Application of Fourier transform infrared (FTIR) spectroscopy coupled with multivariate calibration for quantitative analysis of curcuminoid in tablet dosage form

1.ABSTRACT
Curcuminoid, mainly curcumin (CUR) and desmethoxycurcumin (DMCUR), with chemical structures as shown in Figure 1, has been reported to have some biological activities including antioxidant, anticancer and anti-inflammatory (Rohman, 2012). Curcuminoid has been used as chemical markers during biological activity studies related to Curcuma genus. Some pharmaceutical products containing Curcuma extracts has been commercially available in Indonesian markets such as Curcuma syrup (Wahyono and Hakim, 2007), capsule, and tablet formulations (Rajashree et al., 2013). Therefore, determination of curcuminoid in those formulations was needed to assure the quality of curcuminoid contained in pharmaceutical products. Chromatographic methods including chromatography with ultraviolet-visible detector (Syed et al., 2015), photo-diode array detector (Zhang and Acworth, 2013), and electrochemical detector (Long et al., 2014) has been reported for analysis of curcuminoid due to its capability to provide separation of individual curcuminoid (Siregar et al., 2017). However, chromatographic methods need more time and efforts, therefore, some simple methods based on spectroscopic methods have been introduced to overcome these obstacles. UV spectrophotometry is a method of choice for determination of curcuminoid in a formulation containing pure curcuminoid (Sharma et al., 2012), but this method is not suitable for products containing curcuminoid in plant extracts. Due to much peaks obtained to be used as variables, Fourier transforms infrared (FTIR) spectroscopy has been proposed for the analysis of analytes in a complex composition including curcuminoid in the extracts.

FTIR spectroscopy, based on the interaction between electromagnetic radiation currently in infrared region and samples, in combination with several chemometrics techniques, has emerged as powerful analytical tools in the pharmaceutical application (Chakraborty, 2016) due to its property as fingerprint spectra (Sim et al., 2004). In herbal medicine application, the combination of FTIR spectroscopy and chemometrics have been used for quantification of active pharmaceutical ingredients (Rohman, 2013), for discrimination between wild-grown and cultivated Ganoderma lucidum, an expensive herbal component commonly used in Chinese traditional medicine (Zhu and Tan, 2015), authentication of geographical origin of Gentiana rigescens commonly used as liver protective in traditional Chinese medicine (Wu et al., 2017) and for quality assurance of herbal medicine (Rohman et al., 2014). FTIR spectroscopy combined with partial least square and principal component regression has been used for quantification of curcuminoid in extracts of Curcuma longa (Rohman et al., 2015) and Curcuma xanthorrhiza (Lestari et al., 2017). The reported publication regarding curcuminoid analysis, so far, was in extracts or powder and using literature review, there are no reports related to the quantitative analysis of curcuminoid in tablet formulation. Therefore, in this study, FTIR spectroscopy at specific infrared region combined with multivariate calibration was optimized for quantitative analysis of curcuminoid (CUR and DMCUR). MATERIALS AND METHODS Curcumin (CUR) and desmethoxycurcumin (DMCUR) were isolated from commercial curcuminoid purchased from E. Merck (Darmstadt, Germany). Isolation was performed following method as described in Lestari et al. (2017). Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC) were used to check the purity of CUR and DMCUR. The purity of CUR and DMCUR was performed using internal normalization technique. Tablet samples were purchased from several pharmacies around Yogyakarta, Indonesia. The placebo of samples was kindly given by PT. SOHO Pharmaceutical Industry (Jakarta, Indonesia).

FTIR spectroscopy analysis

The powdered tablet samples were placed on Smart iTR™ Attenuated Total Reflectance (ATR) accessory composed of diamond crystal as sample handling technique at a controlled ambient temperature (25°C). Samples were scanned using Nicolet iS10 FTIR spectrophotometer (Thermo Fisher Scientific Inc, Madison, USA) equipped with deuterated triglycine sulfate (DTGS) detector and potassium bromide (KBr)/Germanium as a beam splitter. The instrument was connected to software OMNIC ver.9.7 and spectra were scanned at wavenumbers of 4000-650 cm−1, recorded for 32 scans at a resolution of 8 cm−1. The air spectrum was used as background. Each data point was recorded in three replicates using absorbance mode to facilitate quantitative analysis (Rohman et al., 2014). Chemometric analysis Multivariate analyses consisted of partial least square regression (PLSR) and principal component regression (PCR) were performed using software TQ Analyst ver.9.7 (Thermo Fisher Scientific Inc., Madison, WI) included in Nicolet iS10 FTIR instrument. PLSR and PCR were used to build a predictive model which correlated the actual values of CUR and DMCUR from HPLC determination and FTIR predicted values. Statistical parameters namely coefficient determination (R2 ), Root Mean Square Error of Calibration (RMSEC) and Root Mean Square Error of Predicted (RMSEP) were computed using TQ Analyst software.

RESULTS AND DISCUSSION

HPLC, due to its capability to be used for qualitative, quantitative and preparative analyses, is a standard method for analysis of active components in herbal medicine including determination of curcumin (CUR) and desmethoxycurcumin (DMCUR) in plant extracts (Prabaningdyah et al., 2017). Figure 2 revealed HPLC chromatogram for separation and quantification of CUR and DMCUR in some tablet samples containing C. xanthorrhiza in its formulation. However, HPLC is timeconsuming and needs skillful analysis. Therefore, in this study, FTIR spectroscopy was developed for routine analysis of CUR and DMCUR in any pharmaceutical products. The levels of CUR and DMCUR in tablet formulation used as actual values of CUR and DMCUR were determined by HPLC using photo-diode array detector at λ 425 nm, and their results obtained were compiled in Table 1. The variation of CUR and DMCUR compositions in evaluated tablets was coming from the addition of tablets with placebo to facilitate calibration models during FTIR spectroscopic analysis. FTIR spectra of a tablet containing extract of C. xanthorrhiza with curcuminoid as active components were depicted in Figure 3. Each peak was corresponding to a functional group present in two main curcuminoids present in C. xanthorrhiza, namely CUR and DMCUR (Lestari et al., 2017). The clear and broad peak at wavenumbers (1/λ) of 3200 cm−1 corresponded to stretching vibration of hydrogen-bonded (-OH) present in curcuminoid, while peaks at 2950 and 2900 cm−1 originated from stretching vibrations of methyl (CH3 ) and methylene (CH2 -) groups, respectively. The bending vibrations of CH3 and CH2 were also observed at 1/λ 1339 and 1423 cm−1, respectively. Conjugated carbonyl group was observed at 1/λ 1655 cm−1, lower than 1/λ in unconjugated carbonyl (Prabaningdyah et al., 2018). Table 2 compiled the functional groups responsible for IR absorption of tablet placebo spiked with Curcuma xanthorrhiza. The presence of these functional groups as indicated in each peak in FTIR spectra proved that the studied tablet contained Curcuma extract.

CONCLUSION

FTIR spectroscopy in combination with multivariate analysis can be used as an alternative technique for quantitative analysis of CUR and DMCUR in table dosage form. The accuracy and precision of FTIR spectroscopy assisted with PLSR were acceptable. This developed method was rapid and suitable for routine analysis. However, if the composition of tablet used was different, a new model must be developed, and indeed the model was also validated.