Selective and sensitive determination of urinary total proline and hydroxyproline by gas chromatography with flame photometric detection.

A selective and sensitive method for the determination of total urinary proline (Pro) and hydroxyproline (Hyp) by gas chromatography (GC) was developed. After acid hydrolysis of urine, primary amino compounds were eliminated by reaction with o-phthaldialdehyde. Subsequently, Pro and Hyp were converted into their N-dimethylthiophosphoryl methyl ester derivatives and then determined by GC with flame photometric detection using a DB-5 capillary column. The derivatives were volatile and stable, giving single and symmetrical peaks. The detection limits for Pro and Hyp were 0.1 and 0.2 pmol injected, respectively. 3,4-Dehydroproline was used as an internal standard. The calibration curves for Pro and Hyp in the range 0.4-10 nmol were linear and sufficiently reproducible for quantitative determination. Overall recoveries of Pro and Hyp added to urine samples ranged from 93% to 103%. By using this method, Pro and Hyp in a small urine sample could be accurately and precisely determined without any influence from other constituent substances.