Sumber : Determination of free and total levels of phenytoin in human plasma from patients with epilepsy by

MEKC: An adequate alternative to HPLC Phenytoin (5,5-diphenylhydantoin sodium)

Larutan stok
50 mM Tris buffer was prepared by dissolving 0.3029 g of Tris in a 50 mL volumetric flask with deionized water and
diluting to volume

Preparasi sampel untuk total fenitoin

Kontrol : Drug-free human plasma samples.
Sampel : 100 microL aliquot plasma pasien + 10microL mL 500 mg/mL of 2-(2-naphthoxy) ethanol sbg internal
standar

Kondisi Kromatografi
: Dynamax autosampler
Detektor : Detector UV 1000
Fase diam : A Microsorb-MVs RP-18e
Suhu :?
Volume injeksi : ?
Deteksi sinyal : 200nm
Fase gerak : Air dan ACN (58:42; v/v)
Flow rate : 1.4 mL/min
Kalkulasi : Rf
Dilakukan degassing sebelum percobaan

BAHAN
Internal standard (IS) :
Ketorolac
2-(2-naphthoxy) ethanol
chloramine T (TCI)

Methanol (MeOH)
Etil asetat
SDS
NaOH

Methanol (MeOH),
ethyl acetate,
SDS,
NaOH
Tris
CAN (Cerric Ammonius Nitrate)
EG
Phosphoric acid (H3PO4, 85%)

The stock solution of.

ALAT
The polyethersulfone membranes (Amicon, USA, 30 kDa, NMWL, 63.5 mm)
Milli-Q (Millipore) treated water was used for the prepara- tion of buffer and related drugs.
EKSTRAKSI
Sumber : Simultaneous determination of antiepileptic drugs and their two active metabolites by HPLC

Untuk setiap sampel :

IS working solution (50 microL) was mixed with
50 microL of patient serum
50 microL of 1 mol/L NaH2PO4
1 mL diethylether
PROSEDUR :

Konsentrasi kalibrasi dan kurva kalibrasi

For total-level phenytoin determination, the tested concentrations were in the range of 3.050.0 mg/mL with a
fixed concentration of 25.0 mg/mL of ketorolac (IS)

IS (mg/mL) Konsentrasi (mg/mL) Respon

Ketorolac
25 1
25 5
25 10
25 15
25 20
25 25
25 30
25 35
25 40
25 45
25 50

For free-level phenytoin determination, the tested concentrations were in the range of
0.35.0 mg/mL with a fixed concentration of 2.0 mg/mL of chloramine T (IS).

IS (mg/mL) Konsentrasi (mg/mL) Respon

Chloramine T
2 0.1
2 0.6
2 1.1
2 1.6
2 2.1
2 2.6
2 3.1
2 3.6
2 4.1
2 4.6
2 5.1

The samples were analyzed with the HPLC method for five replicates within the same day to obtain the intraday
accuracy and precision and for three replicates on five consecutive days to obtain the inter-day accuracy and
precision of analyte, expressed as relative error (RE) % values and RSD % values.
Metode II diambil dari :
Simultaneous determination of antiepileptic drugs and their two active metabolites by HPLC

Larutan stok
Larutkan 1g/L fenitoin ke dalam methanol

Kondisi Kromatografi
: P 1500
: Autosampler AS 1000,
Detektor : Detector UV 1000
Fase diam : Glass column (36150 mm) with stationary phase Separon SGX C18, 5 microm
Suhu : Suhu ruang
Volume injeksi : 20 microL
Deteksi sinyal : 220nm
Fase gerak : Air/ACN/methanol dengan ratio volume 72 : 23 : 5
with addition of TEA as a modificator in the concentrations 10, 40, 100, and 300 lL TEA/100 mL.
pH fasa gerak diatur dengan asam fosfat pada rentang 3.5 7.0 by 0.5 units.
Flow rate : 1 mL/min
Kalkulasi : Rf

Untuk setiap sampel :

IS 5-ethyl-5-p-tolylbarbituric acid 50 microL dicampur dgn 50 microL serum pasien 50 microL of 1 mol/L NaH2PO4
dan 1 mL diethylether

50microL IS + 50microL serum pasien (a)

(a) + 50microL NaH2PO4 1 mol/L

Ekstaksi dengan 1 mL dietilether

Ambil fasa eter - bagian atas

Disentrifugasi

Evaporasi engan Rotav pada suhu 80oC

Residu di rekonstitusi dgn metanol 20microL +

200microL water for HPLC

The samples were analyzed with the HPLC method for five replicates within the same day to obtain the intraday
accuracy and precision and for three replicates on five consecutive days to obtain the inter-day accuracy and
precision of analyte, expressed as relative error (RE) % values and RSD % values.
Pembuatan kurva kalibrasi

Total-level fenitoin

No Volume (micro L) IS Konsentrasi V Respon

(mg/mL) (mg/L)
1 50 1
2 50 10
3 50 20
4 50 30
5 50 40
6 50 50

Pelarutan
1.
2. 10 mg = x
1000ml 20.10^-3

Ringkasan bahan :

5-ethyl-5-p-tolylbarbituric acid 300mikroL -> 1mL

Volume injeksi 6 x 20 microL = 120microL

Stok = 1g/L mau buat ->1mg/mL

1 mg =
1 ml =
Metode ekstraksi diambil dari : Simultaneous determination of lamotrigine, phenobarbitone, carbamazepine and
phenytoin in human serum by high-performance liquid chromatography
Metode kromatogradi diambil dari : Simultaneous determination of antiepileptic drugs and their two active
metabolites by HPLC

Larutan stok
Larutkan 1g/L fenitoin ke dalam methanol

Kondisi Kromatografi
: P 1500
: Autosampler AS 1000,
Detektor : Detector UV 1000
Fase diam : Glass column (36150 mm) with stationary phase Separon SGX C18, 5 microm
Suhu : Suhu ruang
Volume injeksi : 20 microL
Deteksi sinyal : 220nm
Fase gerak : Air/ACN/methanol dengan ratio volume 72 : 23 : 5
with addition of TEA as a modificator in the concentrations 10, 40, 100, and 300 lL TEA/100 mL.
pH fasa gerak diatur dengan asam fosfat pada rentang 3.5 7.0 by 0.5 units.
Flow rate : 1 mL/min
Kalkulasi : Rf

Preparasi dan Ekstraksi sampel :

200L serum

Masukkan ke microtube polipropilene (1.5ml)

tambahkan 200L IS

Vortex selama 30s

Sentrifuga : 11.000 x 20"

Ambil supernatan ke tube lain

Sentrifuga : 11.000 x5'

Ambil supernatan

Filtrasi denga 0.2m mebran filter

(modifikasi) 20L filtrat di injeksikan ke kolom