A simple method for simultaneous RP-HPLC determination of indolic compounds related to bacterial biosynthesis of indole-3-acetic acid
In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (λex = 280/λem = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.0625–125 μg mL−1 (r2 ≥ 0.998) and the limits of detection were below 0.015 μg mL−1. The applicability of the method was confirmed by analysis of Pseudomonasputida culture supernatants.
KeywordsHPLC Indolic compounds IAA biosynthesis
Among the available methods to determine IAA and related compounds, spectrophotometric (Akbari et al. 2007; Karnwal 2009; Sahasrabudhe 2011), thin layer chromatography (TLC) (Hartmann et al. 1983; Robinson et al. 1998; Swain et al. 2007) and HPLC assays are used most frequently. Spectrophotometric methods, which are based on the reaction of indoles with the Salkowski reagent and compare the color development of the reaction mixture with an appropriate reference, are nonspecific and provide only quantitative determination of the total indoles content, rather than each of the analytes individually (Glickmann and Dessaux 1995). TLC methods, on the other hand, are used only for qualitative analysis. For both qualitative and quantitative determination of IAA and related 3-substituted indoles, several HPLC methods have been developed. However, these methods are complex and time- and reagent-consuming. First, sample preparation consists of several steps involving the repeated organic solvent extraction of an acidified bacterial culture supernatant (Garcia-Tabares et al. 1987; Sergeeva et al. 2007; Khakipour et al. 2008; Fedorov et al. 2010). It is worth noting that the extraction of TAM and indole-3-ethanol (TOL) from acidic solutions is difficult, and these compounds have been independently extracted from neutral or basic solutions (Crozier et al. 1988; Furukawa et al. 1996; Carreno-Lopez et al. 2000). Second, for HPLC separation of the studied group of indolic compounds, two independent runs with two different sets of eluents have been conducted (Manulis et al. 1994; Carreno-Lopez et al. 2000; Reineke et al. 2008).
Thus, the aim of the present study was to develop a fast, simple, and reliable method for the simultaneous preparation and a single-run RP-HPLC determination of indolic compounds related to bacterial IAA production. In our study, we included IAA (product), Trp (precursor) and all five known and stable intermediates of IAA biosynthesis: IAM, IAN, indole-3-lactic acid (ILA), TAM and TOL. ILA and TOL are products of the enzymatic reduction of IPyA and indole-3-acetaldehyde (IAAld), respectively. These latter two compounds are unstable and do not accumulate in bacterial cultures (Carreno-Lopez et al. 2000).
Materials and reagents
Indole-3-acetic acid and l-tryptophan were purchased from Roth (Karlsruhe, Germany). Indole-3-acetamide, indole-3-acetonitrile, tryptamine, DL-indole-3-lactic acid, indole-3-ethanol and 3-kDa cut-off membrane centrifugal filters (Amicon Ultra 0.5 ml centrifugal filters Z677094) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol were purchased from POCH SA (Gliwice, Poland). All aqueous solutions were prepared using ultra-pure Milli-Q water.
Preparation of calibration curves and spike recovery analysis
The standard stock solutions (15 mg mL−1) of IAA, IAM, IAN, ILA, TAM and TOL were prepared in methanol. The standard Trp stock solution (15 mg mL−1) was prepared in water. A series (n = 13) of working solutions for each standard, ranging in concentration from 0.0625 to 125 μg mL−1, were prepared by appropriate dilution of the stock solution with methanol. All standard solutions were analyzed in triplicate. Calibration curves were constructed by performing a linear regression analysis of the peak area versus the analyte concentration. The limit of detection of each analyte was calculated from the chromatograms at a signal-to-noise ratio of 3.
To determine the recovery of the studied indolic compounds from bacterial broth during sample preparation, standards were spiked into sterile King B liquid medium (King et al. 1954) at three different concentrations. Bacterial broth was processed with the sample preparation procedure described below.
Bacterial strain and culture conditions
Pseudomonas putida strain A, used in this study, was obtained from the collection of the Department of Biochemistry, SGGW, and was identified using the ribotyping method by Blirt S.A. DNA (Gdańsk, Poland). The 16S rRNA gene sequence of this strain is deposited in the DDBJ database under accession number AB667903. The bacteria were cultivated for 72 h in King B liquid medium with 0.5, 1.0, 2.0, 3.5 and 5.0 mM Trp supplementation. Bacterial cultures were then centrifuged, and the bacterial culture supernatants were processed with the sample preparation procedure described below.
Sample preparation consisted of a single centrifugal filtration step using 3-kDa cut-off membrane centrifugal filters. For this purpose, 0.5 mL of bacterial culture supernatants or spiked sterile bacterial broths were transferred to the sample chamber of a 0.5 mL centrifugal filter tube and centrifuged at 14,000×g (relative centrifugal force) at 4 °C for 30 min. The filtrates were directly analyzed by HPLC.
Instrumentation and chromatographic conditions
The HPLC system was composed of a binary pump (Model 1525, Waters Corporation, Milford, MA, USA), a fluorimetric detector (Model 474, Waters), an autosampler (Model 717plus, Waters) and a personal computer with Breeze data acquisition and integration software (Waters). Chromatographic separations were performed at ambient temperature on a C8 column (Symmetry 4.6 × 150 mm, 5 μm, Waters) fitted with a C8 guard column (Symmetry 3.9 × 20 mm, 5 μm, Waters) using gradient elution. Eluent A consisted of 2.5 : 97.5 % (v/v) acetic acid : H2O, pH 3.8 (the pH was adjusted by addition of 1 mol L−1 KOH) and eluent B consisted of 80 : 20 % (v/v) acetonitrile : H2O. The mobile phase started with eluent A : eluent B at 80 : 20 %, changing to 50 : 50 %, 0 : 100 % and 80 : 20 % in 25, 31 and 33 min, respectively. The total run time was 36 min. The flow rate of the mobile phase was 1 mL min−1, the injection volumes were 20 μL, and the fluorimetric detector was set to excitation and emission wavelengths of 280 and 350 nm, respectively.
Results and discussion
Calibration curves parameters and the limits of detection of studied indolic compounds
Retention time (min)
The linear regression equation
Coefficient of determination (r2)
Limit of detection
3.5 ± 0.04
y = 1090.1x + 469.0
5.9 ± 0.04
y = 1547.4x + 495.1
7.7 ± 0.06
y = 1751.9x – 1044.8
9.3 ± 0.07
y = 2548.4x + 1442.3
13.8 ± 0.09
y = 2346.1x − 1116.3
15.5 ± 0.10
y = 3437.4x + 1618.7
24.1 ± 0.10
y = 4132.5x + 2080.0
Recoveries of the indolic compounds from the spiked King B liquid medium (n = 4)
98.83 ± 2.1
104.67 ± 1.9
97.45 ± 1.6
94.24 ± 3.2
99.44 ± 2.0
98.38 ± 3.5
96.08 ± 4.5
99.74 ± 2.1
100.26 ± 2.4
96.70 ± 4.6
102.44 ± 2.3
99.77 ± 1.9
100.30 ± 4.1
99.84 ± 2.8
101.4 ± 2.7
94.09 ± 4.2
99.87 ± 3.1
98.62 ± 2.6
92.43 ± 4.0
94.77 ± 2.3
95.53 ± 2.9
The method described here, compared to those applied previously, significantly simplifies both the sample preparation and HPLC analysis of IAA and related indolic compounds in bacterial culture supernatants. First, a simple one-step sample preparation procedure, taking advantage of the fluorescent properties of indoles, provides adequate sample purity, selectivity and sensitivity. Second, the developed and optimized chromatographic conditions allow for determination of a investigated range of indolic compounds in a single chromatographic run using one set of eluents. Therefore, the presented method should be especially useful for routine assays.
This work was supported by the Polish Ministry of Science and Higher Education (Grant No. N N310 304639).
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