Dian Sampson

A rapid gas chromatographic method for the quantitation of underivatised individual free fatty acids in plasma

A rapid method for quantitating individual free fatty acids (FFA) in plasma has been developed. An internal standard is added to plasma, the FFA are extracted into an organic solvent, concentrated and injected into a gas chromatograph. For routine estimations, extraction and assay of a single sample takes about 30 min and up to 30 samples can be assayed in a day. Seven FFA (myristic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids), are routinely quantitated. This method can be used to monitor individual FFA levels, to determine the percentage composition of individual FFA in plasma or to investigate metabolic disorders involving fatty acids, for example Refsums disease.

The estimation of plasma valproate by gas-liquid chromatography.

A gas chromatographic method for the plasma assay of the anticonvulsant, sodium valproate, is described. Derivatization is not necessary. 200 microliter plasma are required for a single estimation. The method involves a chloroform extraction of valproate and the internal standard, cyclopentane carboxylic acid, from acidified plasma. Gas-liquid chromatography using the stationary phase 10% SP-216-PS gives complete separation of valproate and the internal standard in eight minutes. The limit of detection is 20 mumol valproate/1 plasma (equivalent to 40 pmol on column). This is well below the lower therapeutic plasma level. The between-run precision of the method indicates a variation for each sample within+/-3% of its mean value.

Economics and the choice of anticonvulsant assay methods

Plasma levels of anticonvulsant drugs correlate well with the clinical status of epileptic patients and are assayed in many biochemistry laboratories. Five major techniques can be used for assay: manual gas chromatography, automated gas chromatography, liquid chromatography, radioimmunoassay and enzyme immunoassay. Technical considerations favour gas chromatography; precision is equal or superior to that of immunoassay methods and chromatographic methods allow more flexibility so that less common anticonvulsants such as clonazepam, sulthiame and methoin can be assayed. We have costed these 5 major techniques using experience gained in our laboratory and material published in current literature. In addition, we have used price lists and assay protocols distributed by the manufacturers of immunoassay kits. The major factors involved in costing are reagents, staff salaries and instrument depreciation. Automated gas chromatography was found to be the least costly method even for small numbers of anticonvulsants.

Drug estimation techniques

The need for drug estimations in the modern laboratory is well-established; drug assays are useful for patient management both for therapy and overdose. The techniques available fall into 4 main groups based on colorimetry, spectrophotometry, chromatography and immunological methods. The choice of a technique for a particular assay or screen depends on many factors: (1) The type of drugs being assayed; (2) Whether a general answer, e.g. barbiturates, is sufficient or a more specific answer, e.g. phenobarbitone, is required; (3) Whether quantitation is needed: (4) How exhaustive must be the search to identify unknown compounds; (5) How much time is available for the assay; (6) What finance is available for equipment. Our approach, based on experience with a large workload, will be presented and possibilities for the smaller laboratory will be discussed.

Evaluation of a semiautomated enzyme immunoassay for digoxin

An automated digoxin assay was assessed and compared with a manual radioimmunoassay (RIA) method which has been used for several years. The Syva enzyme multiplied immunoassay technique (EMIT) was modified slightly to be run economically on an Abbott ABA 100 fitted with a single syringe Cavro pump for the addition of the second reagent. The manual RIA procedure used was the Wellcome Lanoxitest γ. The automated method was evaluated with regard to bias between the two methods, precision, convenience and assay time and cost for both batched and urgent specimens. We consider that the EMIT system offers significant advantages in time and cost with adequate precision.

The use of gas chromatography as part of a general drug overdose screen

Gas chromatography has been used in this laboratory as a screening method to identify and quantitate drugs in the plasma of overdosed patients. The screen takes 2-2½ hr and routinely identifies and quantitates 9 common barbiturates, salicylate, acetylsalicylate, diphenylhydantoin, glutethimide, three tricyclic antidepressants, methaqualone, diazepam, ethanol and methanol. Four samples of plasma are used. The alcohols are estimated without extraction using a column of Poropak Q. Acidic drugs are extracted from acidified plasma, separated into stronly and weakly acidic drugs and identified and quantitated using two columns—one of 10% Apiezon L and one of 3% OV 17. The tricyclic antidepressants are released from their binding sites on plasma proteins and extracted at neutral pH. Both tricyclic antidepressant drugs and alkali-extractable drugs are chromatographed using a column of 3% OV –17. An internal standard is used for each estimation.

Rapid identification and ouantitation of overdose levels of amltrlptyllne in plasma

Arnitriptyline (Tryptanol) is a commonly prescribed antidepressant of which about 95% is bound to plasma proteins. A rapid (45 min.) gas liquid chromatographic assay was set up for the identification and determination of arnitriptyline and two other tricyclic antidepressants (imipramine and nortriptyline) in plasma of overdosed patients. In some cases, the nature of the drug was unknown. An internal standard of triphenylamine was added to the plasma and amitriptyline was released from plasma proteins by formamide. Free arnitriptyline and triphenylamine were extracted into hexane and dried ; the residue was then partitioned between ethanol and hexane under alkaline conditions. The fmal hexane fraction was dried and the residue suspended in a small vol. of chloroform for injection into the gas chromatograph. Extraction efficiencies were 75% for triphenylamine, 70% for amitriptyline and irnipramine and 32% for nortriptyline.