**6. Fourier transform infrared spectrum and 2DIR correlation spectroscopy for complex mixture analysis**

Today, use of combo medicinal contents has been reported to be more efficient compared to single compound when treating viral diseases. Research on the effects of combination of different substances has been reported *in vitro* and *in vivo*. This method is claimed to be effective against mutating virus. Several medicinal products are known to kill selectively and prevent the spread of the infection virus. Many possibility of combination of medicinal products exist [10], e.g. raw and the fractions, fractions from different plants, fractions plus compound and raw with compounds. Identification of this kind of the contents in this type of combination is not a priority as long as the efficacy is enhanced and patient acceptance is good. On the other hand, analysis of the interaction of components in the combination is crucial to generate information for the design of new medicinal product in future. The 2DIR correlation spectroscopy plays a vital role in the combo medicine interpretation.

### **6.1. Difficulty in interpretation**

Analysis of the complex mixture of samples required pre-knowledge of the main compounds involved. The addition of different types of standards is needed to determine the assignment of bond in 1D FTIR. There could be no actual matching of standards peak with the complex mixture because of the overlapping and interruption of the bonds from each other. Consequently, more standards or fractions need to be used for different scope of comparison. The additional library in the software could be helpful in this aspect. When the peaks have been confirmed, then the next step will be carried out as usual. The 2DIR with synchronous spectrum is the best option to investigate the interaction of mixture under the appropriate perturbation.

### **6.2. Level of sensitivity**

It is often difficult to address the interaction of a mixture as it may contain totally dissimilar types of compounds. However, they can react concurrently when perturbation is applied. The degree of sensitivity, either from the user or the system is very much relied on the experience and practice, e.g. two possible interpretations based on the similar spectrum. Therefore, the analysis on the mixture with FTIR and 2DIR must be accurate and detailed initially to prevent subsequent misinterpretation.

### **6.3. Strengthening of data**

It is not advisable to use the asynchronous spectrum in 2DIR for the mixture due to the tremendous overlapping of assignment which is confusing. Other types of data generated from HPTLC, HPLC, GCMS, LCMS and NMR could be additional evidences for mixture complex analysis.
