ANALYTICAL METHODS FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF OXITURMERIC® CURCUMINOIDS
OxiTurmeric® curcuminoids are analyzed by HPLC-DAD coupled to ESI-tandem mass spectrometry. The accurate quantitation of the curcuminoids curcumin (CUR), demethoxycurcumin (DMC), and bis-demethoxycurcumin (BDMC) of OxiTurmeric® is based on the AOAC Official Method 2016.16. After extraction with methanol and due dilution, the extract is analyzed by HPLC by setting the detector to 425 nm, the C18 (2.6 μm, 2.1 × 30 mm) column to 55°C temperature, using an injection volume of 0.8 μL and a flow rate of 1.4 mL/min. The actual concentration of the curcuminoids is then calculated based on calibration curves obtained on pure standards. The identification of the curcuminoids is carried out by HPLC-DAD-ESI-MS / MS.
Very often we are used to see claims of 95-98% curcuminoids. Here we report an example of a turmeric sample claiming 98% curcuminoids that eventually was found to possess a real percentage of 63% total curcuminoids. In a few step we will show how a typical analysis of curcuminoids is done.
The typical chromatogram obtained from a commercial turmeric extract that claims 98% curcuminoids is reported in Figure 1
Figure 1. Typical chromatogram from a turmeric sample claiming 98% curcuminoids
By reading at a fixed wavelength (425 nm) it is expected that only curcuminoids appear in the chromatogram. Therefore, if we calculate the total percentage of the curcuminoids shown in Figure 1 we obtain the results of Table 1, where actually the total curcuminoids percentages is even higher than the declared 98%.
Table 1. Total percentage of peak areas from Figure 1
|Compound||Retention Time (min)||Calculated Area||Area Percentage|
A common mistake is to consider the percentages of Table 1 as a quantitative measure of the turmeric curcuminoids. In order to quantify the real content of curcuminoids quantitation is performed with external calibration against reference standards for CUR, DMC, and BDMC, according to the AOAC Official Method 2016.16. In order to obtain these curves, the analyte concentration (x-axis) is plotted versus individual integrated peak area (y-axis) for CUR, DMC, and BDMC. The least-squares analysis is used to determine the slope, intercept, and determination coefficient (R2) of the best-fit line for each analyte.
Figures 2-4 show the typical calibration curves obtained on pure CUR, DMC, and BDMC standards, respectively.
Figure 2. calibration curve obtained with pure Curcumin standard.
Figure 3. calibration curve obtained with pure Demethoxycurcumin standard.
Figure 4. calibration curve obtained with pure Bis-demethoxycurcumin standard.
Based on these external calibration curves, the amount of curcuminoids in the original sample is calculated as follows:
where C = concentration of the analyte from the standard curve; V = extract volume; W = weight of the test portion (g); and D = dilution factor.
Taking in consideration the above calculation, the data of Table 1 are then transformed to the quantitative data of Table 2
Table 2. Quantitative analysis of data from Table 1 and Figure 1 based on calibration curves of Figures 2-4.
|Compound||Retention Time (min)||Calculated Area||Area Percentage||μg from calibr. curve||mg/g in sample||% in sample|
Therefore, the correct use of external calibration curves shows that the total percentage of curcuminoids in the sample claiming a 98% curcuminoids it is actually 62.4%
A further confirmation of the identity of curcuminoids is given by coupling the HPLC with an electrospray source ionization and tandem mass spectrometry. The typical mass spectra of CUR, DMC, and BDMC are depicted in Figures 5-7.
Figure 5. Mass fragmentation of Curcumin
Figure 6. Mass fragmentation of Demethoxycurcumin
Figure 7. Mass fragmentation of Bis-demethoxycurcumin
ANALYTICAL METHODS FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF OXITURMERIC® VOLATILE BISABOLANE SESQUITERPENES
The bisabolane sesquiterpens (e.g., ar-turmerone, α-turmerone, β-turmerone) present in OxiTurmeric® are identified by gas chromatography coupled to mass spectrometry (GC-MS) and quantified by gas chromatography coupled to Flame Ionization Detector (GC-FID). These are the only reliable methods for the analysis of turmeric volatile bioactive compounds. Other methods (including TLC, UV-Vis, etc.) do not provide neither the precise identification of compounds (only possible through GC-MS) nor their precise quantification (which is obtained by using GC-FID with external calibration curves of pure standards and internal standards).
The typical GC-FID profile of OxiTurmeric® is depicted in Figure 8.
Figure 8. GC-FID chromatogram of OxiTurmeric® that shows the presence of several sesquiterpenes. This analysis is used for the quantitative determination of the bioactive volatile sesquiterpens of OxiTurmeric® by using both external calibration curves with pure standards and internal standards (e.g., trans-nerolidol).
The identification of the main sesquiterpenes of OxiTurmeric® is provided by GC-MS as shown in Figure 9.
Figure 9. Mass spectra of the main bisabolane sesquiterpens contained in OxiTurmeric®. Every single batch is subjected to strict GC-FID and GC-MS controls to provide a standardize product.