Epilepsiu. 18(2), 1 9 7
Raven Press, New York
Rapid Determination of Valproate Sodium in Serum by Gas-Liquid Chromatography W. Loscher Lnhorritoririrn j i i r Pharmrtkologir und Toxikologie, Fachherc.icii Vetc,ririurniediziri Frcie Univrrsitur Berlin, Koserstrajlr 20, 0-1000 Bprlin 33
INTRODUCTION occurring in kinetic studies in small laboratory In recent years, valproate sodium (sodium animals. di-n-propylacetate) has found increasing interest MATERIALS AND METHODS in the treatment of primary generalized epilepsy, Valproate sodium was used in the form of especially of the petit ma1 type in children, and-in combination with other anticonvulsants the commercial 30% solution (Ergenyl@,Labaz +f more difficult cases (for a review see Simon GmbH, Dusseldorf). Chloroform and sulfuric and Penry, 1975). The therapeutic range of val- acid were all reagent grade and were obtained proate sodium was considered to be between from Merck (Darmstadt). 2-Ethyl-2-methyl50 and 100 pgiml plasma (Schobben et al., 1975). caproic acid (internal standard) was obtained The routine analysis to monitor plasma concen- from Fluka (Neu-Ulm). trations in patients treated with this drug as well Extraction Procedure as analysis in laboratory animals during experiTo 0.2 ml serum in a 1 ml tube (Eppendorf mental studies requires specific and rapid methrnicrotubes) were added 50 pI 1 2 sulfuric ~ ods for the determination of valproate. A few 100 pl chloroform containing 2-ethylacid and assays of valproate sodium using gas-liquid chromatography were described in the litera- 2-methyl-caproic acid as internal standard (1: 10, ture; they used partly micro-extraction methods 000 v/v). The tube was shaken for 1 min (Eppento prevent loss of the volatile valproate during dorf shaker 5432) and centrifuged at 12,000 rpm the usual extractionievaporation procedures for I min (Eppendorf centrifuge 5412). T h e (Meijer and Hessing-Brand, 1973; Cremers and upper layer and the protein layer were removed Verheesen, 1974: Dijkhuis and Vervloet, 1974; and 2 PI of the organic phase was then taken Schobben and van der Kleijn, 1974; Schulz and from the bottom of the tube and analyzed by gas chromatography with FID detection. Toseland, 1975). The aim of this study was to develop a rapid and simple micro-extraction method followed by Gas Chromatography gas chromatographic determination sensitive Analysis was carried out with a Varian 1200 enough to determine low concentrations of val- gas chromatograph equipped with a glass colproate in small volumes of serum, a problem umn (6 ft, 2 mm internal diameter) packed with 10% Carbowax 6000 o n Chromosorb WAW (801100). The temperature of the column was Received January 19, 1977. 125°C; injector block and F I D were kept at 175°C. Nitrogen, hydrogen, and air flow rates Key words: Vcrlprocite sodircin-Serum--Gus-liquid chroinutogruphy. were 46,45, and 300 mlimin, respectively. When 225
w.LOSCHER
226
lyzing 10 samples from the same stock-solution (50 p g valproateiml serum.) This gave a coefficient of variation of 2.5%. At low valproate concentrations (< 5 pgml serum), 0.5 ml serum can be extracted with 100 pl chloroform after addition of 100 pl 1 2 sul~ furic acid. If 2 pl of the organic layer is injected, serum concentrations of 2 p d m l can be determined accurately. However, to eliminate inaccuracies due to the altered partition coefficient, it is recommended that the same procedure be repeated with a valproate standard serum of approximately the same concentration. To check the specificity of the method we have analyzed other antiepileptic drugs: phenobarbital, primidone, phenytoin, carbamazepine, ethosuximide, trimethadione, dimethadione. No interference was observed in the chromatograms.
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SUMMARY
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A rapid and simple gas-liquid chromatographic procedure is described for the measurement of valproate sodium (sodium di-n-propylacetate) in serum. Valproate is extracted from serum by a micro-extraction method and chromatographed on a 10% Carbowax 6000 column using 2-ethyl2-methyl-caproic acid as internal standard. The method is capable of measuring at least 2 p g of valproate per milliliter serum.
6min
FIG. 1. Gas chromatogram of valproate on 10% Carbowax 6000. 2-Ethyl-2-methyl-caproic acid was used as internal standard after extraction from serum. Attenuation 2 x IO-"'.
calculating the concentrations of valproate, the peak area was compared with that of the internal standard. Peak areas were calculated as the product of peak height and width at half height and converted to pgiml of drug using standard curves obtained by adding known amounts of drug to drug-free samples. RESULTS AND DISCUSSION The described method is easily practicable, has high capacity, and is suited for clinical routine analysis as well as for experimental studies. The peaks of both valproate and the internal standard in the chromatogram are well separated and almost symmetrical (Fig. I). The calibration curves obtained from standard serum samples were linear in the range of 5-200 pdrnl. Reproducibility of the method was evaluated by ana-
REFERENCES Cremers HMHG and Verheesen PE. A gas chromatographic determination of di-n-propylacetic acid (DPA) in serum. Pharm Weekblod 109: 1-3. 1974. Dijkhuis ID and Vervloet E. Rapid determination of the antiepileptic drug di-n-propylacetic acid in serum. Pharm Weekhlud 109:42-45, 1974. Meijer JWA and Hessing-Brand L. Determination of lower fatty acids, particularly the anti-epileptic dipropyl-acetic acid, in biological materials by means of micro diffusion and gas chromatography. Clin Chim Actu 42:215-222, 1973. Schobben F and van der Kleijn E. Determination of sodium di-n-propylacetate in plasma by gas-liquid chromatography. P h u r m Waekhlad 109:30-33, 1974. Schobben F, van der Kleijn E, and Gabreels FJM. Pharmacokinetics of di-n-propylacetate in epileptic patients. Eur J CIin Pharmucol 8:97-105, 1975. Simon D and Penry JK. Sodium di-n-propylacetate in the treatment of epilepsy. A review. Epi/~~p.sicr 16:549-573, 1975. Schulz H U and Toseland PA. Determination of the anticonvulsant drug sodium dipropylacetate in human plasma by gas chromatography, V. Congress of the Polish Phnrtnac~ologic.olSOC.~C,I.Y. Szczecin, Sept. 24-27, 1975.
GLC OF VALPROATE SODIUM I N SERUM
ZUSAMMENFASSUNG E~ wird cine schnelle und einfache gaschromatographische Methode fur die Bestimmung Valproat (Di-n-propylacetat) im serum beschfieben, Valproat wird aus dem Serum mit einer Mikro-Extraktionsrnethode extrahiert und auf einer 10% Carbowax 6000
22 7
Saule chromatographiert. Als interner Standard wird 2-Athyl-2-methyl-capronsaurebenutzt. Mit der beschriebenen Methode ist e s moglich, ValproatKonzentrationen von bis zu 2 pLg/mlim Serum zu bestimmen. (W. Loscher, Berlin)
Raven Press, New York
Rapid Determination of Valproate Sodium in Serum by Gas-Liquid Chromatography W. Loscher Lnhorritoririrn j i i r Pharmrtkologir und Toxikologie, Fachherc.icii Vetc,ririurniediziri Frcie Univrrsitur Berlin, Koserstrajlr 20, 0-1000 Bprlin 33
INTRODUCTION occurring in kinetic studies in small laboratory In recent years, valproate sodium (sodium animals. di-n-propylacetate) has found increasing interest MATERIALS AND METHODS in the treatment of primary generalized epilepsy, Valproate sodium was used in the form of especially of the petit ma1 type in children, and-in combination with other anticonvulsants the commercial 30% solution (Ergenyl@,Labaz +f more difficult cases (for a review see Simon GmbH, Dusseldorf). Chloroform and sulfuric and Penry, 1975). The therapeutic range of val- acid were all reagent grade and were obtained proate sodium was considered to be between from Merck (Darmstadt). 2-Ethyl-2-methyl50 and 100 pgiml plasma (Schobben et al., 1975). caproic acid (internal standard) was obtained The routine analysis to monitor plasma concen- from Fluka (Neu-Ulm). trations in patients treated with this drug as well Extraction Procedure as analysis in laboratory animals during experiTo 0.2 ml serum in a 1 ml tube (Eppendorf mental studies requires specific and rapid methrnicrotubes) were added 50 pI 1 2 sulfuric ~ ods for the determination of valproate. A few 100 pl chloroform containing 2-ethylacid and assays of valproate sodium using gas-liquid chromatography were described in the litera- 2-methyl-caproic acid as internal standard (1: 10, ture; they used partly micro-extraction methods 000 v/v). The tube was shaken for 1 min (Eppento prevent loss of the volatile valproate during dorf shaker 5432) and centrifuged at 12,000 rpm the usual extractionievaporation procedures for I min (Eppendorf centrifuge 5412). T h e (Meijer and Hessing-Brand, 1973; Cremers and upper layer and the protein layer were removed Verheesen, 1974: Dijkhuis and Vervloet, 1974; and 2 PI of the organic phase was then taken Schobben and van der Kleijn, 1974; Schulz and from the bottom of the tube and analyzed by gas chromatography with FID detection. Toseland, 1975). The aim of this study was to develop a rapid and simple micro-extraction method followed by Gas Chromatography gas chromatographic determination sensitive Analysis was carried out with a Varian 1200 enough to determine low concentrations of val- gas chromatograph equipped with a glass colproate in small volumes of serum, a problem umn (6 ft, 2 mm internal diameter) packed with 10% Carbowax 6000 o n Chromosorb WAW (801100). The temperature of the column was Received January 19, 1977. 125°C; injector block and F I D were kept at 175°C. Nitrogen, hydrogen, and air flow rates Key words: Vcrlprocite sodircin-Serum--Gus-liquid chroinutogruphy. were 46,45, and 300 mlimin, respectively. When 225
w.LOSCHER
226
lyzing 10 samples from the same stock-solution (50 p g valproateiml serum.) This gave a coefficient of variation of 2.5%. At low valproate concentrations (< 5 pgml serum), 0.5 ml serum can be extracted with 100 pl chloroform after addition of 100 pl 1 2 sul~ furic acid. If 2 pl of the organic layer is injected, serum concentrations of 2 p d m l can be determined accurately. However, to eliminate inaccuracies due to the altered partition coefficient, it is recommended that the same procedure be repeated with a valproate standard serum of approximately the same concentration. To check the specificity of the method we have analyzed other antiepileptic drugs: phenobarbital, primidone, phenytoin, carbamazepine, ethosuximide, trimethadione, dimethadione. No interference was observed in the chromatograms.
Q)
Y
0
0
L
a 0 >
d
SUMMARY
-
L 1
0
'
1
3
A rapid and simple gas-liquid chromatographic procedure is described for the measurement of valproate sodium (sodium di-n-propylacetate) in serum. Valproate is extracted from serum by a micro-extraction method and chromatographed on a 10% Carbowax 6000 column using 2-ethyl2-methyl-caproic acid as internal standard. The method is capable of measuring at least 2 p g of valproate per milliliter serum.
6min
FIG. 1. Gas chromatogram of valproate on 10% Carbowax 6000. 2-Ethyl-2-methyl-caproic acid was used as internal standard after extraction from serum. Attenuation 2 x IO-"'.
calculating the concentrations of valproate, the peak area was compared with that of the internal standard. Peak areas were calculated as the product of peak height and width at half height and converted to pgiml of drug using standard curves obtained by adding known amounts of drug to drug-free samples. RESULTS AND DISCUSSION The described method is easily practicable, has high capacity, and is suited for clinical routine analysis as well as for experimental studies. The peaks of both valproate and the internal standard in the chromatogram are well separated and almost symmetrical (Fig. I). The calibration curves obtained from standard serum samples were linear in the range of 5-200 pdrnl. Reproducibility of the method was evaluated by ana-
REFERENCES Cremers HMHG and Verheesen PE. A gas chromatographic determination of di-n-propylacetic acid (DPA) in serum. Pharm Weekblod 109: 1-3. 1974. Dijkhuis ID and Vervloet E. Rapid determination of the antiepileptic drug di-n-propylacetic acid in serum. Pharm Weekhlud 109:42-45, 1974. Meijer JWA and Hessing-Brand L. Determination of lower fatty acids, particularly the anti-epileptic dipropyl-acetic acid, in biological materials by means of micro diffusion and gas chromatography. Clin Chim Actu 42:215-222, 1973. Schobben F and van der Kleijn E. Determination of sodium di-n-propylacetate in plasma by gas-liquid chromatography. P h u r m Waekhlad 109:30-33, 1974. Schobben F, van der Kleijn E, and Gabreels FJM. Pharmacokinetics of di-n-propylacetate in epileptic patients. Eur J CIin Pharmucol 8:97-105, 1975. Simon D and Penry JK. Sodium di-n-propylacetate in the treatment of epilepsy. A review. Epi/~~p.sicr 16:549-573, 1975. Schulz H U and Toseland PA. Determination of the anticonvulsant drug sodium dipropylacetate in human plasma by gas chromatography, V. Congress of the Polish Phnrtnac~ologic.olSOC.~C,I.Y. Szczecin, Sept. 24-27, 1975.
GLC OF VALPROATE SODIUM I N SERUM
ZUSAMMENFASSUNG E~ wird cine schnelle und einfache gaschromatographische Methode fur die Bestimmung Valproat (Di-n-propylacetat) im serum beschfieben, Valproat wird aus dem Serum mit einer Mikro-Extraktionsrnethode extrahiert und auf einer 10% Carbowax 6000
22 7
Saule chromatographiert. Als interner Standard wird 2-Athyl-2-methyl-capronsaurebenutzt. Mit der beschriebenen Methode ist e s moglich, ValproatKonzentrationen von bis zu 2 pLg/mlim Serum zu bestimmen. (W. Loscher, Berlin)
