Regular Article
Development of a Novel TLC Identification Test for “Rosemary Oil” and “Rosemary Water” Listed in the Japanese Standards of Quasi-Drug Ingredients
2025 年 73 巻 9 号 p. 843-851
詳細
2025 年 73 巻 9 号 p. 843-851
Rosemary oil (RO) and rosemary water (RW) are widely used ingredients in cosmetics. According to the Japanese Standards of Quasi-Drug Ingredients (JSQDI), RO is defined as an essential oil obtained by the steam distillation of fresh rosemary (Rosmarinus officinalis L.) leaves, branches, and flowers, whereas RW is the water layer obtained from the steam distillation of the leaves. Although the JSQDI outlines a specific TLC identification test for RW, this test is time-consuming (15-cm development distance) and requires the use of hazardous reagents (toluene). Additionally, no identification tests are available for RO. This study therefore aimed to establish a new TLC-based identification test for RO in JSQDI, while also improving the RW identification test. Using cyclohexane/methyl tert-butyl ether/acetonitrile (20 : 10 : 1) as the developing solvent and 4-methoxybenzaldehyde sulfuric acid as the visualization reagent, 1,8-cineole and borneol were universally detected in 19 RO market products. Thus, 1,8-cineole was selected as the primary marker compound and borneol as the secondary marker compound. Additionally, the extraction process was optimized by applying these TLC conditions to RW, significantly simplifying the process. TLC analysis of the RW market products confirmed the presence of 1,8-cineole in all samples. Thus, in addition to representing a novel method for the identification of RO, these results indicate the suitability of the new extraction conditions for use as an alternative method to identify RW.
The Japanese Standards of Quasi-Drug Ingredients (JSQDI) lists the ingredients approved for use in quasi-drugs and cosmetics, excluding those in the Japanese Pharmacopoeia (JP), Japan’s Specifications and Standards for Food Additives, and Japanese Industrial Standards. The JSQDI aims to ensure the quality of quasi-drugs in Japan at the raw-material level.1) First established in 1991, the JSQDI was later merged with the “Japanese Standards of Cosmetics Ingredients” and the “Japanese Standards of Cosmetic Ingredients for Specific Types of Cosmetics,” forming the latest JSQDI 2021.1,2) Following various revisions, the JSQDI now includes over 2600 synthetic and natural products.1) However, owing to the integration of various standards, differences exist among them, with many lacking the essential criteria. In addition, it is necessary to revise traditional analytical methods, such as colorimetric analysis, using the latest analytical equipment. Notably, TLC-based identification and purity tests are still widely employed, especially in the context of natural products, as described in the “Crude Drug” section of the 18th Edition of the JP.3–5) Indeed, TLC remains popular owing to its low cost, minimal equipment requirements, ease of use, specificity, and suitability for multiple sample analyses. As indicated above, the JSQDI includes many items derived from natural products, and consequently, various TLC-based test methods are available.1)
Rosemary oil (RO) is the essential oil obtained by the steam distillation of fresh rosemary leaves, branches, and flowers (Rosmarinus officinalis L.), whereas rosemary water (RW) is an aqueous component obtained by the steam distillation of rosemary leaves.1) Steam distillation generally separates essential oils into an oil phase and floral water.6) Previous reports have indicated that rosemary essential oil contains approximately 95% monoterpenes and their derivatives, with the remaining components being sesquiterpenes.7–9) In the context of RW, oxygenated monoterpenes such as 1,8-cineole and borneol comprise over 70% of the floral water components.10)
JSQDI 2021 established a TLC-based identification test for RW, which involves hexane extraction and 1,8-cineole detection using a hazardous toluene-based developing solvent.1) Furthermore, this method is time-consuming because of the 15-cm development distance that is required to achieve separation. Similarly, the European Pharmacopoeia (EP) specifies a TLC identification test for RO using 1,8-cineole, borneol, and bornyl acetate as marker compounds.11) However, similar to the JSQDI, the EP method also relies on the use of toluene, and requires extended analysis times because of its comparable 15-cm development distance.
With the above considerations in mind, this study aims to establish a new TLC identification test for RO using essential oil components as marker compounds, in addition to improving the current identification test for RW. Furthermore, in this study, we evaluated the validity of the designed identification tests using commercially available RO- and RW-based products.
1,8-Cineole (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan; purity 85%), camphor (FUJIFILM Wako Pure Chemical Corporation; purity 98%), bornyl acetate (Sigma-Aldrich Co., LLC, St. Louis, MO, U.S.A.; purity 95%), borneol (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan; purity 95%), linalool (Tokyo Chemical Industry Co., Ltd.; purity 96%), isoborneol (Tokyo Chemical Industry Co., Ltd.; purity 90%), verbenone (Tokyo Chemical Industry Co., Ltd.; purity 95%), and 4-methoxybenzaldehyde (FUJIFILM Wako Pure Chemical Corporation; purity 97%) were employed in this study. All solvents were of special grade, pesticide residue grade, poly chlorinated biphenyl (PCB) test grade, or LC-MS grade (FUJIFILM Wako Pure Chemical Corporation). TLC was performed using silica gel 60 F254 plates (Merck, Darmstadt, Germany), and visualization was performed using a TLC reagent spray manufactured by CAMAG (Muttenz, Switzerland), combined with a CAMAG TLC Visualizer 2 (CAMAG). Silica gel 60N neutral (40–100 μm) was used for purification (Kanto Chemical Co., Inc., Tokyo, Japan).
Samples and TLC AnalysisThe RO and RW market products were purchased from the Internet, as detailed in Table 1. The TLC conditions for the EP method and the method developed herein are listed in Tables 2 and 3, respectively. The TLC identification test conditions for the finally constructed RO and RW are shown in Table 4.
| Rosemary oil | Rosemary water | ||||
|---|---|---|---|---|---|
| Sample no. | Production area | Parts*) | Sample no. | Production area | Parts*) |
| RO1 | Spain | Whole plant | RW1 | Japan (Hyogo) | Unknown |
| RO2 | Morocco | Leaves/flower | RW2 | Japan (Shizuoka) | Unknown |
| RO3 | Unknown | Leaves/flower | RW3 | Japan (Hyogo) | Unknown |
| RO4 | Japan (Shizuoka) | Unknown | RW4 | Japan (Hyogo) | Unknown |
| RO5 | Morocco | Leaves/flower/stem | RW5 | France | Leaves |
| RO6 | Spain | Leaves | RW6 | Unknown | Unknown |
| RO7 | Morocco | Leaves/flower | RW7 | France | Unknown |
| RO8 | Morocco | Flower | RW8 | France | Leaves |
| RO9 | Spain | Leaves/flower | |||
| RO10 | France | Leaves/flower | |||
| RO11 | Spain | Leaves/flower | |||
| RO12 | Morocco | Leaves/flower | |||
| RO13 | Spain | Leaves/flower | |||
| RO14 | Morocco | Leaves | |||
| RO15 | Morocco | Whole plant | |||
| RO16 | Japan (Hyogo) | Leaves/flower/stem | |||
| RO17 | Japan (Kagoshima) | Leaves | |||
| RO18 | Spain | Whole plant | |||
| RO19 | Spain | Leaves/flower | |||
*) Parts indicate the marketed products.
| Preparation of analytical samples | Dissolve RO (0.5 mL) in toluene (10 mL) |
| Reference solution | Dissolve 1,8-cineole (0.1 mL) in toluene (10 mL) |
| Dissolve borneol (50 mg) in toluene (10 mL) | |
| Dissolve bornyl acetate (50 mg) in toluene (10 mL) | |
| Developing solvent | Toluene/ethyl acetate = 95 : 5 |
| Development distance | 15 cm |
| Visualization reagent | Vanillin reagent (EP) |
| Heating time | 105°C for 10 min |
| Spot amount | Analyte 10 μL, reference solution 10 μL |
| Marker compounds | 1,8-Cineole, borneol, bornyl acetate |
| Condition 1 | Condition 2 | |
|---|---|---|
| Preparation of analytical samples | Dissolve 0.2 mL RO in hexane (10 mL) | |
| Reference solution | Dissolve 1,8-cineole (0.1 mL) in hexane (10 mL) | Dissolve 1,8-cineole (0.1 mL) in hexane (10 mL) |
| Dissolve linalool (10 mg) in hexane (10 mL) | Dissolve borneol (10 mg) in hexane (10 mL) | |
| Developing solvent | Hexane/ethyl acetate/2-propanol = 20 : 1 : 1 | Cyclohexane/methyl tert-butyl ether/acetonitrile = 20 : 10 : 1 |
| Development distance | 7 cm | |
| Visualization reagent | 4-MBA (JP) | |
| Heating time | 105°C for 5 min | |
| Spot amount | Analyte 5 μL, reference solution 2 μL | |
| Marker compounds | 1,8-Cineole, linalool | 1,8-Cineole, borneol |
| RO | RW | |
|---|---|---|
| Preparation of analytical samples | Dissolve RO (0.2 mL) in hexane (10 mL) | Add hexane (1 mL) and NaCl (0.6 g) to RW (10 mL) and shake. After centrifugation, spot the supernatant onto the TLC plate |
| Reference solution | Dissolve 1,8-cineole (0.1 mL) in hexane (10 mL) | Dissolve 0.1 mL 1,8-cineole in hexane (10 mL) |
| Dissolve borneol (10 mg) in hexane (10 mL) | ||
| Developing solvent | Cyclohexane/methyl tert-butyl ether/acetonitrile = 20 : 10 : 1 | |
| Development distance | 7 cm | |
| Visualization reagent | 4-MBA (JP) | |
| Heating time | 105°C for 5 min | |
| Spot amount | Analyte 5 μL, reference solution 2 μL | |
| Marker compounds | 1,8-Cineole, borneol | 1,8-Cineole |
Silica gel was suspended in hexane/ethyl acetate (98 : 2) and packed into a column. A sample of RO (250 μL) was mixed with 500 μL of hexane/ethyl acetate (98 : 2) and loaded onto the column. Elution was performed using hexane/ethyl acetate (98 : 2), followed by hexane/ethyl acetate (90 : 10). The fractions were monitored and identified using TLC and GC-MS, as detailed below.
Current JSQDI Identification Test for RWA mixture of RW (50 mL), ultra-pure water (150 mL), hexane (50 mL), and sodium chloride (3 g) was stirred and allowed to stand, and the hexane layer was collected in a glass test tube. Subsequently, the resulting hexane layer was dried under reduced pressure, and the obtained residue was redissolved in methanol (0.5 mL) for TLC analysis. An aliquot (10 μL) of this solution was spotted onto the TLC plate and developed over a distance of 15 cm using a toluene/ethyl acetate (93 : 7) solvent mixture. After development, the plate was sprayed with an ethanol (95) solution of sulfuric acid (1 → 20), followed by vanillin–ethanol reagent, and heated at 110°C for 5 min.
New Protocols for the Extraction and TLC Analysis of RW RW Extraction 1A mixture of RW (20 mL), ultra-pure water (60 mL), hexane (20 mL), and sodium chloride (2 g) was shaken for 3 min. After allowing to stand, the hexane layer was collected in a glass test tube, and the hexane layer was dried under reduced pressure and re-dissolved in methanol (0.5 mL) for TLC analysis. TLC was performed under condition 2 (Table 3) using a 2 μL sample spot.
RW Extraction 2A mixture of RW (20 mL), hexane (20 mL), and sodium chloride (2 g) was shaken for 3 min. After allowing to stand, the hexane layer was collected in a glass test tube, and the hexane layer was dried under reduced pressure and re-dissolved in methanol (0.5 mL) for TLC analysis. TLC was performed under condition 2 (Table 3) using a 2 μL sample spot.
RW Extraction 3A mixture of RW (10 mL), hexane (1 mL), and sodium chloride (0.6 g) was shaken for 3 min and subjected to centrifugation at 1740 × g for 5 min. The resulting supernatant was used for TLC analysis under condition 2 (Table 3) using a 5 μL sample spot.
GC-MS Analysis GC ConditionsAn 8890 gas chromatograph system (Agilent Technologies Inc., Santa Clara, CA, U.S.A.), a PAL3 RTC120 autosampler (Agilent Technologies Inc.), and a DB-5MS UI column (30 m, 0.25 mm, i.d., 0.25 μm; Agilent Technologies Inc.) were used for the GC setup. Analysis was performed using an initial oven temperature of 50°C (2 min hold), followed by heating to 200°C at a rate of 3.5°C/min (2.5 min hold), then heating to 280°C at a rate of 20°C/min (5 min hold). Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The split injection mode was employed (100 : 1), with an injection volume of 1 μL and an inlet temperature of 250°C.
MS ConditionsA 5977B mass spectrometer (Agilent Technologies Inc.) was employed under electron ionization conditions (70 eV), with an ion source temperature of 230°C, an interface temperature of 300°C, and an m/z scan range of 40–400.
Initially, TLC analysis of the marketed RO was performed under the EP identification test conditions11) (Table 2). Figure 1 shows the analyzed compounds, including 1,8-cineole (1), borneol (2), and bornyl acetate (3), which are marker compounds for RO in the EP. Upon examination of the TLC results (Fig. 2a), it was evident that some marketed RO samples exhibited weak or undetectable bornyl acetate (3) spots. We also selected a visualization reagent different from that used in the EP and examined the use of 4-methoxybenzaldehyde sulfuric acid (4-MBA) due to its ease of preparation and its inclusion in the JP. The color obtained using 4-MBA provided a superior spot visibility than the vanillin reagent, revealing red spots in the borneol (2) region for some samples (Fig. 2b). Therefore, 4-MBA was selected as the visualization reagent for TLC in this study.
(a) Vanillin sulfate (EP), and (b) 4-MBA (JP).
GC-MS analysis of the EP marker compounds was conducted using commercially available RO products. The percentages of 1,8-cineole (1), borneol (2), and bornyl acetate (3) were determined relative to the total peak area (Figs. 3a–3c), averaging 32% for 1,8-cineole (1), 1.3% for borneol (2), and 0.7% for bornyl acetate (3). Borneol (2) generally ranged between 1 and 2% across all the samples (Fig. 3b). In contrast, bornyl acetate (3) represented <1% of the total peak area in most cases, except for 3 samples, RO15–17 (Fig. 3c). In addition, among the essential oil components other than the EP marker compounds, camphor (4) tended to be an abundant component (Supplementary Fig. S1a). Based on a combination of the GC-MS and TLC results, 1,8-cineole (1), borneol (2), and camphor (4) were selected as candidate marker compounds for the purposes of this study.
Initially, solvent systems such as hexane and ethyl acetate mixtures were tested for TLC conditions; however, compounds such as borneol (2), with low Rf values, were not separated. Subsequently, the TLC developing solvent was examined, and the conditions mentioned in Table 3 (condition 1) were devised without the use of toluene. The eluotropic strength (ɛ°) of the developing solvents used in condition 1 of Table 3 was 0 for hexane, 0.45 for ethyl acetate, and 0.63 for 2-propanol.12,13) The examined developing solvents enabled excellent separation in commercial RO samples, exhibiting Rf values of 0.56 and 0.27 for spots A and B, respectively, even at a development distance of 7 cm (Fig. 4). These spots were therefore selected as the marker compounds. Following their isolation and comparison with GC-MS standards, spot A was identified as a mixture of 1,8-cineole (1) and camphor (4), while spot B was identified as linalool (5) (Supplementary Figs. S2a and S2b). Additionally, 4-MBA was found to color 1,8-cineole (1), but not camphor (4) (Supplementary Fig. S2a), consistent with the result of Raul et al. that camphor (4) does not react with 4-MBA.14) Based on these results, camphor (4) was excluded as a candidate marker compound. In addition, a faint dark blue spot was also observed slightly above spot B (Fig. 4). This spot was also isolated from the commercial product RO and identified as terpinen-4-ol (6) based on a comparison with a standard compound using GC-MS analysis (Supplementary Fig. S3a). Furthermore, simultaneous TLC analysis of the standard and isolated products of linalool (5) and terpinen-4-ol (6) confirmed that these compounds did not overlap on the TLC plate (Supplementary Fig. S3b).
Notably, the analysis of various commercial RO samples revealed that the dark blue spot B, corresponding to linalool (5), was not detected in sample RO19. Instead, the yellow-green spot C was observed at the same Rf value (Fig. 4), suggesting an overlap of the 2 spots. The color differences at these spots were considered to result from differences in the chromogenic reaction between the compounds in each spot and 4-MBA. Thus, spots C and D were isolated from sample RO19 (Fig. 4) and identified using GC-MS as isoborneol (7: spot C) (Supplementary Fig. S4) and borneol (2: spot D) (Supplementary Fig. S5). In sample RO19, a comparison of the GC-MS peak area values between linalool (5, corresponding to spot B) and isoborneol (7, corresponding to spot C) showed that isoborneol (7) was approximately 52.6 times more abundant (Supplementary Figs. S1b and S1c). According to previous reports, the composition and content of rosemary essential oil vary with climate conditions and harvest time,15,16) and the compositional changes of linalool (5) and isoborneol (7) in RO19 may have been influenced by these factors as well. In the context of the TLC identification test, the marker compounds should be as universally present as possible in the RO samples.
Based on the experimental results, linalool (5) was considered unsuitable as a marker compound because it may not be detected in some samples. Therefore, we examined the developing solvents based on their eluotropic strengths and selected the solvents—cyclohexane (ɛ° = 0.03), methyl tert-butyl ether (ɛ° = 0.29), and acetonitrile (ɛ° = 0.50)—that have lower eluotropic strengths than those tested under condition 1.12,13) As shown in Fig. 5a, the selected solvents enabled clear separation of spots C and D, exhibiting lower Rf values than those exhibited when using linalool (Table 3, condition 2). Based on the obtained results, 1,8-cineole (1) (Rf = 0.62 for spot A) and borneol (2) (Rf = 0.41 for spot D) were selected as marker compounds (Fig. 5a).
Next, the universality of these 2 compounds was evaluated in conjunction with GC-MS analysis of samples RO15–19, with both 1 and 2 being detected in all samples (Figs. 5a and 5b). Furthermore, the cineole (1) and borneol (2) standards were analyzed under the TLC conditions listed in Table 3 (condition 2), along with the cineole (1) and borneol (2) components isolated from the commercial products, and the commercial products themselves (Supplementary Fig. S6a). Notably, spots A and D exhibited identical Rf values for the standards, isolated compounds, and commercial products.
Moreover, spot B (linalool, 5), which was selected as a marker compound under condition 1 (Table 3), appeared just above spot D (borneol, 2) in the TLC analysis performed under condition 2 (Table 3 and Supplementary Fig. S6b). Similarly, the terpinen-4-ol (6) that appeared near linalool (5) under condition 1 (Table 3 and Fig. 4) was observed slightly above linalool (5) under condition 2 (Table 3 and Supplementary Fig. S6b). TLC analysis performed under condition 2 (Table 3) demonstrated that the marker compounds of spots A (1,8-cineole, 1) and D (borneol, 2) did not overlap with spots B (linalool, 5) and terpinen-4-ol (6) (Fig. 5a). Additionally, GC-MS analysis detected spot B (linalool) in samples RO15–18 (Fig. 5b). By contrast, spot B (linalool) was hardly detected in the GC-MS analysis of sample RO19, whereas spot C (isoborneol) was identified instead (Fig. 5b). Furthermore, the red spot observed below spot D (borneol, 2) in samples RO16–18 (Fig. 5a) was estimated to be verbenone (8) based on comparison with a standard TLC sample (Supplementary Fig. S6c). GC-MS analysis confirmed the presence of a verbenone (8) peak exclusively in samples RO16–18 (Fig. 5b and Supplementary Fig. S1d). These findings indicate a strong correlation between the TLC and GC-MS results, suggesting that 1,8-cineole (1, spot A) and borneol (2, spot D) were universally present in the examined RO samples. To confirm this, TLC analysis under condition 2 (Table 3) was performed on all the commercial RO samples purchased for this study, and both spot A (1,8-cineole, 1) and spot D (borneol, 2) were detected in all cases (Supplementary Fig. S7).
Subsequently, the descriptions (characteristics) and optical rotations of the commercially purchased RO samples were evaluated in accordance with the JSQDI monographs, excluding the purity tests (such as the heavy metal test). According to the monographs of RO, its descriptions are that it is a colorless to pale yellow liquid with a distinctive odor, and its optical rotation ranges from
Additionally, the major components of the essential oil in the RO product purchased in this study were largely consistent with previously reported GC-MS data for RO.7–9) The TLC conditions established herein were considered to be superior to those of the EP due to the superior component visibility and efficient 7-cm development distance. Consequently, 1,8-cineole (1, dark blue spot) and borneol (2, gray–green spot) were selected as the primary and secondary marker compounds, respectively.
Note that 1,8-cineole (1) and borneol (2) are biosynthesized in rosemary, which can be classified into several chemotypes—such as cineole type, camphor type, and verbenone type—based on the proportion of essential oil components.15,17) The compositional differences among rosemary chemotypes result from genetic variation in biosynthetic enzymes and environmental factors that influence their expression levels.15) Notably, borneol (2) is known as a biosynthetic precursor of camphor (4), which is formed through enzymatic oxidation.17) In conclusion, rosemary contains the necessary enzymes for biosynthesizing 1,8-cineole (1) and borneol (2), which can be utilized as reliable and stable markers for essential oil components in plants.
Improvement of the RW Identification TestInitially, the JSQDI RW identification test was conducted on the marketed products, as outlined in “Current JSQDI Identification Test for RW.” TLC analysis showed a poor spot visibility after coloration, with broad spots being observed, including for the marker compound 1,8-cineole (Fig. 6). This was attributed to the high component concentration that resulted from concentrating the hexane extract to dryness, followed by redissolution in methanol (0.5 mL).1)
Analysis was performed according to the conditions outlined in the jsqdi (15-cm development distance).
Thus, with the aim of improving the analytical appropriateness, the conditions outlined in “RW Extraction 1” were examined following the JSQDI process but using a reduced sample concentration. Additionally, the TLC conditions established for RO were applied, and Fig. 7a shows the corresponding results. Notably, reducing the concentration factor from 100- to 40-fold significantly improved the spot shape (Figs. 6 and 7a), for marker compound 1,8-cineole (1) but also for borneol (2) and verbenone (8).
Subsequently, the “RW Extraction 2” protocol was devised, wherein a direct extraction was performed using hexane (Fig. 7b). As shown in Figs. 7a and 7b, the TLC coloration was similar, and GC-MS analysis confirmed that the extracts obtained using both the “RW Extraction 1” and “RW Extraction 2” methods were compositionally similar (Fig. 7d).
In an attempt to simplify the process, the conditions outlined in “RW Extraction 3” were employed without a concentration step. This method involved the addition of hexane (1 mL) and salt directly to the RW sample (10 mL), followed by liquid–liquid partitioning and direct spotting of the supernatant onto a TLC plate. As shown in Fig. 7c, comparable results were obtained compared to those of the “RW Extraction 1” and “RW Extraction 2” methods, and GC-MS analysis showed similar chromatographic patterns for all 3 extracts (Fig. 7d). These results clearly indicate that the extraction method did not affect the extracted components. Thus, the TLC conditions corresponding to “RW Extraction 3” were considered to be simple and efficient because they eliminated the concentration step while yielding comparable results (Table 4).
Survey of RW-Based Market ProductsFinally, TLC analysis was performed on various RW-based market products using the optimized extraction conditions (Table 4). As shown in Supplementary Fig. S8, 1,8-cineole (1) was detected in all examined products. The descriptions of RW-based market products were assessed according to the JSQDI,1) and it was confirmed that all products satisfy the required criteria.
Next, GC-MS analysis was conducted to obtain complementary data because 1,8-cineole (1) is also present in other essential oils, such as eucalyptus and sage. GC-MS analysis showed a high content of oxygenated monoterpenes such as 1,8-cineole (1), borneol (2), and camphor (4) in the examined RW-based products, consistent with previous reports obtained after steam distillation of RW10) (Fig. 7d).
Note that only 1,8-cineole (1) is designated as a marker compound in the JSQDI for performing tests to identify RW. Since 1,8-cineole (1) is also present in other essential oils, it is necessary to consider adopting analytical methods with excellent separation capabilities, such as GC, as test methods in the future. GC is an analytical method that offers excellent separation capabilities. It allows for the identification and quantification of multiple oxygenated monoterpenes that can serve as marker compounds for RW analysis.
The improved TLC identification method presented in this study for RW analysis offers several practical advantages: high visibility of separated components using visualization reagents, use of safer developing solvents, reduced developing distance, and simplified extraction conditions. The method developed in this study can potentially serve as an alternative to the current identification test for RW.
New TLC conditions were devised for the analysis of RO from the JSQDI using 1,8-cineole (1) and borneol (2) as marker compounds, with GC-MS being used to confirm the obtained results. Both compounds were detected in all 19 commercial RO samples by TLC, indicating their ubiquity throughout these samples. The establishment of these conditions is of particular importance, since no standardized identification test has previously been reported. The same conditions were applied to improve the TLC identification protocol for RW. The new extraction procedure produced clear TLC spots and offered a simplified process, thereby providing a potential alternative to the current JSQDI identification tests.
Although TLC is an inexpensive and straightforward technique for separating compounds, its effectiveness in separating monoterpenes with similar structures is limited. Since 1,8-cineole (1) is also present in other essential oils, the adoption of test methods with high separation capabilities, such as GC, may need to be considered. In this study, we examined the conditions for identification tests using commercially available RO- and RW-based products. In the future, we plan to verify the test method established by this study using JSQDI-compliant products.
This study was supported by a Grant-in-Aid for Research on the Regulatory Science of Pharmaceuticals and Medical Devices from the Ministry of Health, Labor, and Welfare, Japan (Grant No. 24KC2009).
The authors declare no conflict of interest.
This article contains supplementary materials.
