João Benhur Mokochinski1, Daniely Xavier Soares2, Roy E. Bruns2, Paulo Mazzafera1,
Alexandra C.H.F. Sawaya1
[email protected]
Departamento de Biologia Vegetal, Programa BTPB , Instituto de Biologia, UNICAMP
Instituto de Química, UNICAMP
Amino acids (AA) are fundamental structures of cellular metabolism as building blocks of proteins.
Thus, they are directly related with important metabolic functions, which result in plant growth
and development. Several studies have reported on the determination of AA content in plants.1-3
Although they are soluble in aqueous solutions, there is not a well established method for the
efficient extraction of these substances. Thus, the aim of this work was to optimize the extraction
conditions of free amino acids in plants by a 23 factorial design. We verified the simultaneous
effect of the solvent (methanol and water), pH (4,5 to 8,5), time of extraction (15 to 45 min) and
their interactions. All extraction assays were randomly performed to determine which procedure
was the most efficient to extract free AA from leaves and roots of Coffea arabica. The answer of
the factorial design was the sum of the areas of all AA quantified in the extracts. The conclusion of
this optimization procedure can be applied to extract AA from other plant material. Ground
samples were extracted according the conditions cited in the factorial design using ultrasonic bath.
The extract was centrifuged at 8,000 rpm for 10 min and the supernatant was collected into a vial
and analyzed directly on a Waters Acquity UPLC-MS. Chromatographic separation was carried out
by a Waters Acquity C18 BEH analytical column (150 mm × 2.1 mm i.d., 1.7 µm) at 30ºC. Methanol
(A) and 0.1% formic acid in water (B) were used as mobile phases in a flow rate of 0.2 ml/min.
The gradient was 1% of A and 99% of B for 2.5 min, followed by change to 50% A and 50% of B,
until 5 min and then returning at the initial condition and stabilizing in 8 min. ESI(+)-MS and
tandem ESI(+)-MS/MS were obtained under the following conditions: capillary 3.5 KV, cone 30 V,
source and desolvation temperature were 150 and 300ºC, respectively. For ESI(+)MS/MS, the
energy for the collision induced dissociations (CID) was 15 eV. Data were acquired in the m/z 50 300 range. Ions were identified by the comparison of their m/z, retention time and ESI(+)MS/MS
dissociation patterns with pure standards. We constructed confidence intervals to assess the
significance of the effects of variables by the standard error and the Student distribution. No
significant effect of the variables tested and their interactions was observed for the leaf extracts
(on 95% of CI). For root extracts, solvent and pH were significant. The best result was achieved
using methanol as solvent extractor and at a neutral pH. This can be explained by the efficiency of
methanol in permeating plant tissues, which exposed the constituents for extraction. As time was
not a significant variable, ultrasonic bath can be used for only 15 min allowing a rapid extraction.
Furthermore, leaf extracts showed a higher response than the root extracts, as higher
concentrations of amino acids are present in leaves than roots due to differences in the
metabolism of both plant organs.
[1] Nimbalkar, M.S., Pai, S.R., Pawar, N.V., Oulkar, D., Dixi, G.B. Food Chemistry. 2012, 2565-2569.
[2] Gu, L., Jones, A.D., Last, R.L. Analytical Chemistry. 2007, 79, 8067-8075.
[3] Özcan, S., Senyuva, H.Z., (2006). Journal of Chromatography A. 2006, 1135, 179-185.