A modification of a method to determine adenosine nucleotides in forest organic layers and mineral soils by ion-paired reversed-phase high-performance liquid chromatography

Tn the past, difficulties in the determination of microbial adenylate in forest soils were due to acidity of soils and high humus content. Therefore, a method was developed that allows separation of adenylates from impurities on highperformance liquid chromatography and quantitative extraction from soil. For extraction, samples were stirred with dimethyl sulfoxide and a 0.01 M Na3PO4/0.02 M EDTA buffer (adjusted to pH 12) was added. An aliquot of soil suspension was mixed with an equal volume of nucleotide releasing reagent and then sonified. The suspension was passed through a membrane filter (45 mu m). The extracts were reacted with chloroacetaldehyde at pH 3.5 to form the fluorescent 1,N-6-etheno-derivatives for fluorometric determination. The separation of the 1,N-6-etheno-adenylates (E-adenylates) was performed using a reversed-phase high-performance liquid chromatography. The adenylate derivatives were eluted on a ODS column with 0.05 M ammonium acetate, 1.0 mM EDTA and 0.4 mM tetrabutylammonium hydrogen sulfate (pH 6.3) mixed with methanol (water phase-methanol 89.5:10.5 v/v) as a mobile phase. Column temperature was set to 26-28 degrees C. For quantitative determination, the fluorometric emission was measured at 410 nm with 280 nm as excitation wavelength. This procedure resulted in recovery rates of 91%, 91% and 93% for added AMP, ADP and ATP, respectively in acidic soils as well as in clay rich soils and organic layers. The soil microbial adenylate can be determined with an accuracy of 10%. (C) 1997 Elsevier Science B.V.