Method for evaluating quality of honeysuckle flower distillate based on gas phase characteristic map

Abstract

This invention discloses a method for evaluating the quality of honeysuckle dew based on gas chromatographic characteristic spectroscopy, belonging to the field of pharmaceutical quality analysis. Through comparative analysis of a large number of samples, five volatile characteristic components in the GC chromatogram of honeysuckle dew were identified: n-hexanol, phytol, linalool, furfural, and α-terpineol. Based on this, a GC characteristic chromatogram of honeysuckle dew was established. This invention also identified two characteristic components with a strong positive correlation in content. By utilizing the GC characteristic chromatogram and the peak area ratio of the two positively correlated characteristic components, accurate detection of the quality of honeysuckle dew was finally achieved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical quality analysis, specifically relating to a method for evaluating the quality of honeysuckle dew based on gas phase characteristic spectra. Background Technology

[0002] Honeysuckle dew is made by steam distillation of honeysuckle, a plant belonging to the Caprifoliaceae family. It has the effects of clearing heat and detoxifying, and can be used clinically for symptoms such as heatstroke and sore throat. This preparation mainly contains volatile components, including hydrocarbons, alcohols, ketones, and terpenes. The current Chinese Pharmacopoeia only measures the content of linalool in the preparation, making it difficult to comprehensively and effectively evaluate its quality. The volatile components of honeysuckle are complex and numerous; nearly 200 components can be separated using the NPLCC-GC method. Furthermore, the volatile components vary depending on the origin, development stage, variety, and drying method.

[0003] Currently, there are numerous studies on the GC characteristic spectra and fingerprint spectra of honeysuckle medicinal materials. Since the origin, variety, and processing technology of honeysuckle all affect the characteristic components of honeysuckle dew preparations, this invention uses gas chromatography-mass spectrometry (GC-MS) to identify the volatile components in honeysuckle dew and screens five characteristic components to establish the GC characteristic spectra of honeysuckle dew. This invention also uses metabolomics and chromatographic analysis to screen the ratio of two components and establish a quality evaluation standard, aiming to more comprehensively and accurately control the quality of honeysuckle dew preparations. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing honeysuckle dew quality standards, such as their simplicity and inability to comprehensively and accurately control product quality, by providing a method for evaluating the quality of honeysuckle dew based on gas phase characteristic spectra.

[0005] To achieve the above objectives, the present invention employs the following technical means:

[0006] A method for evaluating the quality of honeysuckle dew based on gas phase characteristic spectra includes the following steps:

[0007] S1. Prepare a mixed reference solution containing n-hexanol, phytol, linalool, furfural, and α-terpineol;

[0008] S2. Take the honeysuckle dew sample to be tested, extract the volatile oil, and use it as the test solution;

[0009] S3. The mixed reference solution and the test solution are separately detected by gas chromatograph to obtain the gas chromatograms of the mixed reference solution and the test solution;

[0010] S4. Compare the gas chromatograms of the mixed reference solution and the test solution. Evaluate the product quality of honeysuckle dew by analyzing the presence or absence of five chromatographic peaks (n-hexanol, phytol, linalool, furfural, and α-terpineol) in the chromatogram of the test solution, as well as the ratio of the peak areas of linalool and α-terpineol.

[0011] The gas chromatograph contains a capillary column, a programmed temperature ramp, a split injection mode, and the injection port and detector temperatures are both 220°C.

[0012] Preferably, the capillary column is an Agilent HP-INNOWax with dimensions of 30m × 0.25mm × 0.25μm.

[0013] Preferably, the specific steps of the programmed temperature rise are as follows: hold at 70°C for 1 minute, increase the temperature to 100°C at 2°C / min, increase the temperature to 130°C at 3°C / min, increase the temperature to 200°C at 50°C / min, and hold for 10 minutes.

[0014] Preferably, the split ratio of the split mode injection is 5:1.

[0015] More preferably, if the chromatogram of the test sample shows five chromatographic peaks simultaneously: n-hexanol, phytol, linalool, furfural, and α-terpineol, and the peak area ratio of linalool and α-terpineol is between 1.0 and 10.0, then it is judged as a qualified product; if any one of these conditions is not met, then it is judged as a non-qualified product.

[0016] The method for preparing the mixed reference solution is as follows: take 5 mg each of n-hexanol, phytol, linalool, furfural, and α-terpineol reference standards, place them in a volumetric flask, and add ethyl acetate to make up to 100 ml.

[0017] The extraction method of the volatile oil is as follows: 100 ml of the sample to be tested is placed in a round-bottom flask, 20 g of sodium chloride is added, and the sample is extracted with a volatile oil extractor. 2 ml of ethyl acetate is added to the volatile oil extractor, the mixture is heated to boiling and kept at a gentle boil for 2 hours, cooled, and the ethyl acetate layer is dehydrated with anhydrous sodium sulfate. The supernatant is taken as the test solution.

[0018] The beneficial effects of this invention are:

[0019] This invention, through comparative analysis of a large number of samples, identified five volatile characteristic components in the GC chromatogram of honeysuckle dew and established a GC characteristic chromatogram for honeysuckle dew based on these components. These five components are mainly low-boiling-point components that can effectively reflect the quality differences of honeysuckle dew, and also include characteristic components with the same metabolic pathways. Furthermore, this invention utilizes software to perform correlation analysis on the peak areas of characteristic components in multiple batches of honeysuckle medicinal materials, ultimately identifying two characteristic components with a strong positive correlation in content. The quality control and detection of honeysuckle dew are achieved by using the GC characteristic chromatogram and the peak area ratio of the two positively correlated characteristic components.

[0020] The quality analysis method established in this invention has few indicator components (only five components), and the reference standard has a simple structure (molecular weight not exceeding 200) and is easy to obtain, thus facilitating its widespread application. This method also boasts advantages such as high detection efficiency (gas chromatography time not exceeding 40 minutes), convenient result analysis (comparing only the retention times of the five components and performing simple peak area calculations), accuracy, reliability, and ease of traceability. Therefore, it provides a new and high-value means for guiding and supervising drug production and ensuring the safety of medication for the public. Attached Figure Description

[0021] Figure 1 Total ion chromatogram of honeysuckle dew.

[0022] Figure 2 GC characteristic spectrum of honeysuckle dew (1: n-hexanol, 2: phytol, 3: furfural, 4: linalool, 5: α-terpineol).

[0023] Figure 3 Schematic diagram of the monoterpene synthesis pathway.

[0024] Figure 4 Correlation analysis results of linalool and α-terpineol.

[0025] Figure 5 Scatter plot of peak area ratios of linalool and α-terpineol.

[0026] Figure 6 : Abnormal sample - Chromatogram of sample with missing peak (1: furfural, 2: linalool, 3: α-terpineol).

[0027] Figure 7 Abnormal sample - abnormal peak ratio (linalool peak / α-terpineol > 10.0; 1: linalool, 2: α-terpineol).

[0028] Figure 8 Abnormal sample - abnormal peak ratio (linalool peak / α-terpineol <1.0; 1: linalool, 2: α-terpineol). Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments.

[0030] Example 1

[0031] 1. Materials

[0032] An Agilent 7890B gas chromatograph and an Agilent 7890C gas chromatograph-mass spectrometer (Agilent Technologies, USA) were used. α-Terpineol (batch number: 111859-201503), benzaldehyde (batch number: 111650-201303), and linalool (batch number: 111503-201603) were purchased from the China National Institutes for Food and Drug Control. Phytophthol (lot number: Lot 124630) and n-hexanol (lot number: Lot 165935) were purchased from Dr. Ehrenstorfer GmbH, Germany. Furfural was analytical grade (obtained by redistillation); all other reagents were analytical grade; and water was primary purified water. A total of 24 batches of honeysuckle were purchased from the main producing areas of Shandong, Henan, and Hebei provinces, and were identified as genuine products by experts (see Table 1 for details). A total of 217 batches of honeysuckle dew were collected from the national evaluative sampling samples of the National Medical Products Administration in 2018.

[0033] Table 1 Information on honeysuckle medicinal samples

[0034]

[0035]

[0036] 2. Methods and Results

[0037] 2.1 Analysis Conditions

[0038] 2.1.1GC

[0039] Agilent HP-INNOWax capillary column (30m × 0.25mm × 0.25μm); temperature programmed (70℃ for 1 min, ramp to 100℃ at 2℃ / min, ramp to 130℃ at 3℃ / min, ramp to 200℃ at 50℃ / min, hold for 10 min); split injection mode, split ratio 5:1; injector temperature 220℃; detector temperature 220℃.

[0040] 2.1.2 GC-MS

[0041] Agilent HP-INNOWax capillary column (30m × 0.25mm × 0.25μm); temperature programmed (70℃ for 1 min, ramp to 100℃ at 2℃ / min, ramp to 130℃ at 3℃ / min, ramp to 200℃ at 2℃ / min); split injection mode, split ratio 5:1; injection port temperature 220℃; auxiliary line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; ionization mode EI: electron energy 70eV; electron multiplier tube voltage 1347kV; scan range m / z 50~500.

[0042] 2.2 Sample Preparation

[0043] 2.2.1 Reference Solution: Take 5 mg each of n-hexanol, phytol, linalool, furfural, and α-terpineol reference standards, place them in a volumetric flask, and dilute to volume with 100 ml of ethyl acetate.

[0044] 2.2.2 Medicinal Material Solution: Take 6.25g of honeysuckle, place it in a round-bottom flask, add 100ml of water, and extract using a volatile oil extractor. Add 2ml of ethyl acetate to the volatile oil extractor, heat to boiling and maintain a gentle boil for 2 hours, cool, add an appropriate amount of anhydrous sodium sulfate to the ethyl acetate layer to dehydrate, and take the supernatant to obtain the solution.

[0045] 2.2.3 Test Solution: Take 100 ml of this product and place it in a round-bottom flask. Add 20 g of sodium chloride and extract using a volatile oil extractor. Add 2 ml of ethyl acetate to the volatile oil extractor, heat to boiling and maintain a gentle boil for 2 hours. Cool, add an appropriate amount of anhydrous sodium sulfate to the ethyl acetate layer to dehydrate, and take the supernatant.

[0046] 2.2.4 Simulated Preparation Based on market research, a batch of honeysuckle medicinal materials with large market demand were purchased from the main honeysuckle producing areas. The honeysuckle dew was prepared by Hubei Wushi Pharmaceutical Co., Ltd., and the test solution was prepared according to the method in section 2.2.3.

[0047] 2.3 GC Feature Map Establishment

[0048] 2.3.1 Characteristic Peak Confirmation

[0049] Volatile components were analyzed using GC-MS. Based on library searches and n-alkane retention index results, chromatographic peaks with an area >5% and a matching degree higher than 80% were assigned. A total of 31 components were identified, mainly including alcohols, aldehydes, and esters, as detailed in Table 2.

[0050] Table 2 Volatile Components of Honeysuckle Dew

[0051] Serial Number Retention time / min name CAS number Molecular formula relative molecular weight Matching score / point 1 5.33 (Z,Z)-3-hexenyl-2-methyl-2-butenoate 74645-87-7 <![CDATA[C 12 H 24 ]]> 168.2 72.85 2 5.52 2-n-pentylfuran 3777-69-3 C9H14O 138.1 91.95 3 7.58 n-Hexyl alcohol 111-27-3 <![CDATA[C6H 14 O]]> 102.2 70.8 4 9.87 Phytophthol 928-96-1 <![CDATA[C6H 12 O]]> 100.2 80.18 5 12.57 1-Octen-3-ol 3391-86-4 <![CDATA[C8H 16 O]]> 128.1 83.15 6 13.18 furfural 98-01-1 <![CDATA[C5H4O2]]> 96.1 79.44 7 13.59 4-Vinylpyridine 100-43-6 <![CDATA[C7H7N]]> 105.1 91.13 8 14.35 trans-2,4-heptadienal 4313-03-5 <![CDATA[C7H 10 O]]> 110.1 77.01 9 15.54 benzaldehyde 100-52-7 <![CDATA[C7H6O]]> 106 80.44 10 16.91 Linalool 78-70-6 <![CDATA[C 10 H 18 O]]> 154.1 85.96 11 18.59 9-Dodecyn-1-ol 71084-08-7 <![CDATA[C 12 H 22 O]]> 182.2 79.5 12 20.76 phenylacetaldehyde 122-78-1 <![CDATA[C8H8O]]> 120.1 79.79 13 22.72 3,4-Dimethoxyphenol 2033-89-8 <![CDATA[C8H 10 O3]]> 154.1 75.52 14 23.14 α-Terpineol 10482-56-1 <![CDATA[C 10 H 18 O]]> 154.1 78.69 15 26.07 methyl nicotinic acid 93-60-7 <![CDATA[C7H7NO2]]> 137 89.4 16 27.41 trans, trans-2,4-decadienal 25152-84-5 <![CDATA[C 10 H 16 O]]> 152.1 80.77 17 27.67 Damaskone 23726-93-4 <![CDATA[C 13 H 18 O]]> 190.1 90.95 18 29.00 hexanoic acid 142-62-1 <![CDATA[C6H 12 O2]]> 116.1 78.3 19 29.16 Geraniol 106-24-1 <![CDATA[C 10 H 18 O]]> 154.1 80.52 20 30.01 benzyl alcohol 100-51-6 <![CDATA[C7H8O]]> 108.1 94.8 21 31.11 β-Phenylephethanol 60-12-8 <![CDATA[C8H 10 O]]> 122.1 93.05 22 38.01 cis-3-hexenol benzoate 25152-85-6 <![CDATA[C 13 H 16 O2]]> 204.1 85.36 23 38.29 Phytosterol 502-69-2 <![CDATA[C 18 H 36 O]]> 268.3 88.28 24 38.79 5-Pentylresorcinol 500-66-3 <![CDATA[C 11 H 16 O2]]> 180.1 81.64 25 39.87 2-Methyl aldehyde benzoate 4122-56-9 <![CDATA[C9H8O3]]> 164 90.34 26 41.16 Methyl palmitate 112-39-0 <![CDATA[C 17 H 34 O2]]> 270.3 90.57 27 42.28 Ethyl palmitate 628-97-7 <![CDATA[C 18 H 36 O2]]> 284.3 90.5 28 44.03 n-Trisane 646-31-1 <![CDATA[C 24 H 50 ]]> 338.4 83.79 29 45.23 Farnesol 4602-84-0 <![CDATA[C 15 H 26 O]]> 222.2 87.2 30 47.04 Tetracosane 143-07-7 <![CDATA[C 12 H 24 O2]]> 200.2 91.94 31 51.76 benzyl benzoate 120-51-4 <![CDATA[C 14 H 12 O2]]> 212.1 89.59

[0052] Because the production process of honeysuckle dew involves steam distillation followed by collection of the distillate, the enrichment rate of water-soluble volatile components is relatively high. This experiment found that the peak responses of n-hexanol (7.58 min), phytol (9.87 min), furfural (13.18 min), benzaldehyde (15.54 min), linalool (16.91 min), and α-terpineol (23.14 min) were relatively high; see the total ion chromatogram (…). Figure 1 Meanwhile, after studying the characteristic spectra of multiple batches of honeysuckle dew, it was found that the compositional differences were small in the high-boiling-point region and large in the low-boiling-point region. The compositional differences in the low-boiling-point region can better reflect the differences in product quality.

[0053] Benzoic acid, a preservative in honeysuckle dew, easily decomposes into benzaldehyde at high temperatures, and the latter has weak specificity. Based on the above analysis, five components with high response in the low boiling point region—n-hexanol, phytol, and furfural—were selected to construct GC characteristic spectra, as shown below. Figure 2 .

[0054] 2.3.2 Selection of Judgment Criteria

[0055] Of the five characteristic components, linalool and α-terpineol both belong to the monoterpenoid class. In plants, they are mainly synthesized in plastids via the methyl-erythritol-4-phosphate pathway (MEP). They share the same metabolic pathway and exhibit a certain correlation within the same plant. (See metabolic pathways below.) Figure 3 .

[0056] Correlation analysis of the peak areas of linalool and α-terpineol in 24 batches of honeysuckle medicinal materials was performed using SPSS 24.0 software. The results are shown in the figure. Figure 4 The results show a positive correlation between the two (correlation coefficient of 0.994). Through metabolic and spectral analysis, this experiment revealed a strong positive correlation between the accumulation of linalool and α-terpineol in honeysuckle dew. Therefore, the peak area ratio of linalool to α-terpineol is proposed to evaluate the quality.

[0057] All 24 batches of medicinal materials exhibited 5 characteristic peaks, among which linalool and α-terpineol had peak area ratios of [missing information].

[0058] Within the range of 1.92 to 6.34, see details. Figure 5 The ratio range for normal samples is defined as 50% of the lower limit and 150% of the upper limit, i.e., 1.0 to 10.0.

[0059] Applying the above criteria to the simulated formulation, five characteristic peaks were detected. The peak area ratio of linalool and α-terpineol was 3.18, all within the acceptable range. Ultimately, the criteria for a normal sample were determined to be the detection of five chromatographic peaks: n-hexanol, phytol, furfural, linalool, and α-terpineol, with the linalool / α-terpineol peak area ratio between 1.0 and 10.0.

[0060] 2.4 Methodological Examination

[0061] 2.4.1 Repeatability test: Six samples of this product (batch number 18014025) were taken and the test solution was prepared according to the method in section 2.2.3. The determination was carried out under the chromatographic conditions in section 2.2.2. The peak areas RSD of n-hexanol, phytol, furfural, linalool and α-terpineol were 4.37%, 4.26%, 3.54%, 2.12% and 3.24%, respectively, indicating that the method has good repeatability.

[0062] 2.4.2 Stability Test: Take an appropriate amount of this product (batch number 18014025), prepare the test solution according to the method in section 2.2.3, and determine the stability at 0, 2, 4, 8, 10, and 12 h under the chromatographic conditions in section 2.2.2. The peak areas RSD of n-hexanol, phytol, furfural, linalool, and α-terpineol were 3.98%, 3.18%, 2.40%, 2.09%, and 4.23%, respectively, indicating that the solution has good stability within 12 h.

[0063] 2.5 Results Analysis

[0064] Twenty-seven batches of honeysuckle dew samples had problems, mainly manifested as missing peaks and abnormal peak ratios. For example, three batches of samples from manufacturer 5 only showed three characteristic peaks, and five batches of samples from manufacturer 6 had linalool / terpineol chromatographic peak areas exceeding the ratio range of 1.0 to 10.0. Specific abnormal samples are shown in Table 3.

[0065] Table 3. Characteristic Peak Area of ​​Abnormal Samples

[0066] factory batch number Number of characteristic peaks Linalool peak area terpineol peak area Linalool / α-terpineol Manufacturer 1 20170415 3 17.1 7.5 2.28 Manufacturer 2 170611-2 5 83.5 283.5 0.29 Manufacturer 3 20170745 5 1633.4 155.7 10.49 Manufacturer 4 170939 5 1212.3 101 12.00 Manufacturer 4 170763 5 2202.4 169.4 13.00 Manufacturer 4 170767 3 3694.7 291.8 12.66 Manufacturer 4 171044 3 3024.4 163.4 18.51 Manufacturer 4 170866 5 1903.4 142.9 13.32 Manufacturer 4 170916 5 4899.2 369.3 13.27 Manufacturer 4 180111 5 2354 171.7 13.71 Manufacturer 4 180118 5 2841.9 200.4 14.18 Manufacturer 4 180443 5 1550.8 144.2 10.75 Manufacturer 4 180454 5 2186.3 146.6 14.91 Manufacturer 5 171109 3 89.1 26.2 3.40 Manufacturer 5 180101 3 148.2 35.6 4.16 Manufacturer 5 170917 3 135.6 65.6 2.07 Manufacturer 6 170671 5 788.9 18.7 42.19 Manufacturer 6 170830 5 1125.5 98.8 11.39 Manufacturer 6 170749 5 722.1 68.7 10.51 Manufacturer 6 170843 5 1156.2 108.8 10.63 Manufacturer 6 180308 5 948.1 88 10.77 Manufacturer 7 1708919 3 56.6 74.4 0.76 Manufacturer 7 1707932 3 89.2 63.2 1.41 Manufacturer 7 1707902 3 83.1 116.8 0.71 Manufacturer 7 1803908 3 252 35 7.20 Manufacturer 7 1804934 3 214.2 43 4.98 Manufacturer 7 1804935 3 203 19.7 10.30

[0067] 3. Discussion

[0068] This experiment found that the abnormal samples of honeysuckle dew mainly manifested in two aspects, as follows.

[0069] First, chromatographic peaks are missing, mainly n-hexanol and phytol ( Figure 6 These two compounds are low-boiling-point compounds in honeysuckle dew. The packaging materials for this preparation include soda-lime glass bottles and plastic bottles. The permeability of these materials varies, and differences in distillation processes, sterilization processes, and storage environments can all lead to the loss of these components.

[0070] Second, there are abnormal peak ratios, the first type of which is a linalool / α-terpineol peak area ratio >10.0. Figure 7 The first category of samples had abnormally high linalool peaks. Studies of honeysuckle from different origins and varieties revealed that the linalool / α-terpineol ratio in normal samples did not exceed this value. Tracing the source, the abnormal linalool peaks were found to be caused by some manufacturers illegally adding linalool. The second category of samples had a linalool / α-terpineol peak area ratio <1.0. Figure 8 The main manifestation was that the peak areas of volatile components, including linalool, were all low. After tracing the source, it was found that the honeysuckle used in the process was of inferior quality.

[0071] Currently, there are many manufacturers of honeysuckle dew, but the existing quality standards for this preparation are relatively simple, leading to inconsistent quality among products on the market. Problems such as manufacturers using inferior materials, improper ingredient addition, and tampering with process parameters occur frequently. The quality analysis method established in this experiment has advantages such as fewer indicator components, simple and easily obtainable reference standards, high detection efficiency, accurate and reliable analytical results, and easy traceability. Therefore, it provides a high-value means for guiding and supervising drug production and ensuring the safety of medication for the public.

Claims

1. A method for evaluating the quality of honeysuckle dew based on gas phase characteristic spectra, characterized in that... Includes the following steps: S1. Prepare a mixed reference solution containing n-hexanol, phytol, linalool, furfural, and α-terpineol; S2. Take the honeysuckle dew sample to be tested, extract the volatile oil, and use it as the test solution; S3. The mixed reference solution and the test solution are separately detected by gas chromatograph to obtain the gas chromatograms of the mixed reference solution and the test solution; S4. Compare the gas chromatograms of the mixed reference solution and the test solution. Evaluate the product quality of honeysuckle dew by analyzing the presence or absence of five chromatographic peaks (n-hexanol, phytol, linalool, furfural, and α-terpineol) in the chromatogram of the test solution, as well as the ratio of the peak areas of linalool and α-terpineol. The extraction method of the volatile oil is as follows: 100 ml of the sample to be tested is placed in a round bottom flask, 20 g of sodium chloride is added, and the sample is extracted with a volatile oil extractor. 2 ml of ethyl acetate is added to the volatile oil extractor, the mixture is heated to boiling and kept at a gentle boil for 2 hours, cooled, and the ethyl acetate layer is dehydrated with anhydrous sodium sulfate. The supernatant is taken as the test solution. The gas chromatograph contains a capillary column, uses a programmed temperature ramp, and employs split injection mode. The injector and detector temperatures are both 220°C. The capillary column is an Agilent HP-INNOWax, measuring 30m × 0.25mm × 0.25µm. The programmed temperature ramp consists of: holding at 70°C for 1 min, ramping at 2°C / min to 100°C, ramping at 3°C / min to 130°C, ramping at 50°C / min to 200°C, and holding for 10 min. The split ratio for split injection mode is 5:

1.

2. The method for evaluating the quality of honeysuckle dew based on gas phase characteristic spectra as described in claim 1, characterized in that: If the chromatogram of the test sample shows five chromatographic peaks simultaneously: n-hexanol, phytol, linalool, furfural, and α-terpineol, and the peak area ratio of linalool and α-terpineol is between 1.0 and 10.0, it is judged as a qualified product; if any one of these conditions is not met, it is judged as a non-qualified product.

3. The method for evaluating the quality of honeysuckle dew based on gas phase characteristic spectra as described in claim 1, characterized in that: The method for preparing the mixed reference solution is as follows: take 5 mg each of n-hexanol, phytol, linalool, furfural, and α-terpineol reference standards, place them in a volumetric flask, and add ethyl acetate to make up to 100 ml.

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