Determination of isoniazid, acetylisoniazid, acetylhydrazine and diacetylhydrazine in biological fluids by high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the determination of isoniazid, acetylisoniazid, acetylhydrazine and diacetylhydrazine (plasma and urine) was developed. The m-chlorobenzoyl derivatives of isoniazid, acetylhydrazine and the internal standard propionylhydrazine were prepared, separated on a RP-18 column and detected at 220 nm. Acetylisoniazid, diacetylhydrazine and the internal standard dipropionylhydrazine were converted to isoniazid, acetylhydrazine, and propionylhydrazine by acidic hydrolysis and subsequently derivatized with m-fluorobenzoyl chloride, separated on a RP-18 column and detected at 220 nm. The lower limits of detection in plasma are acetylhydrazine 0.5 nmol/ml, isoniazid 1.0 nmol/ml, diacetylhydrazine 1.0 nmol/ml and acetylisoniazid 2.0 nmol/ml, and in urine, acetylhydrazine 10 nmol/ml, isoniazid 15 nmol/ml, diacetylhydrazine 20 nmol/ml and acetylisoniazid 40 nmol/ml. This method is sensitive, reproducible, accurate and precise; therefore, it is well suited for detailed pharmacokinetic studies.