Jurnal Kimia Sains dan Aplikasi

Thin layer chromatography (TLC) fingerprint profile analysis can be used for quality control of herbal medicinal raw materials through identification, authentication


Introduction
Tempuyung (Sonchus arvensis L.) plant belongs to the Asteraceae family and is a non-nitrophilic species that can grow in nutrient-poor soil. Tempuyung is widespread in several continents, including Asia, Europe, and Africa. The distribution of tempuyung in Indonesia is found widely in Sumatra, Java, Bali, Papua, Kalimantan, and Sulawesi [1]. The content of tempuyung compounds in these areas is different [2] due to several factors, such as growing location [3], soil nutrients, temperature, and weather. Tempuyung, which contains a large number of bioactive compounds [4], has been widely used as a raw material for herbal medicine due to its wide range of pharmacological properties, including antioxidant [5], anti-inflammatory [6], diuretic, antibacterial [7], and anticancer [1]. These bioactive compounds come from alkaloids, flavonoids, saponins, triterpenes, and steroids [8]. Examples of flavonoid compounds include orientin, quercetin, catechins, rutin, myricetin, luteolin, apigenin, and kaempferol [9]. Luteolin is one of the flavonoid molecules that is a distinctive component of tempuyung [10]. Among the many benefits of tempuyung, Xia and Liang [11] found that the plant can be an insecticide. According to Seal [12], Indians also reportedly eat tempuyung leaves as a salad.
Quality control must be developed as tempuyung is increasingly used in pharmaceutical products, particularly when providing premium raw materials. This quality control is necessary for ensuring the product's suitability for the purpose, consistency, efficacy, and safety [13] and for guaranteeing the pharmacological activity of the products [14]. Additionally, quality control is practiced to prevent the counterfeiting of raw materials using plants that are morphologically or closely related to one another. The absence of quality control can cause inconsistent efficacy and safety, harming human health. One of the analytical techniques that can be used to control tempuyung quality is the fingerprint analysis approach using thin layer chromatography (TLC) [15]. This fingerprint profile analysis provides information about the compounds contained in the sample. This analysis can be utilized to identify, authenticate, and discriminate herbal medicinal raw materials. This analysis provides information on the broad and detailed chemical compound profile that can be used as a reference for quality control [16].
Thin layer chromatography (TLC) is a simple chromatographic analysis technique widely used for plant fingerprint analysis. This method is widely utilized as a separation method in quality control because of its simplicity of usage, accessibility, and affordability [17]. TLC can be used for qualitative analysis by examining the separated fingerprint profile based on the position, color, quantity, intensity, and Rf (retardation factor) of the resulting bands. The fingerprint TLC method has been widely used in several studies, such as the analysis of metabolite profiles of the rhizome of intersection mango (Curcuma mango) [18], differentiating simplicia powders of turmeric, bangle, temulawak [15], and Dutch teak leaves (Guazuma ulmifolia). Several previous related studies have not reported the fingerprint analysis of tempuyung plants using the TLC method.

Sample Extraction
The extract was obtained by extracting 1 gram of sample with 10 mL of methanol using an ultrasonicator for 30 minutes at room temperature. The extraction results were filtered (gravity filtration), put into another bottle, and then covered with aluminum foil.

Plate Preparation and Sample Application
TLC plates were cut into three different sizes: 13 × 10 cm, 10 × 10 cm, 4 × 10 cm, and 2 × 10 cm. The horizontal lines of about 1 cm each were drawn on plates from the bottom of the plate as the starting line and from the top as the finishing line. The plate was eluted to the finishing line using methanol solvent in a chamber saturated for 10 minutes. The plate was then dried in the oven at 100°C for 30 seconds and utilized for a sample application. Sample spotting was performed using a TLC applicator with a spotting speed of 80 nL/s, a sample volume of 10 µL and a standard of 5 µL, a bandwidth of 8 mm, and a spacing of 1.2 mm between spots.

Mobile Phase Selection
The sample extract was applied on the dried plate using the TLC applicator. The plate was then put into 10 mL of a single solvent in a chamber saturated for 10 minutes to elute until it reached the finishing line. After the elution, the plates were removed, dried at room temperature, and observed under UV lights (254 and 366 nm). The plates were derivatized using 10% sulfuric acid, dried in an oven at 100°C for 5 minutes, then observed under UV light at 366 nm. The solvents used were nhexane, diethyl ether, chloroform, ethyl acetate, dichloromethane, acetone, acetonitrile, ethanol, and methanol. The best chromatogram profile obtained from a single mobile phase was then formulated to obtain the optimum mobile phase and compared with the luteolin standard.

Method Validation for the Best Mobile Phase Mixture
The best mobile phase obtained was validated with some parameters, including stability, specificity, precision, robustness of chamber, and development distance [16]. The stability of the analyte was determined by developing two-dimensional chromatography. In contrast, the analyte in the plate was determined by comparing the fingerprint pattern resulting from the difference in the delay time of the plate before and after the sample was applied. Specificity was done by comparing the samples with plants with similar morphology, namely tapak liman. Precision was performed by repeating the process thrice and on different days. Robustness was measured by comparing the separation results using twin-trough and flat-bottom chambers.

Application of TLC Separation Method
The developed TLC separation method was applied by measuring tempuyung plants from three locations with three repetitions for each analysis. The obtained fingerprint patterns were then processed using the ImageJ application program to determine the differences in intensity of the fingerprint patterns of the tempuyung in the three locations. This application can change the bands on the chromatogram into a densitogram, a graph of the relationship between the intensity and the Rf value. format could be obtained by pulling all the data on the line graph. The data was then processed using the Origin software to form further curves.

Best Mobile Phase
The ideal mobile phase was selected to obtain the optimal separation quality, which started with developing several solvents with various properties, ranging from non-polar to polar. This selection was done so that analytes with various polarity levels could be separated appropriately. It is necessary to pay attention to the resolution value and the number of bands, as both are good separability parameters for the TLC profile. The resolution value equal to one (=1) is the minimum value [16], and more than 1.5 is the best value. The results of developing a single solvent with various polarity indexes were then detected with UV lights at 254 and 366 nm to achieve maximum visualization of the TLC profile (Figures 1 & 2).  The TLC profile with the best visualization was obtained from UV detection at 366 nm. This can be seen from the appearance of the band stains produced on each plate, which are more clearly visible ( Figure 2). Detection for subsequent experiments was carried out under 366 nm UV light. The single solvent that produces the best separation is obtained from the single solvent chloroform, ethyl acetate, and dichloromethane ( Figure  2), where the three bands produce good separation resolution with the highest number of bands. These three solvents were then selected to be formulated with various comparisons to obtain optimal separation results. The composition of the mobile phase mixture used was chloroform: dichloromethane with a ratio of (A) 5:5, (B) 6:4, (C) 7:3, (D) 8:2, (E) 9:1, chloroform: ethyl acetate with a ratio of (F) 7:3, (G) 8:2, (H) 9:1, and chloroform: ethyl acetate:dichloromethane: formic acid with a ratio of (I) 6:3:2:0.1, (J) 6.5:2:1.5:0.1, (K) 7:2:1:0.1, (L) 7.5:2:0.5:0.1, (M) 8:1.5:0.5:0.1. The TLC profile resulting from the use of a mixture of solvents with various comparisons can be seen in Figure 3. The separation between the bands, the number of bands, and the resolution resulting from the use of various mobile phase mixture compositions in each TLC profile differed from each other due to the difference in the degree of polarity of the mobile phase mixture for the separation of flavonoid compounds from tempuyung. The formic acid solvent was added to the mobile phase mixture code I to M to reduce the tailing effect on the band [10]. Data on the number of bands produced from the several mobile phase compositions can be seen in Figure 4.
The highest number of bands obtained from using a solvent composition with code L is 11 bands. Another parameter to determine the best mobile phase can be seen from the resolution value. The resolution value produced by the mixed mobile phase with code K is considered good because it exceeds 1.5. However, the number of bands produced is not optimal; thus, derivatization was performed to produce the best separation. The best mobile phase composition was obtained using an eluent mixture with code L, which produced the highest number of bands (11 bands) with a resolution value greater than 1.5. These results were obtained using 366 nm UV irradiation after derivatization ( Figure 5). The calculation results for the Rf value and resolution for the mobile phase mixture K and L from the TLC profile can be seen in Table 1.   The standard luteolin band and the sample luteolin glow differently due to the difference in concentration. The standard concentration of luteolin was 200 ppm, whereas a previous study reported that the concentration of the luteolin compound in the tempuyung plant was 12.57 ppm [9]. Standard luteolin with a concentration of 200 ppm is used as a comparison so that the resulting band is clearer, making it easier to calculate the Rf value and resolution. Luteolin was chosen as the target compound because, according to the pharmacopeia, it is a characteristic compound of tempuyung. The luteolin component is also utilized as a quality control marker in herbal medicines containing tempuyung components. The mobile phase with the L code was validated further, as it is a method development in fingerprint analysis for quality control.

Stability
The TLC separation method is an open system, and each step requires a particular delay time. This can affect the separation results due to the many external confounding factors that require stability testing. The first stability test parameter is the stability of the analyte during the chromatography process by developing twodimensional chromatography. The TLC chromatogram x y shows stable results because the stain is located on a diagonal line connecting the application position with the intersection of the two solvent fronts in both development directions (Figure 7).

Figure 7. TLC chromatogram of analyte stability test after derivatization under UV 366 nm
The second stability parameter is the stability of the analyte resulting from visualization, which is determined by observing the visual chromatogram results at several time intervals. The experimental results were stable because there was no significant change in the band's color and the number of TLC stains (Figure 8). The third stability parameter is the stability of the analyte on the plate and sample solution. The test was conducted by comparing the (a) samples applied to the plate and left for 3 hours, (b) fresh samples, (c) samples left in the solution for 3 hours, and (d) fresh samples. The fingerprint pattern, color, quantity, and intensity of stains on the band did not change significantly, indicating that the separation remained stable (Figure 9).

Precision
The precision test was conducted using three plates tested on the same and different days. The results of the precision and intermediate precision tests showed fingerprint patterns, colors, numbers, and intensities that were not significantly different (Figure 10). The intraplate precision test yielded a mean Rf value for the luteolin (X) compound of 0.22 with a standard deviation of 0.0063. The mean Rf value for the luteolin compound obtained from the three-day precision testing results was 0.23, with a standard deviation of 0.0055. Based on these results, the precision and intermediate precision tests meet the acceptance requirements where the standard deviation Rf value for precision tests is less than 0.02 and for intermediate precision is less than 0.05 [16].

Specificity
Specificity was tested by comparing the fingerprint patterns of tempuyung and tapak liman plants because both have similar morphologies, such as shape, color, and texture. The test is conducted because the method is considered specific when testing samples with the same identity gives similar separation results, and vice versa [15]. TLC separation results showed differences in fingerprint patterns between tempuyung and tapak liman plants ( Figure 11). The TLC stain band was visible in the tempuyung plant but not in the tapak liman at Rf = 0.78 (x). In contrast to tempuyung, tapak liman plants have strong red and bright blue stain bands at Rf 0.44 (z) and 0.64 (y), respectively. However, there are similarities in the bands of luteolin compounds, with an Rf value of 0.21 in both plants marked with a yellow line ( Figure 11). These similarities make the development of this method not specific to the separation of luteolin compounds.

Robustness
The robustness test was conducted with two parameters: the robustness of the chamber type and the robustness of the development distance. Robustness tests on chamber types were performed using twin-trough and flat-bottom chambers. The results of the robustness test met the acceptance requirements. This can be seen from the resulting fingerprint patterns, which were not significantly different for the two types of chambers (Figures 12a & 12b), and the standard deviation Rf values obtained for both were less than 0.05. A robustness test on development distance was carried out by comparing the results of separation at 8 cm and 7 cm floating distances. The results of separating the two development distances met the acceptance requirements because the fingerprint patterns of the two are not significantly different (Figures  12a & 12b) with a standard deviation Rf of less than 0.05.

TLC Separation Method Application
The validated TLC separation method was then used to separate tempuyung from three different locations, namely Malang (a1) (a2) (a3), Solo (b1) (b2) (b3), and Yogyakarta (c1) (c2) ( c3). This determines whether this development method can be applied to tempuyung from different locations. The separated fingerprint profile ( Figure 13) shows a pattern with the same bands and colors. The TLC fingerprint patterns from the three regions produced eleven bands with good and uniform separability. Luteolin-characterizing compounds are shown in band number 3 with an Rf value of 0.20. The difference is only found in the color intensity of the tempuyung fingerprint bands from Yogyakarta numbers 4 and 7, where the colors appear brighter than bands from other regions. The difference is thought to be due to differences in the concentration of a compound contained therein. Some factors causing these differences include different natural conditions, such as temperature, soil nutrients, postharvest treatment, and harvest period [19]. The TLC fingerprint patterns of the three areas were then compared using a densitogram ( Figure 14) to determine the level of component content in the samples. The resulting densitogram shows peak uniformity from three different locations. However, at peaks number 4 and 7 for the Yogyakarta area (d), the intensity was higher than for the other two regions (Malang (a) and Solo (b)). This shows that the compound content contained in sample (d) is much higher. This result corresponds to the band on the fingerprint pattern (Figure 13), where the band's color appears lighter.

Conclusion
The TLC fingerprint analysis method development for tempuyung plants has been successfully implemented and applied to tempuyung from Malang, Solo, and Yogyakarta. This method has been validated, and the experimental results for all test parameters have met the established criteria, allowing it to be utilized for tempuyung quality control. The optimum mobile phase chosen was a mixture of chloroform: ethyl acetate:dichloromethane: formic acid (7.5:2:0.5:0.1) because it could separate eleven bands with good separation results. These results were obtained using UV light at 366 nm and 10% sulfuric acid for derivatization.