Determination of p-aminobenzoic acid and its metabolites in rabbit plasma by high-performance liquid chromatography with fluorescence detection

Dah Jing Song, Kuang Yang Hsu

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

A simple, accurate and sensitive high-performance liquid chromatographic method with fluorescence detection was used for measuring plasma concentrations of p-aminobenzoic acid (PABA) and its three metabolites: p-acetaminobenzoic acid (PAABA), p-aminohippuric acid (PAHA) and p-acetaminohippuric acid (PAAHA). A Cosmosil MS-C18 column (250X4.6 mm, 5μm) was used under temperature control at 40°C. The mobile phase was H2O-CH3CN-CH3COOH (100:3:1, pH 4.0) with a flow-rate of 1.5 ml/min. The excitation and emission wavelengths for fluorescence detection were set at 270 and 350 nm, respectively. Plasma samples (200 μl) were acidified by the addition of 150 μl of 1 M HClO4 solution containing salicylic acid (SA) as the internal standard. After centrifugation, 30 μl of the supernatant were injected onto the column. Using this method, PABA and its three metabolites could be determined within 25 min. Within the investigated concentration ranges of PABA (0.1-50 μg/ml), PAABA (0.2-50 μg/ml), PAHA (0.1-50 μg/ml) and PAAHA (0.5-50 μg/ml), good linearity (r>0.99) for the standard curves was obtained. The validation of this method showed coefficient of variance (C.V.) that was well below 15% for all compounds. After intravenous (i.v.) administration of PABA (20 mg/kg) to rabbits (n=7), PABA followed a one-compartment open model elimination with a half-life of 10.90 ± 1.03 min. The mean half-lives for PAABA, PAHA and PAAHA were 24.61 ± 6.42, 12.81 ± 6.04 and 11.27 ± 2.77 min, respectively.

Original languageEnglish
Pages (from-to)69-75
Number of pages7
JournalJournal of Chromatography B: Biomedical Applications
Volume677
Issue number1
DOIs
Publication statusPublished - Feb 23 1996

Fingerprint

4-Aminobenzoic Acid
High performance liquid chromatography
Metabolites
p-Aminohippuric Acid
Fluorescence
Plasmas
Acids
Salicylic Acid
Centrifugation
Temperature control
Flow rate
Wavelength
Liquids

Keywords

  • p-Acetaminobenzoic acid
  • p-Acetaminohippuric acid
  • p-Aminobenzoic acid
  • p-Aminohippuric acid

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Determination of p-aminobenzoic acid and its metabolites in rabbit plasma by high-performance liquid chromatography with fluorescence detection. / Song, Dah Jing; Hsu, Kuang Yang.

In: Journal of Chromatography B: Biomedical Applications, Vol. 677, No. 1, 23.02.1996, p. 69-75.

Research output: Contribution to journalArticle

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abstract = "A simple, accurate and sensitive high-performance liquid chromatographic method with fluorescence detection was used for measuring plasma concentrations of p-aminobenzoic acid (PABA) and its three metabolites: p-acetaminobenzoic acid (PAABA), p-aminohippuric acid (PAHA) and p-acetaminohippuric acid (PAAHA). A Cosmosil MS-C18 column (250X4.6 mm, 5μm) was used under temperature control at 40°C. The mobile phase was H2O-CH3CN-CH3COOH (100:3:1, pH 4.0) with a flow-rate of 1.5 ml/min. The excitation and emission wavelengths for fluorescence detection were set at 270 and 350 nm, respectively. Plasma samples (200 μl) were acidified by the addition of 150 μl of 1 M HClO4 solution containing salicylic acid (SA) as the internal standard. After centrifugation, 30 μl of the supernatant were injected onto the column. Using this method, PABA and its three metabolites could be determined within 25 min. Within the investigated concentration ranges of PABA (0.1-50 μg/ml), PAABA (0.2-50 μg/ml), PAHA (0.1-50 μg/ml) and PAAHA (0.5-50 μg/ml), good linearity (r>0.99) for the standard curves was obtained. The validation of this method showed coefficient of variance (C.V.) that was well below 15{\%} for all compounds. After intravenous (i.v.) administration of PABA (20 mg/kg) to rabbits (n=7), PABA followed a one-compartment open model elimination with a half-life of 10.90 ± 1.03 min. The mean half-lives for PAABA, PAHA and PAAHA were 24.61 ± 6.42, 12.81 ± 6.04 and 11.27 ± 2.77 min, respectively.",
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