m-methylhippuric acids in urine by high-speed liquid chromatography

High-speed liquid chromatography employing an ultraviolet photometric detector has been applied to the simultaneous determination of hippuric and m-methylhippuric acids in urine. Reversed-phase partition chromatography is carried out on a ,tBondapak C18 column with methanol-water as the eluent system. This method obviates the necessity for isolation or reaction of these acids before assay. The only pretreatment necessary is extraction of samples with ethyl acetate. A linear relationship is obtained between the peak heights and the hippuric or m-methylhippuric acid concentrations. Mean recovery of hippuric and m-methylhippuric acids in urine is 99-8 % and 99.3 % respectively. The determination of hippuric acid by this method gives lower concentrations in normal urine than does the colorimetric method of Umberger and Fiorese (1963). It has been observed that the concentration of hippuric and m-methylhippuric acids in urine correlates well with exposure of the subject to toluene and mxylene (Pagnotto and Lieberman, 1967; Ikeda and Ohtsuji, 1969; Ogata et al., 1970). Many methods have been used for the determination of these metabolites using colorimetry (Gaffney et al., 1954; Umberger and Fiorese, 1963), fluorometry (Ellman et al., 1961), and ultraviolet spectrophotometry (Pagnotto and Lieberman, 1967). However, some constituents in the urine interfere with the assay and have to be removed by liquid/ liquid extraction and chromatography (Ogata et al., 1962). Where the urine contains both hippuric and m-methylhippuric acids, they can be separated using paper or thin-layer chromatography, with subsequent conversion to azlactones and assay of each constituent (Ogata et al., 1969). Recently, a gas-liquid chromatographic method (Buchet and Lauwerys, 1973) has been reported based on the conversion of hippuric and m-methylhippuric acids for their specific determination. This report describes a high-speed liquid chromatographic method which obviates the necessity for isolating these acids or for converting them before assay. Received for publication 30 June 1976 Accepted for publication 16 June 1977 Materials and methods CHROMATOGRAPHIC SYSTEM AND OPERATING CONDITIONS A Waters ALC 202 high-speed liquid chromatograph equipped with a low-pressure mercury (254 nm) photometric detector was used in this study. Samples were introduced on to the column through a Waters U6K septumless injector. A ,uBondapak C18 prepacked column (Waters Assoc.), which is a reversed-phase column (30cm x 4mm I.D.) containing small-diameter porous silica particles chemically bonded to aliphatic hydrocarbon groups, was used. The column was eluted at 1 ml/min with methanol and 0-01 mol/l KH2PO4 containing 0 5% acetic acid (20/80 by volume) at room temperature. Injections were performed with a 10 ,ul syringe (Hamilton 701N). URINE TREATMENT Extraction of hippuric and m-methylhippuric acids from urine was carried out according to the method described by Ogata et al. (1969). One millilitre of urine, 0 04 ml of concentrated HCI, 0 3 g of NaCl and 4-0 ml of ethyl acetate were shaken vigorously in a glass-stoppered tube for two minutes. After centrifugation for five minutes at 2500 rpm, 0-2 ml of the supernatant ethyl acetate was transferred to a test tube and evaporated to dryness on a water bath 310 group.bmj.com on August 14, 2017 Published by http://oem.bmj.com/ Downloaded from