Quantification of trans-zeatin in corn wastes and liquid organic fertilizers by HPLC chromatography

The trans-zeatin and other phytohormones are used as growth regulators to be induced in plants. Their presence has been determined in corn grains and some plant species, but not in their waste or liquid organic fertilizers, so the objective of this investigation was to quantify the compound by high performance liquid chromatography (HPLC). Ethanol extracts of corn waste and bioinsumos were prepared. Were identified by thin layer chromatography on aluminum chromatoplates Merck 60 F254 10x10cm as stationary phase and methanolacetonitrile (80:20) as mobile phase. Their quantification was performed by HPLC on a C18 column, 250mm x 4 mm (5 μm), and methanol-water (40:60) acidified 5% acetic acid as mobile phase, 0.8 mL.min-1 flow rate and wavelength of 280 ηm. The extract of corn grains was taken as control. Statistical analysis to validate the results obtained was performed in the InfoStac software. The control sample was found to have the highest concentration of trans-zeatin (0.730 mg / g). These results have not been referenced in the literature and allow adding ecological and economic utility of this type of agricultural waste.


Introduction
The trans-zeatin is a natural phytohormone as auxins, abscisic acid, gibberellins, ethylene and other cytokinins are synthesized in plants regulating very important physiological functions of the plant cells (Ló pez et al., 2003).This cytokinin has the ability to accelerate cell division, inducing callus formation, promoting the formation of cotyledons and delaying senescence in leaves (Taiz et al., 2006).
Procedures in plant samples for quantification of trans-zeatin require various preliminary purification steps since phytohormone levels are very low relative to other compounds which copurify with them (Olivella et al., 2001).
Despite the presence of this cytokinin in plants and the use of synthetic fertilizers, actually there is a strong interest in the preparation and use of organic fertilizer with characteristics that improve agricultural productivity.One of them, are liquid organic fertilizers which are a mixture of substances able to improve the physical, chemical and biological properties of soil (INN-Chile, 2004).Are produced by anaerobic fermentation and have a high content of organic matter and microorganisms (Garcí a and González, 2005).
Work performed by bioassays of mass of radish cotyledons, ELISA and high performance liquid chromatography HPLC showed positive results for the quantification of trans-zeatin and other phytohormones in buds of potato tubers (Garcí a-Fló rez et al., 2009), leaves developed of Gerbera jamesonii Bolus cv (Olivella et al., 2001), tuber of Solanum tuberosum L. and Solanum phureja Juz.et Buk (Ortiz et al., 2008).
Studies of organic liquid fertilizers of local production has been quantified other phytohormones such as indole acetic acid (1020.88ppm) and kinetin (3.091 ppm).The results were positive for these phytohormones but are not referenced to quantify trans-zeatin in this type of fertilizers (Manzano et al., 2010).
The trans-zeatin was isolated from extracts of embryos and immature fruits of corn, however there are no studies for corn waste as corn leaves and corncobs (Chong-Maw, 1998), or organic liquid fertilizer produced locally.For this reason, the objective of present investigation was to determine the concentration of trans-zeatin by HPLC chromatography in corn waste and liquid organic fertilizers produced locally.

Materials and Methods
The corn samples used were obtained from corn collected in a supply center of Guayaquil city in March 2011 and three organic liquid fertilizers produced locally which were prepared with different inoculums: rumen content, bovine manure and commercial microorganisms.Evaluations were performed in triplicate for each sample, both of the organic liquid fertilizers and corn waste.

Extraction
Protocols to extraction modified and used in this research were described by (Olivella et al., 2001) and (Ortiz et al., 2008).Extraction was performed by soaking with 0.5 g of each dry sample liquid organic fertilizer and 3.0 g dry weight of corn´s samples.This process included the addition of 50 mL of 0.2M acetic acid-methanol (80:20) and 100 mg/mL of butylated hydroxytoluene solution for 24 hours in absence of light and -17°C.Before to 24 hours at rest the samples were homogenized for 1 hour.The extracts were carried to gravity filtration and centrifugation at 5000 rpm, tempered at 5°C for 10 minutes and then concentrated on a rotary evaporator at 50°C, 600 mmHg vacuum and 120 rpm.Each extract were adjusted to pH=3 with 1N sulfuric acid and liquid-liquid extraction was performed with ethyl acetate to remove pigments.The aqueous extract was recovered by decantation and was brought to a water bath for 48 hours and 70°C to remove traces of solvent.Liquid extracts were diluted in a solution of methanol-acetic acid (30:70) and purified with nylon membrane syringe filters of 25 mm and 45 um.
The extracts obtained for each sample were designated as follows: EAB1 (aqueous extract of fertilizer with rumen contents), EAB2 (aqueous extract of fertilizer with bovine manure), EAB3 (aqueous extract of fertilizer with commercial microorganisms), EAMG (aqueous extract of corn grains), EAMT (aqueous extract of corncobs) and EAMH (aqueous extract of leaves of maize).

Trans-zeatin identification by TLC
The thin layer chromatography TLC was performed in a glass chamber of 28x28 cm, aluminum chromatoplates Merck 60 F254 10x10cm were used as stationary phase where were placed 20 µL of each sample and the chemical standard of trans-zeatin Sigma with No. CAS.1637-39-4.As mobile phase a mixture of methanol-acetonitrile (80:20) was used.The chromatoplates were dried at 40°C for 1 hour, were sprayed with a solution of sulfuric acid-ethanol (95:5) as developer solution and identified after 72 hours using UV light.

Calibration curve of trans-zeatin
For the calibration curve was used the methodology described by (Ortiz et al., 2008), five solutions were prepared with known concentration (500ppm, 400ppm, 200ppm, 100ppm and 50ppm) of chemical standard of trans-zeatin.These solutions were read on the Hewlett Packard HPLC chromatograph with UV detector equipped with a C18 250mm x 4 mm (5 µm) was used as mobile phase methanol-water (40:60) acidified (5% acetic acid) with a rate flow of 0.8 mL.min-1, wavelength of 280 ηm and analysis time of 10 min.With the chromatographic peaks of each solution and their corresponding areas a concentration vs. area graphic was generated.This graphic was used to determine the concentration of each sample and was adjusted by linearization analysis where the correlation coefficient R2 = 0.998 with polynomial trendline of order two was determined.

Trans-zeatin quantitation by HPLC
To determine the chromatographic peaks of the samples the same equipment and conditions in the previous section were employed.

Statistical analysis
The results of each sample were subjected to analysis of variance and Tukey test for comparison of average values between samples with 5% significance, using the InfoStat statistical software, version 2008.

Results
The results for identification by TLC showed the presence of trans-zeatin in each sample of corn waste and liquid organic fertilizers.The solvent mixture used differs from that described by (Garcia et al., 2009), because proportions of solvents referenced not allowed to identify the compound, in fact these quantities were modified to vary the polarity of the mobile phase, but was not obtained positive results.
The Rf average values for each sample have not been referenced and helped identify the presence of the compound (see table 1), because the chemical standard values are relatively close considering an error of ± 0.02 between trans-zeatin and liquid organic fertilizer samples, and an error of ± 0.03 between the trans-zeatin and samples of corn.Figure 1 shows the chromatograms of each samples analyzed by high resolution liquid chromatography HPLC.The chromatograms of each liquid organic fertilizer samples had two overlapping chromatographic peaks without presence of valleys, but not allowed to observe a correct separation of the compounds.In the three extracts the first peak corresponds to trans-zeatin with the following retention times: EAB1 (2.950 min), EAB2 (2.866 min) and EAB3 (2.983 min).The second chromatographic peak corresponding to another compound which co-purified possibly with this phytohormone (Olivella et al., 2001) and each extract had the following retention times EAB1 (3.200 min), EAB2 (3.033 min) and EAB3 (3.183 min).
For corn samples, chromatograms had larger integration areas with respect to liquid organic fertilizers extracts.Overlapping chromatographic peaks with shoulders presence for each sample was detected.In extracts of corn grains the greater amount of trans-zeatin was observed, EAMG (2.666 min) demonstrating where was isolated this phytohormone (Chong-Maw, 1998).In the chromatograms of corn waste the trans-zeatin presence was also observed EAMT (2.750 min) and EAMH (2,800 min).However, in these two extracts the major component is represented by another compound that co-purified too with trans-zeatin EAMT (3,083 min) and EAMH (3.033 min).
Table 2 shows the average concentrations values of trans-zeatin for samples of liquid organic fertilizers and corn waste determined with the peak area of the chromatograms and the equation of the calibration curve (y =-1E-05x2 + 0.185x-9.545).No concentration of the sample EAB1 is presented because the value of area under the peak in the chromatogram for the replicas was less than 51.74 and the result for this sample was negative.
These results showed there are no significant differences between the trans-zeatin concentrations of leaves (0.176 mg / g) and corncobs (0.296 mg / g).However, differences between the samples of liquid organic fertilizers were found and the most efficient bioinsumo was the fertilizer with commercial microorganisms (0.057 mg / g).In both cases, these values have not been referenced and differ to those reported by (Taiz et al., 2010), who explained that increase in the concentration of zeatin with very low molarities (10 M) is directly proportional to the growth of callus of snuff.

Conclusions
The liquid organic fertilizer extract with commercial microorganisms EAB3 presented the highest concentration of trans-zeatin (0.057 mg/g).The presence of trans-zeatin in liquid organic fertilizers extracts set allowed the quantification of this phytohormone as an additional indicator for determining the quality of this type of bioinsumo.Extracts of corn waste had concentrations of transzeatin EAMH (0.296 mg / g) and EAMT (0.176 mg / g) that can be useful for agriculture because of the physiological functions that brings this cytokinin as natural growth regulator in plants.

Table 1 .
Rf average values of trans-zeatin determined by thin layer chromatography TLC

Table 2 .
Trans-zeatin concentrations of liquid organic fertilizer and corn samples obtained by high resolution liquid chromatography HPLC.