QUANTIFICATION OF 5-AMINOLEVULINIC ACID IN SWINE MANURE EXTRACT BY HPLC-FLUORESCENCE

Abstract

A modified high performance liquid chromatography (HPLC)-fluorescence method for determination of the 5-aminolevulinic acid (ALA) concentration in swine waste was studied. The method included digestion of the sample by heating in aqueous HCl and derivatization by acetyl acetone/formaldehyde reagent. The derivative was isolated and detected by fluorescence with excitation at 370 nm and emission at 460 nm. Chromatographic separation of ALA was carried out using the optimum mobile phase of a methanol/water/glacial acetic acid (49.5:49.5:1, v/v/v). The method produced a limit of detection of the ALA concentration of 0.10 µg/mL and a limit of quantification of the ALA concentration of 0.31 µg/mL. The method was used for determination of the ALA concentration in concentrated swine manure extract (CSME) fermented for 0, 7, 14, 21, and 28 d. The ALA concentrations in the CSME fermented for 0, 7, and 14 d were low and ranged from 0.216 to 0.399 µg/mL, but increased to a level ranging between 1.240 and 1.295 µg/mL at 21 and 28 d of fermentation. The results of the study confirmed the presence of ALA in the swine waste. The ability to quantify the ALA concentration in swine waste developed by the study was confirmed.

Keywords:

• 5-aminolevulinic acid
• analysis
• derivatization
• high performance liquid chromatography-fluorescence
• method validation
• swine manure extract

ACKNOWLEDGMENT

The authors wish to thank the Animal Nutrition Research and Development Center, the Suwanvajokkasikit Animal Research and Development Institute, Kasetsart University, Kampaeng Saen campus, Nakhon Pathom; the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Kasetsart University, Bangkok for financially support; and the Central Laboratory and Greenhouse Complex, Kasetsart University, Kampaeng Saen, Nakhon Pathom for chemical analysis of the swine manure extract.

Notes

*Significant difference between two AA reagent formulae (P ≤ 0.05) by statistical t-test (n = 6).

ALA, 5-aminolevulinic acid; CSME, Concentrated swine manure extract.

^a Samples submitted to the hydrolysis step.

ALA, 5-aminolevulinic acid; RSD, Relative standard deviation.

^a Samples submitted to the hydrolysis step.

RSS, Residual sum of squares; LOD, Limits of detection; LOQ, Limits of quantification.

CSME, Concentrated swine manure extract; RSD, Relative standard deviation.

*Significant difference between two AA reagent formulae (P ≤ 0.05) by statistical t-test (n = 3).

CSME, Concentrated swine manure extract.

^a,b Means in the same row with different superscripts differ significantly (P ≤ 0.05) (n = 6).