# Identification and Isolation Techniques for Plant Growth Inhibitors in Rice
## 6 Current Status, Existing Limitations, and Future Perspectives
### Current Status and Existing Limitations
#### 6.1.2 Identification, Qualitative and Quantitative Analyses  
After the extraction steps, the identification and quantification steps of rice allelochemicals are characterized by the use of a variety of techniques including spectroscopic techniques (UV-Vis, IR, NMR, X-ray crystallography, MS) and chromatography techniques (TLC, CC, HPLC, UPLC, HILIC). UV-vis spectroscopy is a commonly used method for not only polyphenols and flavonoids, but also other various biologically active compounds due to their wide range of wavelengths, short duration, and economical nature. However, the PGIs in the rice plant extract usually remain as an integration of a mixture of bioactive compounds with different types of extract solvent, and the use of spectroscopic analysis is not selective and sensitive for the qualification and quantification of individual PGI compounds. As a result, these factors cause the separation, identification, and characterization provided by UV-vis spectroscopy to become a significant issue. To address this issue, mass spectrometry was developed as a widely used and reliable technique. In addition, the combination of MS with chromatography techniques such as LC or GC has become one of the most powerful options for the identification and quantification of PGIs in rice due to its sensitive and selective nature. Specifically, even if the pure standard is unavailable, the application of LC with tandem mass spectrometry can provide abundant information for the elucidation of the compound structure. However, the requirement for the sample preparation to be in a gas or liquid form for the ionization sources is one of the MS drawbacks. Furthermore, mass spectrometric equipment is expensive and requires highly competent operators. Among the chromatography techniques, HPLC is the most frequently used technique for the isolation and analysis of polyphenols and organic acids found in rice, since it is the most high-resolution, efficient, high-precision, and robust technique, with no derivatization and no restrictions on the sample volatility technique when compared to other methods. Currently, the demand for environmentally friendly and robust techniques is significantly increasing. This led to the development of ultrahigh-performance liquid chromatography (UHPLC). Results from UHPLC show that it produces higher peak efficiency and chromatographic resolution than traditional HPLC. Additionally, since UHPLC techniques usually require 80% less chemical solvent than traditional HPLC techniques, they may be considered more economical. Both HPLC and UHPLC can be conveniently connected to a range of detectors, such as UV-Vis, DAD, and MS. On the other hand, a limitation of the LC system is that it requires a high-cost strategy. In addition, a large amount of organics is needed for LC operation. Muddled situations can occur in investigating issues or developing new methods that need multiple replicated confirmations. In addition, the UV-vis detector is able to identify only chromophoric compounds. In the case of LC-MS, the matrix's complexity entering the MS ion source cannot be controlled by the operators. Therefore, fluctuations in the matrix composition can lead to a reduction in ion yield. Accordingly, matrix complexities are important determinants in LC-MS analysis since they may occur in individual samples and can consequently lead to inaccurate results in these specimens. Therefore, validation of their occurrence and absence must be indicated in applying relevant assays. The obtained results should also be carefully confirmed. Further strategies should focus on minimizing their occurrence through appropriate solutions.