

Lacosamide 0,1 – 30 mg/l


0,2 – 100 µg/ml 0,003 – 5 µg/ml

CV% : Levetiracetam 4,36% intra serie 7,60% inter serie

Lamotrigine 5,40% intra serie 6,50% inter serie Primidone 5,65% intra serie 5,70% inter serie Oxcarbazepine 4,79% intra serie 6,50% inter serie Carbamazepine Epoxide 5,76% intra serie 6,90% inter serie 10,11-Dihydro-10-Hydroxy-Carbamazepine 5,87% intra serie 7,90% inter serie Carbamazepine 4,84% intra serie 7,40% inter serie Phelbamate 5,68% intra serie 6,30% inter serie Rufinamide 6,59% intra serie 7,90% inter serie Desmethylsuximide 5,52% intra serie 9,20% inter serie Phenytoin 5,84% intra serie 9,90% inter serie Methsuximide 5,56% intra serie 9,20% inter serie Topiramate 6,50% intra serie 7,80% inter serie Phenobarbital 6,17% intra serie 6,30% inter serie Valproic Acid 4,79% intra serie 5,40% inter serie Ethosuximide 4,43% intra serie 5,10% inter serie Lacosamide 4,59% intra serie 9,20% inter serie

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4,09% intra serie 4,90% inter serie 4,61% intra serie 4,89% inter serie

Therapeutic range : Levetiracetam 10 – 37 µg/ml

Lamotrigine 3 – 14 µg/ml

Primidone 5 – 12 mg/l

Oxcarbazepine 0 – 4 mg/l

Carbamazepine Epoxide 2 – 4 mg/l 10,11-Dihydro-10-Hydroxy-Carbamazepine 3 – 40 µg/ml

Carbamazepine 6 – 12 mg/l

Phelbamate 10 – 80 mg/l

Rufinamide 1 – 50 mg/l

Desmethylsuximide 10 – 40 mg/l

Phenytoin 10 – 20 mg/l

Methsuximide 10 – 40 mg/l

Topiramate 2 – 25 mg/l

Phenobarbital 10 – 40 mg/l

Valproic Acid 40 – 100 mg/l

Ethosuximide 40 – 100 µg/ml

Lacosamide 1 – 10 mg/l


10 – 38 µg/ml 0,1 – 1 µg/ml

Accuracy: Levetiracetam 96,4%

Lamotrigine 100,7%

Primidone 116,8%

Oxcarbazepine 100,1%

Carbamazepine Epoxide 89,3% 10,11-Dihydro-10-Hydroxy-Carbamazepine 101,8%

Carbamazepine 91,3%

Phelbamate 95,3%

Rufinamide 105,5%

Desmethylsuximide 110,7%

Phenytoin 94,3%

Methsuximide 105,9%

Topiramate 100,1%

Phenobarbital 91,3%

Valproic Acid 103,1%

Ethosuximide 97,1%

Lacosamide 96,3%


116,6% 103,5%

Recovery: 100%

Components of the kit (100 tests): All reagents are ready to use and stable 3 years at 2 - 8 °C, except Reagent A.

Reagent A – Deproteinization Solution with Internal Standards, 1 x 50 ml

Store at -20 °C

Reagent B – Stabilizing Solution, 1 x 50 ml

Plasma Calibrator – Level 0, 1 x 1 ml Code LC04916 (packed separately – see data sheet)

Plasma Calibrator – Level 1, 1 x 1 ml Code LC04916 (packed separately – see data sheet


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Reagent M1 – Mobile Phase A, 1 x 500 ml

Reagent M2 – Mobile Phase B, 1 x 500 ml

Minimum Instrumental equipment required:

LC/MS System with triple quadrupole MRM Work Mode, positive/ negative ESI Operational Computer

Optional Equipment:

Autosampler.

Blood Collection Procedure:

Take 3 ml of whole blood in a test tube without gel or with EDTA as anticoagulant. Centrifuge at 4000 rpm for 5 minutes. Separate the serum and store at – 20 °C. Stable 4 weeks.

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ANALYTICAL PROCEDURE

STEP 1 : Sample Preparation Dispense in an eppendorf:

Calibrator Sample Control

Calibrator 50 µl

Sample 50 µl

Control 50 µl

Reagent A – Deproteinization Sol. with Internal Standards

500 µl 500 µl 500 µl

Warning: deproteinize directly on Vortex for at least 20 sec.

STEP 2 : Incubate at 2-8 °C for 15 minutes. STEP 3 : Centrifuge at 13.000 rpm for 15 minutes

STEP 4 : Put 50 µl of supernatant in a glass vial of 2 ml.

• Add 450 µl of Reagent B – Stabilizing Solution (or an appropriate volume for the instrument sensitivity)

Vortex for 10 seconds

N.B.: at this step, the sample is stable 2 days at 2-8 °C INJECTION :

• Inject 1-10 µl of the eluate into the chromatographic system.

Release N° 002 Antiepileptics in plasma by LC/MS February 2017

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ANTIEPILEPTICS - Warnings

COLUMN CONDITIONING

Install a new analytical column HYPERSIL GOLD C18 50 x 2,1 mm – 1,9 µm. Thermostated at 40 °C. Disconnect the detector and flux a solution of Acetonitrile : H2O ( 95 : 5 v/v ) set flow at 400 ul / minute for 15 minutes. Don’t recycle the washing solutions. Condition further on the column with a solution of

Mobile Phase M1 : Mobile Phase M2 ( 98 : 2 v/v ) set flow at 400 ul / minute for 15 minutes. Making two injections of HPLC grade water before proceeding with the analytical series.

It’s NOT possible to make analysis at recycling phase.

If room temperature is > 20 °C store the Mobile Phase at 2-8 °C between an analytical session and another.

COLUMN CLEANING

Wash with a solution containing Mobile Phase M2: M1 (95: 5 v / v) at a flow of 400 ul / minute for 15 minutes. It recommends flushing the Analytical Column at the end of each run. The column must be stored in a solution containing Mobile Phase M2: M1 (95: 5 v / v).

WASH AUTOSAMPLER SYRINGE

Wash the syringe, before every analytical session, with a solution of Ethanol, Acetontrile or Methanol.

ESI+ PARAMETERS (optimized on triple quadrupole Sciex 4500)

ESI- PARAMETERS (optimized on triple quadrupole Sciex 4500)

GRADIENT

Time (min)

% M1 (PUMP A)

% M2 (PUMP B)

Flow (µl/min)

0

98 2 400

0.50

98 2 400

0.80

75 25 400

2.00

65 35 400

3.00

2 98 400

4.00

2 98 400

4.10

98 2 400

Curtain Gas (CUR): 40 Collision Gas (CAD): 8 Ion Spry Voltage: (IS): 5500 V Temperature (TEM): 600°C Ion Source Gas 1 (GS1): 40 Ion Source Gas 2 (GS2): 30

Curtain Gas (CUR): 40 Collision Gas (CAD): 8 Ion Spry Voltage: (IS): -4500 V Temperature (TEM): 600°C Ion Source Gas 1 (GS1): 30 Ion Source Gas 2 (GS2): 30

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DEPROTEINIZATION SOLUTION WITH INTERNAL STANDARD:

Solution with: Levetiracetam-d6, Zonisamide-d4, Lacosamide-d3, Phelbamate-d4, Ethosuximide-d3, Desmethylsuximide-d5. Store at – 20 °C.

Ionization Analyte Standard Interno consigliato per la calibrazione

Positive

Levetiracetam Levetiracetam-d6

Lamotrigine Lacosamide-d3

Primidone Lacosamide-d3

Oxcarbazepine Lacosamide-d3

Carbamazepine epoxide Lacosamide-d3

OH-carbamazepine Lacosamide-d3

Carbamazepine Lacosamide-d3

Phelbamate Phelbamate-d4

Rufinamide Lacosamide-d3

Desmethylsuximide Desmethylsuximide-d5

Phenitoin Lacosamide-d3

Methsuximide Desmethylsuximide-d5

Topiramate Lacosamide-d3

Lacosamide Lacosamide-d3

Perampanel Lacosamide-d3

Negative

Phenobarbital Zonisamide-d4

Valproic Acid Zonisamide-d4

Ethosuximide Ethosuximide-d3

Zonisamide Zonisamide-d4

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FRAGMENTATIONS (optimized on TRIPLE QUADRUPOLE Sciex 4500)

ANALYTE Q1 Q3 COLLISION ENERGY

Levetiracetam 171.1 69.2 37.0

Lamotrigine 256.1 211.1 – 159.1 30.0 – 39.0

Primidone 219.2 162.2 – 91.2 17.0 – 40.0

Oxcarbazepine 253.2 236.2 – 180.2 18.0 – 38.0

Carbamazepine Epoxide 253.0 210.0 – 180.0 30.0 – 30.0

10,11-Dihydro-10-Hydroxy-Carbamazepine

255.2 237.2 – 194.1 15.0 – 27.0

Carbamazepine 237.2 194.2 26.0

Phelbamate 239.3 178.2 10.0

Rufinamide 239.3 127.2 29.0

Desmethylsuximide 190.2 120.0 – 91.1 12.0 – 36.0

Phenitoin 253.1 104.3 – 182.3 48.0 – 23.0

Methsuximide 204.1 126.2 – 79.2 21.0 – 26.0

Topiramate 357.1 264.1 20.0

Lacosamide 251.2 91.2 – 108.2 30.0 – 14.0

Phenobarbital 231.0 188.1 -18.0

Valproic Acid 143.0 143.0 -12.0

Ethosuximide 140.1 140.1 -12.0

Zonisamide 211.0 119.0 – 147.0 -22.0 - -17.0

Perampanel 350.1 219.0 47.0

Levetiracetam-d6 177.0 132.1 24.0

Phelbamate-d4 265.0 204.0 19

Desmethylsuximide-d5 195.1 120.0 – 125.1 29.0 – 24.0

Lacosamide-d3 255.0 220.0 – 178.0 11.5 – 18.0

Ethosuximide-d3 142.9 142.9 -13.0

Zonisamide- d4 215.0 151.0 – 123.0 -14.0 - -21.0

CODE DESCRIPTION PACKAGING

LC04916 Calibrator for Antiepileptics in plasma 6 x 2 x 1 ml

LC04917 Control per Antiepileptics in plasma – Level 1 5 x 1 ml

LC04918 Control per Antiepileptics in plasma – Level 2 5 x 1 ml

LC04919 Control per Antiepileptics in plasma – Levels 1, 2 and 3 3 x 5 x 1 ml

Z25002052130 Hypersil GoldC18 (50 x 2,1 mm – 1,9 um) Analytical Column 1 Pk

S51843550 Clear glass vials with reduced volume 1 x 100 Pk

S51820717 Caps for clear glass vials with reduced volume 1 x 100 Pk

SK05010 Starter kit for Antiepileptics 1 Pk

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ANTIEPILEPTICS IN PLASMA ( Reference Chromatograms )

Fig. 1 : Plasma Calibrator Fig. 2 : Plasma Calibrator

R.T. 2.0 Levetiracetam-D6 R.T. 2.76 Desmethylsuxmide-D5

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R.T. 2.37 Phelbamate-D3 R.T. 2.35 Lacosamide-D3

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R.T. 2.16 Ethosuximide-D3 R.T. 2.28 Zonisamide-D4

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R.T. 2.34 Lamotrigine R.T. 2.27 Primidone

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R.T. 2.99 Topiramate R.T. 2.44 Rufinamide

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R.T. 2.36 Lacosamide R.T. 3.53 Phenytoin

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R.T. 3.40 Methsuximide R.T. 2.36 Phelbamate

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R.T. 2.36 Ethsuximide R.T. 2.78 Desmethylsuximide

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R.T. 2.29 Zonisamide R.T. 2.70 Phenobarbital

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R.T. 4.20 Valproic Acid R.T. 2.97 Oxacarbazepine

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R.T. 2.82 Carbamazepine Epoxide R.T. 2.57 10,11-Dihydro-10-Hydroxy-Carbamazepine

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R.T. 3.53 Carbamazepine R.T. 2.00 Levetiracetam

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Fig. 25 : Plasma Calibrator

R.T. 4.35 Perampanel

Documents

LC05010 Antiepileptics Rev - eurekakit.com€¦ · Antiepileptics are separated treating the serum/plasma with a suitable precipitant. After addition of a stabilization Reagent the