Sugar cane (Saccharum officinarum
L.), a widely distributed crop in the world, has many economic dimensions, especially for the sugar industry, in many tropical and subtropical countries [1
]. In many countries where sugar cane is cultivated, general population like to drink sugar cane juice as a delicious, refreshing, and popular beverage. Past research works have revealed several interesting biochemical properties of sugar cane, e.g., certain phenolic analytes and flavonoids in sugar cane were considered to have certain antioxidant activity [1
]. In addition, sugar cane juice contains sugar, vitamins, inorganic minerals and plant growth regulators (i.e., phytohormones). Therefore, sugar cane juice has also been used in the plant tissue culture industry [3
], though not as widely used as coconut water [4
]. For the tissue culture work performed in our group (unpublished data), sugar cane juice supplementation is able to support tissue culture growth. Thus, this work aims to provide some evidence on the phytohormone (e.g., auxin) composition of the sugar cane juice, as such work has not been previously reported. Characterization of phytohormone composition of sugar cane could promote its wider applications in the tissue culture industry.
Indole, an aromatic heterocyclic organic compound, is the potent basic pharmacodynamic nucleus that has been reported to possess a wide variety of biological and clinical properties [6
]. All indole compounds have a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring (Figure 1
). The indole structure can be found in many organic compounds like tryptophan, tryptophan-containing proteins, alkaloids, pigments, etc. Some earlier studies reported that plant tissues contain many indole compounds [7
], which included the most important phytohormone, i.e., auxin. Auxins play various important functions in plant growth and development, such as cell division, elongation, embryo formation, and they also function as ‘chemical signals’ among cells, tissues and organs. Indole-3-acetic acid (IAA) is the main active endogenous auxin in most plants [8
]. IAA is bio-synthesized from the tryptophan or indole, via indole-3-pyruvic acid (IPA), indole-3-acetamide, indole-3-acetonitrile, etc. [8
]. In addition, some other indole compounds have been reported to have beneficial effects in biomedical research involving human health: relief from insomnia, enhanced anticonvulsive activity of antiepileptic drugs, some antioxidant activity, as starting materials for some anti-inflammatory drugs, etc. [13
]. For example, IPA is a potential drug used for treating anxiety and insomnia [13
Considering the low concentrations of indole compounds in plant tissues, different sample preparation methods—mainly including liquid–liquid extraction (LLE) and solid phase extraction (SPE)—have been employed for preconcentration and purification of indole compounds [22
]. Compared with LLE, SPE is a simpler, yet more effective and versatile method for different sample matrices.
Analyses of indoles or phytohormones (e.g., anxins) by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) had been previously reported [23
]. Also, there were some reports on the analyses of indoles and/or phytohormones by capillary electrophoresis (CE) [28
]. Compared with the other chromatographic procedures, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS) have several advantages: high efficiency coupled with high sample capacity; rapid speed of analysis; simplicity of sample recovery; ability to analyze non-derivatized samples, etc. Although HPLC and LC-MS have been successfully used to analyze some indoles or phytohormones [30
], few of these studies attempted to simultaneously analyze and detect different kinds of indole compounds within a complex plant matrix using HPLC or LC-MS.
Current work proposes a simple and sensitive HPLC method for analyzing IAA, IPA, 5 indole carboxylic acid isomers, 3-acetylindole, and indole-3-acetamide in a single analysis. The developed methodology was used to screen for the presence of endogenous indole compounds in sugar cane juice after application of a new SPE method for pre-concentration and purification of indole compounds. The presence of four indole compounds, which included IAA, was further confirmed by LC-MS/MS assay based on its characteristic fragmentation pattern. With HPLC and LC-MS based methods, IAA, IPA, 3-acetylindole, and indole-2-carboxylic acid were successfully identified and quantified.
2. Materials and Methods
2.1. Reagents and Materials
All indole standards were purchased from Sigma-Aldrich (Steinheim, Germany) except IAA, which was obtained from PhytoTechnology Laboratories (Shawnee Mission, KS, USA). Figure 1
shows the chemical structures of the 10 indole compounds. All the standards were dissolved in methanol with the concentration ranging from 250 to 500 μM and stored at temperature 4 °C. Methanol and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid (analytical reagent grade) was purchased from Merck (Darmstadt, Germany). Filter paper (12.5 cm, No. 542, Whatman, Maidstone, Kent, England) was used to filter the sugar cane juice before SPE. Ultrapure water (Milford, MA, USA) was used throughout the analysis. The pH of the sample solution was adjusted to 3 by adding formic acid and monitored using a pH meter (CORNING 440, Corning Glass Works, Corning, NY, USA).
2.2. HPLC Instrumental Set-Up and Procedure
The analysis of indole compounds was performed using a high performance liquid chromatography system (Waters 2695 Separations Module, Waters, Milford MA, USA) linked simultaneously to a PDA system (PDA 2996 detector, Waters). Typically, the PDA system was operated at a detection wavelength of 280 nm. Data were processed by the accompanying system software (Millennium32 Software, Data Handling System for Windows, version 4.0).
The initial HPLC running condition was methanol–formic acid buffer (20:80, v/v). Solvent (A) consisted of 0.1% formic acid (w/v); and solvent (B) consisted of methanol. The flow rate was 0.3 mL·min−1 throughout the whole separation. Separations were performed using a C18 reverse-phase column (Zorbax SB-C18 100 Å, 3.5 μm, 2.1 mm×150 mm, Agilent Technologies, Palo Alto, CA, USA), at 30 °C. The injection volume of standards and purified sugar cane juice samples were 5 μL and 10 μL, respectively.
2.3. LC-MS/MS Conditions
LC (Model 1100 Series, Agilent Technologies) linked with LC/MSD Trap XCT (Agilent Technologies, Palo Alto, CA, USA) by an electrospray ionization (ESI) interface was used in scan mode for standards. The same C18 reverse-phase column and solvents as HPLC were employed with an injection volume of 5 μL. The column thermostat was set at 40 °C.
ESI-MS analysis was performed in the negative mode, and the ion trap was scanned at m/z 50–400 in full scan mode. The maximum accumulation time for the ion trap was set at 200 ms and the target count was set at 100,000. The actual accumulation time was controlled by ion charge control (ICC), which was used to prevent ion saturation in the ion trap. The nebulizer gas pressure, drying gas flow rate, drying gas temperature, and capillary voltage for the ESI source were set at 30 psi, 8 L·min−1, 350 °C, and 3500 V, respectively. Other instrument parameters were optimized for generating the highest signal intensities.
The multiple reaction monitoring (MRM) modes were used to monitor the transitions from the precursor ions to the most abundant product ions. MRM were performed in the smart mode with helium as collision gas. Table 1
showed the MRM transition results of different indole compounds. The data were processed using LC/MSD Chemstation software.
2.4. Retention of Indole Compounds on SPE Cartridges
The SPE experiments were conducted using C18 SPE cartridges (J.T. Baker, Phillipsburg, NJ, USA; 500 mg, 3 mL). In the preliminary experiments, standard solutions containing 10 indole compounds (2 µmol for each standard) dissolved in 100 mL acidified ultra-pure water (pH adjusted to 3 by formic acid) were extracted using SPE, which was previously conditioned with 2 mL methanol, 2 mL methanol-water solution (50:50, v/v), 2 mL methanol-water solution (30:70, v/v), and 2 mL acidified water (pH adjusted to 3 by formic acid) to obtain the optimum extraction conditions. Typically, the cartridge was washed with 5 mL acidified water (pH adjusted to 3 by formic acid), and then eluted with 5 mL methanol-water solution (80:20, v/v). The recovery of each indole standard was measured by HPLC method.
2.5. Sample Preparation
Sugar cane juice was crushed from fresh green sugar cane stems (Malaysia), and the analysis was performed within one week after harvest, since the quality of juice would change upon delayed extraction and storage [36
]. Before further pre-concentration and purification, the pH of 500 mL sugar cane juice was adjusted to 3 by adding formic acid and filtered through filter paper (Whatman, 12.5 cm, No. 542) to remove the suspended matters. The optimum novel SPE procedure using C18
SPE cartridges (Section 2.4
) was applied to pre-concentrate and purify the putative indole compounds. The SPE eluate was lyophilized to dryness, and the dried residue was reconstituted in 1 mL methanol. The collected samples were finally analyzed by HPLC and LC-MS.
2.6. Recovery Study of SPE Procedure
The extraction recoveries of 10 indole compounds using the C18 cartridges under optimum SPE conditions were also investigated. Unlabeled 10 indole standards (0.2 μmol each) were added to 50 mL sugar cane juice, and extracted according to the same sample preparation procedure. The spiked extracted sample was then subsequently analyzed using HPLC measurement.
This study reported the development and validation of new and relatively simple HPLC and LC-MS/MS methods for the simultaneous analysis of various indole compounds with high detection sensitivity. The separation of the 10 standards, including 5 isomers (indole-2-carboxylic acid, indole-3-carboxylic acid, indole-4-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid), was completed in 35 min with well-resolved peaks. Running time was decreased and the separation of different indole compounds was optimized by increasing the strength of the organic solvent. The optimum separation conditions developed in this project will be useful for future studies, as typical separation of different indole compounds including isomers and IAA, being one of the most important auxins in a single analysis was rarely, if not never, reported in the literature.
Furthermore, a new SPE method for the pre-concentration and purification of indole compounds was developed by using C18 SPE columns. The pH of water solution used for the washing step and the composition of eluent were also optimized to achieve higher recovery rates by reducing the effects of the interfering substances in the final extraction.
The effectiveness of the new pre-concentration SPE method and analytical HPLC method was evaluated by screening for the putative indole compounds in sugar cane juice. The presence of indole compounds in sugar cane juice, first detected by HPLC, was further confirmed independently by LC-MS/MS experiments. The results from the HPLC and LC-MS/MS indicated the presence of IAA, 3-acetyindole, IPA, and indole-2-carboxylica acid in sugar cane juice samples. This is also the first report of indole compounds in sugar cane juice. The detection of different indole compounds is valuable in plant science and biomedical science, due to the different important biological functions of these indole compounds. The presence of IAA in sugar cane juice can partially explain why sugar cane juice can be used as a growth supplement in plant tissue culture. The presence of IPA in sugar cane juice can partly confirm the relationship between IAA and IPA. In addition, as IPA has medically useful properties such as bringing relief from anxiety and insomnia [13
], sugar cane juice may be a good beverage for people who suffer from sleep related problems. The presence of 3-acetylindole and indole-2-carboxylic acid may provide more potential usage of sugar cane juice besides being the main ingredient for the sugar industry. It is also plausible that some other indole compounds may be present in sugar cane juice, which we were unable to detect due to the current detection limits of current HPLC and LC-MS/MS methods.
Nevertheless, due to the important bioactivities driven by indole compounds, more work should be carried out on indole compounds naturally present in plants. Moving forward, UPLC-TOF-MS analytical approach can also be evaluated in order to develop shorter analytical duration, higher separation resolution, and higher sensitivity for the indole compounds [39
]. Furthermore, the outstanding performances provided by core-shell particles column on a traditional HPLC instruments are comparable to those obtained with a costly UPLC instrumentation, making this novel column a promising tool for separation of indole compounds [40
]. Also, sugar cane juice can be analyzed for the other bioactive compounds, such as other phytohormones.