(Beijing Physical and Chemical Analysis and Testing Center, Domestic Scientific Instrument Application Demonstration Center, Beijing 100089, China)

Abstract: The preliminary qualitative analysis of ethanol extracts from different musk samples was carried out by gas chromatography -mass spectrometry to determine the quality of different musk samples. The results showed that the natural musk samples contained a variety of terpenoids in addition to the active ingredient musk ketone. Some of the commercially available musk samples have only a significant amount of musk ketone, which is almost free of terpenoids; other substances are higher fatty acids. Experiments show that the quality of different musk can be easily, quickly and effectively identified by ethanol extraction-gas chromatography/mass spectrometry.

Key words: musk, musk ketone, gas chromatography , mass spectrometry

Preliminary Identification of Musks for Different Quality by GasChromatography-Mass Spectrometry

Wang Yu, Liu Cong, Chen Shun-cong, Wu Yan-wen

(Beijing Center for Physical & Chemical Analysis, Application & Demonstration Center for Homegrown Scientific Instruments, Beijing 00089, China)

Abstract:

A study for the preliminary identification of different qualitymusks by gas chromatography-mass spectrometry (GC/MS) is presented. The musk samples were extracted using ethanol, and then the muskextracts carried out qualitative analysis by GC/MS. The resultsshowed: as active ingredient , the muscone couldn't be the onlybasis to identify the natural musk; there were various steroids innatural musk besides muscone; In the some musk samples withoutsteroids, the higher fatty acid/ester was detected. Using GC/MSwas an easy, effective and rapid Method for identification ofdifferent quality musks.

Keywords: Musk, Muscone, Gas Chromatography-Mass Spectrometry, Ethanol Extract

Musk is a dried traditional Chinese herbal medicine and a high-grade spice. Due to the high market price of natural musk and the extremely limited source of medicinal materials, there are currently synthetic musk products in various commercial musk products, as well as various adulterated "musk". The methods for identifying musk are generally judged by physical properties (such as microscopic identification, etc.) and methods for determining the content of the main active ingredient muscone (1, 2). These methods all have certain one-sidedness. For example, the microscopic identification method can not effectively judge the specific components of musk; while musk ketone can be added after artificial synthesis [3]. In this paper, the ethanol-ultrasonic extraction of several different musk samples was used to determine the various components in different musk samples by gas chromatography-mass spectrometry, and the quality of different musk was preliminarily identified.

1 Experimental part

1.1 Instrument and working conditions

GC/MS300 gas chromatography/mass spectrometer (Beijing East-West Analytical Instrument Co., Ltd.); GDYQ-721S ultrasonic-assisted fast extraction instrument (Changchun Jida·Little Swan Instrument Co., Ltd.). SE30 capillary column

GC/MS instrument conditions: gas chromatographic partial inlet temperature 250 ° C; oven temperature: initial temperature 60 ° C, held for 10 min, at 5 ° C / min rate to 160 ° C, held for 5 min, at 10 ° C / min rate to 290 ° C, hold for 10 min; no split injection, injection volume 1 μL, open the diverter valve after 1 min; the column is DB-5MS (30m × 0.25mm × 0.25μm); the carrier gas is high purity helium.

Mass spectrometry part electron bombardment ion source (EI), energy 70eV; interface temperature 250 ° C; ion source temperature 150 ° C; quadrupole mass analyzer; solvent delay 2 min, data acquisition time 58 min; scanning mass range 30-500 amu.

1.2 main reagents

Anhydrous ethanol (analytical grade, Beijing Chemical Plant); anhydrous sodium sulfate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); natural musk (provided by the Institute of Chinese Medicine).

1.3 Experimental methods

Accurately weigh 50 mg of musk sample (accurate to 0.0001 g) and place in a stoppered test tube. 4.0 mL of absolute ethanol and 1 g of anhydrous sodium sulfate were added to the test tube, and the mixture was soaked overnight, and then ultrasonically extracted for 30 minutes (extraction temperature 25 ° C, power 400 W). The extract was allowed to stand for clarification, and the supernatant was aspirated through a 0.45 μm organic microfiltration membrane and then measured by GC/MS under the above-mentioned instrument conditions.

2 Results and discussion

2.1 Determination of musk ketone

The main active substance in musk is musk ketone (3-methyl-cyclopentadecanone), which can be artificially synthesized, which is one of the main ways to solve the lack of natural musk source. If the quality of musk is judged only by the content of musk ketone, it is simple but obviously not comprehensive. Because natural musk is a secretion of cockroaches, it contains not only musk ketone but also complex biological components. The results of this experiment also illustrate this point. In the three types of samples tested, musk ketone was found in both natural musk and artificial musk. The standard mass spectrum of musk ketone is shown in Figure 1.

Figure 1 Comparison of musk ketones in the sample with the database standard mass spectrum

Fig.1 Compared the mass spectrum of muscone in samples (red)with standard reference's (blue)

2.2 Determination of multi-components in natural musk samples

The total ion current map of the ethanol extract of the natural musk sample was obtained experimentally (see Figure 2), and the mass spectrum of each component was structurally searched and matched using the NIST database.

Figure 2 Mass spectrometry total ion chromatogram (TIC) of natural musk samples

Fig.2 The total ion chromatogram (TIC) of muscone by full scanin natural musk

The experimental results show that the natural musk has a significant content in addition to the main component musk ketone (retention time 36.61min), and also contains a variety of other substances such as terpenoids, which is consistent with the literature reports [4-6]. Matching the mass spectrum obtained from the experiment with the NIST database, it is possible to determine more than ten kinds of terpenoids, such as deoxyisoindrone-3-acetate, 3-hydroxy-androst-17-one, and episode (甾) ketone, androst-3,17-diol, cholesterol, etc.; and other substances such as cyclopentadecanone. Figure 3-7 compares a partial target mass spectrum with a standard spectrum in the NIST database.

Figure 3 Comparison of deoxyisoindrone-3-acetate in the sample with the standard mass spectrum of the database

Fig.3 Compared the mass spectrum of prasterone acetate insamples (red) with standard reference's (blue)

Figure 4 Comparison of 3-hydroxy-androst-17-one in the sample with the database standard mass spectrum

Fig.4 Compared the mass spectrum of 3-hydroxy-androstan-17-onein samples (red) with standard reference's (blue)

Figure 5 Comparison of the standard mass spectrum of epirubicin with the database

Fig.5 Compared the mass spectrum of epiandrosterone in samples(red) with standard reference's (blue)

Figure 6. Comparison of the standard mass spectra of androst-3,17-diol in the sample with the database.

Fig.6 Compared the mass spectrum of androstane-3,17-diol insamples (red) with standard reference's (blue)

Figure 7 Comparison of the mass spectrometry of cyclopentadecanone in the sample with the database

Fig.7 Compared the mass spectrum of Cyclopentadecanone insamples (red) with standard reference's (blue)

2.3 Determination of components in commercially available musk samples

The experiment conducted a determination of multiple components in three commercially available musk samples, and the results showed that some of the commercially available musk contained only musk ketone; some were free of musk ketone. The two commercially available musk samples in the experiment contained almost no terpenoids, and the obtained mass spectrum matched with the NIST database to determine the saturated/unsaturated higher fatty acid (ester) substances in the sample, such as stearic acid, sub- Oleic acid, methyl linoleate, ethyl palmitate, ethyl oleate and ethyl linoleate. Figure 8-10 compares a partial target mass spectrum with a standard spectrum in the NIST database.

Figure 8 Comparison of the standard mass spectrum of methyl linoleate in the sample with the database

Fig.8 Compared the mass spectrum of methyl linoleate in samples(red) with standard reference's (blue)

Figure 9 Comparison of the standard mass spectrum of ethyl palmitate in the sample with the database

Fig.9 Compared the mass spectrum of ethyl palmitate in samples(red) with standard reference's (blue)

Figure 10 Comparison of the standard mass spectrum of ethyl linoleate in the sample with the database

Fig.10 Compared the mass spectrum of ethyl linoleate in samples(red) with standard reference's (blue)

3.3 Measurement results of commercially available musk samples

The gas chromatographic/mass spectrometric analysis of the natural musk samples and the ethanol extracts of three commercially available musk samples is shown in Table 1. The results showed that there were great differences in the quality of the commercially available musk. Some samples contained only the active (active) component musk ketone. Some samples contained musk ketone, but no scorpion substances contained in natural musk, only high fatty acids/ Ester compounds; and some commercially available "musk" contain only higher fatty acid/esters. It can also be seen from the table that musk ketone cannot be used as the sole criterion for the identification of natural musk, and anthraquinones are an important basis for judgment.

Table 1 Main components in ethanol extracts of different musk samples

Table 1 The main components of ethanol extract in different musksamples

Main substance in ethanol extract
Natural musk samples Muskone, terpenoids (cholesterol), cyclopentadecanone, etc.
Commercially sold 麝香1#
Musk ketone, steroid (cholesterol)

Commercially sold 麝香2#
Ethyl palmitate, ethyl linoleate, ethyl oleate, etc.
Commercially sold 麝香3#
Musk ketone, methyl linoleate, stearic acid, linoleic acid, etc.

3 Conclusion

In this paper, ultrasonic-assisted extraction-gas chromatography/mass spectrometry was used to analyze the various components in ethanol extracts of different musk samples, and the quality of different musk samples was preliminarily identified. The results show that the method can quickly and effectively identify musk with different qualities. Due to the limited amount of sample and the source of the natural musk in the experiment (the comparison of the extract components of different solvents is not possible), and the sensitivity of the instrument and the capacity of the mass spectrometry database are limited, this experiment can only be used as a basis for a feasibility study. The natural musk component is complex, and the content of musk ketone can no longer be used as the sole basis for identification. Gas chromatography/mass spectrometry has strong separation and qualitative ability, which is suitable for the quality identification of musk samples. Through this experiment, it also provides the necessary basis and feasibility for further establishing the gas chromatography-mass spectrometry library of natural musk.

references:

[1] Chen Qinhua, Li Peng, Zhang Zhuo et al. Determination of musk ketone in musk by gas chromatography-mass spectrometry[J], Pharmaceutical Herald, 2009, 28(5): 647-648

[2] Tang Hongmei, Huang Yinghua, Li Detang et al. Determination of musk ketone in artificial musk by gas chromatography[J], Chinese Journal of Experimental Formulation, 2007, 13(9): 4-5

[3] Li Ru, Synthesis of Musk Ketone [J], Synthetic Chemistry, 2004, 12(3): 222-224

[4] Zhang Yibing, Tao Wei, Hong Yukun, et al. Determination of carcass components in musk by gas chromatography/mass spectrometry (GC/MS)[J],中成药,2005,27(1):79-83

[5] Lv Jianguo, Han Enshuo, Research on the aroma components of natural musk[J], Gansu Science and Technology Information, 1994, 10(3): 38-39

[6] Su Guoyi, Wu Ailin, Gan Xiaoni et al. Analysis of musk ketone and anthraquinones in Linzhixiang by gas chromatography/mass spectrometry[J], Sichuan Animals, 2009, 28(4): 507-512

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