A method for simultaneously determining the contents of multiple active ingredients in borneol camphor essential oil

Abstract

The invention discloses a method for simultaneously determining the content of multiple active ingredients in borneol camphor essential oil, wherein the multiple active ingredients are α-terpineol, camphor, p-cymene, myrcene, limonene, α-pinene; borneol camphor essential oil is analyzed by high performance liquid chromatography, and the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler; using a specific mobile phase gradient elution program; flow rate: 1mL / min; column temperature: 35°C; injection solvent is 10μL; detection wavelength selection program is: 0.00min-50.00min selects 208nm. The present invention respectively carries out a linear relationship investigation, stability test, precision test, reproducibility test and sample recovery test to verify the method, proving that this analytical method is accurate and reliable.

Description

Technical Field

[0001] The invention relates to a method for determining the contents of multiple active ingredients in borneol camphor essential oil, in particular to a method for simultaneously determining the contents of alpha-terpineol, camphor, p-cymene, myrcene, limonene and alpha-pinene. Background Art

[0002] Camphor essential oil (Cinnamomum camphora (L.) PresL) is a volatile aromatic substance extracted from the stems, leaves and branches of Camphor trees through washing, drying, distillation, testing and analysis. It has multiple pharmacological activities such as analgesia, anti-inflammatory, antioxidant, improvement of cognitive dysfunction, antibacterial and insecticidal, and has been widely used in the fields of medicine, fragrance and chemical industry. Its main active ingredients contain monoterpenes and sesquiterpenes. The main representative ingredients are α-terpineol, camphor, p-cymene, myrcene, limonene, α-pinene, etc.

[0003] The quality control of the above active ingredients generally adopts ultraviolet-visible spectrophotometry (UV), thin layer scanning (TLCS) and gas chromatography (GC).

[0004] In the prior art, GC is a commonly used analytical method for detecting active ingredients in borneol camphor essential oil, but it can generally only detect one component. We have not yet found a method for simultaneously detecting the contents of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene in borneol camphor essential oil using high performance liquid chromatography. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention adopts a high performance liquid chromatography method and proposes a method for simultaneously determining the contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in borneol camphor essential oil, and the chromatographic conditions adopted are completely different from the methods used in the currently reported relevant literature. The present invention respectively carries out linear relationship investigation, stability test, precision test, reproducibility test and sample recovery test to verify the method, and proves that the analytical method is accurate and reliable.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0007] A method for simultaneously determining the contents of multiple active ingredients in borneol camphor essential oil, wherein the multiple active ingredients are α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene;

[0008] Take 3-5 mg of borneol camphor essential oil, add methanol to make the volume to 5-10 mL, filter with a microporous membrane, analyze the borneol camphor essential oil by high performance liquid chromatography, record the sample peak area, take α-terpineol, camphor, p-cymene, myrcene, limonene, α-pinene reference substances, prepare a mixed reference substance solution with a concentration of 0.1 mg / mL with acetonitrile, filter with a microporous membrane, and calculate the contents of the α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene by an external standard method;

[0009] The analysis conditions are as follows: octadecylsilane bonded silica gel is used as the stationary phase; the mobile phase gradient elution program is as follows: mobile phase A is acetonitrile, and mobile phase B is a phosphoric acid aqueous solution with a mass concentration of 0.1%-0.3%; the volume percentage of mobile phase B is reduced from 50%-54% to 30% according to a first-order linear gradient from 0.00min-20.00min; and maintained at 30% from 20.00min-50.00min; flow rate: 0.9mL / min-1.1mL / min; column temperature: 35℃-38℃; injection volume is 10μL-15μL; and the detection wavelength is 208nm.

[0010] The preferred chromatographic analysis conditions are: using octadecylsilane bonded silica gel as a filler; the mobile phase gradient elution program is: mobile phase A is acetonitrile, mobile phase B is a phosphoric acid aqueous solution with a mass concentration of 0.1%; the volume percentage of mobile phase B is reduced from 50%-54% to 30% according to a first-order linear gradient from 0.00min to 20.00min; and maintained at 30% within 20.00min-50.00min; flow rate: 1mL / min; column temperature: 35°C; injection volume is 10μL; and the detection wavelength is selected as 208nm.

[0011] Through ultraviolet spectrum scanning, we found that α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene have a common ultraviolet absorption wavelength at 208nm, so 208nm was used as the detection wavelength, and the effect was better.

[0012] The mobile phase gradient elution program in the embodiment of the present invention is shown in Table 1:

[0013]

[0014] The relative retention time ranges of the components in the examples set according to the method in Table 1 are shown in Table 2:

[0015]

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] 1. The determination method provided by the present invention can simultaneously determine the contents of multiple active ingredients in borneol camphor essential oil, and can simultaneously determine α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene in borneol camphor essential oil, thereby improving the detection efficiency.

[0018] 2. The present invention has carried out a large number of methodological verification tests, and carried out linear regression equation, stability test, precision test, specificity test, repeatability test and sample recovery test to verify the method, which proves that the determination method is accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HPLC profile of the borneol camphor essential oil sample under the chromatographic conditions of Example 1;

[0020] Figure 2 HPLC diagram of borneol camphor essential oil sample under the chromatographic conditions of Example 2;

[0021] Figure 3 HPLC profile of the borneol camphor essential oil sample under the chromatographic conditions of Example 3;

[0022] Figure 4 HPLC diagram of borneol camphor essential oil sample under the chromatographic conditions of Example 4;

[0023] Figure 5 HPLC profile of the borneol camphor essential oil sample under the chromatographic conditions of Example 5;

[0024] Figure 6 HPLC profile of the borneol camphor essential oil sample under the chromatographic conditions of Example 6;

[0025] Figure 7 HPLC profile of the borneol camphor essential oil sample under the chromatographic conditions of Example 7;

[0026] Figure 8 HPLC profile of the borneol camphor essential oil sample under the chromatographic conditions of Example 8;

[0027] Fig. 9 HPLC profile of α-pinene;

[0028] Fig.10 HPLC profile of camphor;

[0029] Fig.11 HPLC profile of myrcene;

[0030] Fig.12 HPLC profile of α-terpineol;

[0031] Fig.13 HPLC profile of limonene;

[0032] Fig.14 HPLC profile of p-cymene;

[0033] Fig.15 HPLC profile of mixed control sample;

[0034] Fig.16 HPLC chart of blank solvent (acetonitrile)

[0035] in, Figure 1-5 The peaks are labeled as follows: Peak 1 is α-terpineol; Peak 2 is camphor; Peak 3 is p-cymene; Peak 4 is myrcene; Peak 5 is limonene; Peak 6 is α-pinene. DETAILED DESCRIPTION

[0036] The present invention is further described below with reference to the embodiments. Example 1

[0037] An Agilent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) decreased from 50% to 30% according to a first-order linear gradient from 0.00 min to 20.00 min; and remained constant at 30% from 20.00 min to 50.00 min; the flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0038] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0039] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances and prepare a mixed reference solution with a concentration of 0.1 mg / mL using acetonitrile. Filter through a microporous membrane filter, inject the sample, and record the peak area.

[0040]

[0041] (3) Results: The contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in the samples were 0.74%, 14.98%, 0.83%, 1.81%, 18.21% and 10.89%, respectively.

[0042] Method validation experiment of the present invention

[0043] Linear relationship investigation: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, dissolve them in acetonitrile to prepare mixed reference substance solutions of different concentrations as described in Table 3, and inject them separately, record the peak area, and fit the standard curve equation and correlation coefficient. The results show that the linear relationship of each standard curve equation is good.

[0044]

[0045] Based on the operating procedure of Example 1, 5 mg of borneol camphor essential oil was weighed and placed in a 5 mL volumetric flask, 1 mL of 0.1 mg / mL mixed reference solution was added, the volume was fixed, and the detection was carried out according to the method of Example 1. The results are shown in Table 4: The experimental results show that the sample recovery effect of this method is good, ranging between 97% and 105%.

[0046]

[0047] (3) Precision test investigation

[0048] The mixed reference solution described in Example 1 was injected 5 times in succession according to the chromatographic conditions of Example 1, with an injection volume of 10 μL. The peak area and relative retention time (with camphor as 1) of each component were recorded, and the RSD was calculated.

[0049] The test results are shown in Table 5, which show that the RSD (%) of the relative retention time and peak area of ​​limonene, α-pinene, p-cymene, α-terpineol, and myrcene are all <5%, indicating that the instrument has good precision.

[0050]

[0051] (4) Repeatability test investigation

[0052] 6 portions of 5 mg of borneol camphor tree essential oil were accurately weighed and measured according to the method described in Example 1. The test results are shown in Table 6. The results show that the relative retention time and content RSD (%) of α-terpineol, limonene, α-pinene, p-cymene, α-terpineol and myrcene are all <5%, indicating good repeatability.

[0053]

[0054] (5) Stability test investigation

[0055] The samples were prepared according to the method described in Example 1, and injected at 0, 3, 6, 9, 12, and 24 h, respectively, with an injection volume of 10 μL. The chromatographic conditions of Example 1 were used for determination, and the peak areas and retention times were recorded to calculate the content of each component.

[0056] The test results are shown in Table 7. The results show that the relative retention time and content RSD (%) of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene are all <5%, indicating that the solution has good stability within 24 hours and can meet the measurement requirements.

[0057]

[0058] (6) Specificity experiment:

[0059] Referring to Example 1, a mixed reference solution of 0.10 mg / mL α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene was prepared and filtered using a microporous membrane. Acetonitrile was then taken as a blank negative solution and filtered using a microporous membrane. According to the chromatographic conditions of Example 1, 10 μL of each sample was injected to obtain the corresponding chromatogram. Fig.15 The chromatogram of the mixed reference solution is shown in Fig.16 The blank negative solution chromatogram is shown. Figure 1 , Fig.15 and Fig.16 The chromatogram shows that the blank solvent has no interference with the determination of the main substances and the method has good specificity.

[0060] Example 2

[0061] The initial proportion of acetonitrile in mobile phase A is 49%. The steps for detecting the active ingredients in borneol camphor essential oil extract are as follows:

[0062] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) decreased from 51% to 30% according to a first-order linear gradient from 0.00 min to 20.00 min; and remained constant at 30% from 20.00 min to 50.00 min. The volume flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0063] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0064] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, prepare a mixed reference solution with a concentration of 0.1 mg / mL in acetonitrile, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as in Example 1.

[0065] (3) Results The contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in the sample were 0.70%, 14.58%, 0.84%, 1.72%, 17.23% and 10.10%, respectively. See the chromatogram for details. Figure 2 .

[0066] Example 3

[0067] The initial proportion of acetonitrile in mobile phase A was 48% for the detection of active ingredients in borneol camphor essential oil extract

[0068] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) decreased from 52% to 30% according to a first-order linear gradient from 0.00 min to 20.00 min; and remained constant at 30% from 20.00 min to 50.00 min. The volume flow rate was 1.0 mL / min. The detection wavelength was 208 nm. The column temperature was 35 °C.

[0069] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0070] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, prepare a mixed reference solution with a concentration of 0.1 mg / mL in acetonitrile, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as in Example 1.

[0071] (3) Results The contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in the sample were 0.66%, 14.39%, 0.80%, 1.64%, 16.57% and 10.15%, respectively. See the chromatogram for details. Figure 3 .

[0072] Example 4

[0073] The initial proportion of acetonitrile in mobile phase A was 47% for the detection of active ingredients in borneol camphor essential oil extract

[0074] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) decreased from 53% to 30% according to a first-order linear gradient from 0.00 min to 20.00 min; and remained constant at 30% from 20.00 min to 50.00 min. The volume flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0075] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0076] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, prepare a mixed reference solution with a concentration of 0.1 mg / mL in acetonitrile, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as in Example 1.

[0077] (3) Results The contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in the sample were 0.72%, 14.06%, 0.86%, 1.82%, 18.07% and 10.88%, respectively. See the chromatogram for details. Figure 4 .

[0078] Example 5

[0079] The initial proportion of acetonitrile in mobile phase A was 46% for the detection of active ingredients in borneol camphor essential oil extract

[0080] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) decreased from 54% to 30% according to a first-order linear gradient from 0.00 min to 20.00 min; and remained constant at 30% from 20.00 min to 50.00 min. The volume flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0081] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0082] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, prepare a mixed reference solution with a concentration of 0.1 mg / mL in acetonitrile, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as in Example 1.

[0083] (3) Results The contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in the sample were 0.73%, 13.64%, 0.89%, 1.83%, 18.44% and 10.80%, respectively. See the chromatogram for details. Figure 5 .

[0084] Example 6

[0085] The mobile phase A is acetonitrile: the mobile phase B is 0.1% phosphoric acid aqueous solution at a ratio of 50%:50% to detect the active ingredients in the borneol camphor essential oil extract.

[0086] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) remained constant at 50% from 0.00 min to 20.00 min and from 0.00 min to 50.00 min; the volume flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0087] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0088] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, prepare a mixed reference solution with a concentration of 0.1 mg / mL in acetonitrile, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as in Example 1.

[0089] (3) Results: α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene in the sample could not be effectively separated. The chromatogram is shown in Figure 6 The results showed that α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene could not be separated and detected.

[0090] Example 7

[0091] The mobile phase A was maintained at 70% acetonitrile for the detection of active ingredients in borneol camphor essential oil extract

[0092] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) remained constant at 70% from 0.00 min to 20.00 min and from 0.00 min to 50.00 min; the volume flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0093] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0094] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, prepare a mixed reference solution with a concentration of 0.1 mg / mL in acetonitrile, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as in Example 1.

[0095] (3) Results: α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene in the sample could not be effectively separated and detected. The chromatogram is shown in Figure 7 .

[0096] Example 8

[0097] The initial proportion of acetonitrile in mobile phase A was 40% for the detection of active ingredients in borneol camphor essential oil extract

[0098] An AgiLent SB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase program was as follows: the volume percentage of mobile phase B (0.1% phosphoric acid aqueous solution) decreased from 60% to 30% according to a first-order linear gradient from 0.00 min to 20.00 min; and remained constant at 30% from 20.00 min to 50.00 min. The volume flow rate was 1.0 mL / min; the detection wavelength was 208 nm; and the column temperature was 35 °C.

[0099] (1) Preparation of test solution: Weigh 5 mg of borneol camphor essential oil accurately using an analytical balance, place it in a 5 mL volumetric flask, add methanol to make up to volume, filter using a microporous filter membrane, inject 10 μL of the solution, and record the sample peak area.

[0100] (2) Preparation of reference solution: Accurately weigh appropriate amounts of α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene reference substances, dissolve them in acetonitrile to prepare a mixed reference solution with a concentration of 0.1 mg / mL, filter through a microporous membrane filter, inject the sample, record the peak area, and calculate the formula using the same method as Example 1.

[0101] (3) Results The contents of α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene in the sample could not be effectively separated and detected. The chromatogram is shown in Figure 8 .

[0102] Example 9

[0103] Other conditions were the same as those in Example 1, and the detection wavelength was changed to 250 nm. It was found that α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene could not be separated and detected.

[0104] Example 10

[0105] Other conditions were the same as those in Example 2, except that mobile phase A was changed to methanol. It was found that α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene could not be separated and detected.

[0106] Embodiment 11

[0107] Other conditions were the same as those in Example 3, except that mobile phase B was changed to a methanol aqueous solution with a mass concentration of 0.3%. It was found that α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene could not be separated and detected.

Claims

1. A method for simultaneously determining the contents of multiple active ingredients in borneol camphor essential oil, characterized in that: The multiple active ingredients are α-terpineol, camphor, p-cymene, myrcene, limonene and α-pinene; Take 3-5 mg of borneol camphor essential oil, add methanol to make the volume to 5-10 mL, filter with a microporous membrane, analyze the borneol camphor essential oil by high performance liquid chromatography, record the sample peak area, take α-terpineol, camphor, p-cymene, myrcene, limonene, α-pinene reference substances, prepare a mixed reference substance solution with a concentration of 0.1 mg / mL with acetonitrile, filter with a microporous membrane, and calculate the contents of the α-terpineol, camphor, p-cymene, myrcene, limonene, and α-pinene by an external standard method; The analysis conditions are as follows: octadecylsilane bonded silica gel is used as the stationary phase; the mobile phase gradient elution program is as follows: mobile phase A is acetonitrile, and mobile phase B is a phosphoric acid aqueous solution with a mass concentration of 0.1%-0.3%; the volume percentage of mobile phase B is reduced from 50%-54% to 30% according to a first-order linear gradient from 0.00min-20.00min; and maintained at 30% from 20.00min-50.00min; flow rate: 0.9mL / min-1.1mL / min; column temperature: 35℃-38℃; injection volume is 10μL-15μL; and the detection wavelength is 208nm.

2. A method for simultaneously determining the contents of multiple active ingredients in borneol camphor essential oil according to claim 1, characterized in that: High performance liquid chromatography was used to analyze borneol camphor essential oil, with octadecylsilane bonded silica gel as the stationary phase; the mobile phase gradient elution program was as follows: mobile phase A was acetonitrile, and mobile phase B was a phosphoric acid aqueous solution with a mass concentration of 0.1%; the volume percentage of mobile phase B was reduced from 50%-54% to 30% according to a first-order linear gradient from 0.00min to 20.00min; and maintained at 30% from 20.00min to 50.00min; flow rate: 1mL / min; column temperature: 35℃; injection volume: 10μL; detection wavelength: 208nm.

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