The antiepileptic drugs phenobarbital and phenytoin were determined in serum by enzyme immunoassay (Emit, Syva Corp.) and gas-liquid chromatography. The Emit assays were mechanized by the use of an Eppendorf analyzer 5010. The precision of the Emit system was sufficient (coefficient of variation within series 6-13% and from day to day 8-15% with various calibrators and control sera). Moreover the Emit method is rapid, specific and easy to perform. The procedure requires only 10 of serum per determination. A disadvantage however is the high cost of the reagents. A comparison of the results obtained by Emit and gas-liquid chromatography in a series of about 50 patients showed a good correlation between both methods (correlation coefficient r = 0.968 for phenobarbital and 0.978 for phenytoin).
Bestimmung von Phenobarbital und Phenytoin im Serum mit einem mechanisierten Enzymimmuntest (Emit) im Vergleich zu einer gas-liquid chromatographischen MethodeZusammenfassung: Die antiepileptischen Pharmaka Phenobarbital und Phenytoin wurden mit einem Enzymimmuntest (Emit, Syva Corp.) und einer gas-liquid chromatographischen Methode bestimmt. Die Emit Tests wurden durch Verwendung eines Eppendorf Enzymautomaten 5010 mechanisiert. Die Präzision des Emit Systems war ausreichend (Variationskoeffizient in der Serie 6-13% und von Tag zu Tag 8-15% mit verschiedenen Kalibratoren und Kontrollseren). Darüber hinaus ist das Emitverfahren schnell, spezifisch und leicht durchführbar. Für eine Bestimmung werden nur 10 Serum benötigt. Nachteilig sind jedoch die hohen Kosten der Reagenzien. Ein Vergleich der Ergebnisse, welche mit Emit und Gas-Liquid-Chromatographie in einer Serie von etwa 50 Patienten erhalten wurden, zeigte eine gute Über-einstimmung beider Methoden (Korrelationskoeffizient r = 0,968 für Phenobarbital und 0,978 für Phenytoin).Introduction these compounds have been described. These techniques . ~. . . r r ,
A colorimetric method for the determination of glycerides (Short communication) J.-TH. MORSEL and R. HEYERThe quantitative determination of lipids is, a universal and repeating problem of analytical chemistry in different fields, for example food-or biochemistry. Important criterions to value methods are the time needed for analysis, reproducibility and detection limit. The determination of glycerides can be performed by classical methods, for instance by the WEIBULL-STOLDT method [I] or by extraction. HPLC and GLC can be used successfully, too. All these methods are expensive of time or equipment.In this paper a colorimetric method is reported characterized by good reproducibility, great sensitivity and little time needed to perform. The determination, first time reported by FLETCHER [2] is based on the oxidation of glycerol after saponification of the lipid with periodate. The formaldehyde produced is determined by coupling a dye in a HANTZSCH condensation with ammonium ions and 2,4-pentadione. The 3,5-diacetyl-1,Cdihydrolutidin formed is measured in a photometer at 410nm.
MaterialsPotassium hydroxide solution. 5 % in isopropanol/water, 4: 6 (v/v).Sodiummetaperiodate stock solution. 0.025 M in 1 N acetic acid. The working solution is prepared by mixing 12 ml stock solution, 20 ml isopropanol and 68 ml 1 N acetic acid. Acetylacetone solution. 0.75 ml 2,4-pentadion and 2.5 ml isopropanol are completed with 2 M ammonium acetate buffer (pH 6) to 100 ml. The pH should be determined exactly. The solution can be stored in dark bottles in a refrigerator.Water used for preparing the solution should be bidistilled. Isopropanol has to be free of n-alcohols. It has to be distilled before use.Standard solutions of glycerol (0.3 mg/100 ml), triolein (14 mg/100 ml) and tricaproin (3.5 mg/100 ml) are prepared by dissolving the substances in isopropanol/water 9: 1 (v/v). The triglycerides must be free of glycerol. This will be reached by filtration of crude products over a silica column.
MethodsThe determination was performed by the FLETCH~X method [2] with some modifications. 2 ml of sample are shaken with 0.6 ml potassium hydroxide solution in a stoppered test tube and heated for I5 min to 60-70 "C. After cooling in a water bath to room temperature 1 ml of fresh prepared periodate solution is added and after shaking vigorously 0.5 ml acetylacetone solution is added and colour is developed at 50 "C within 30min. The colour is measured in a photometer at 410nm against isopropanol/water 9 : 1 (v/v). The extinction of a blank test (2 ml isopropanol/water and all reagents) is substracted.
ResultsThe method has been tested with glycerol, triolein and tricaproin. Calibration curves are linear in a wide range (Fig. 1). In the experiments the minimum detectable concentration of glycerol was 70 pg/lOO ml. This indica-
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