A green liquid chromatography method for rapid determination of ergosterol in edible fungi based on matrix solid-phase dispersion extraction and a core–shell column

Zhengming Qian ab, Zi Wu a, Chunhong Li a, Guoying Tan a, Hankun Hu c and Wenjia Li *a
aKey Laboratory of State Administration of Traditional Chinese Medicine, Sunshine Lake Pharma Co., Ltd., Dongguan, Guangdong 523850, China. E-mail: liwenjia1111@163.com
bSchool of Rehabilitation, Xiangnan University, Chenzhou, China
cZhongnan Hospital, Wuhan University, Wuhan, China

Received 7th April 2020 , Accepted 31st May 2020

First published on 1st June 2020


Developing a green analytical method for the analysis of components in food samples is an important research aspect of liquid chromatography (LC). The traditional LC method usually consumes a lot of toxic solvent for sample extraction and LC separation. In the current study, a green analytical method for the rapid determination of ergosterol in edible fungi was established. The sample was extracted and purified by matrix solid-phase dispersion (MSPD) with a green solution (ethanol and water). The LC separation was performed using a Poroshell 120 SB-C18 (4.6 × 30 mm, 2.7 μm) column with a green mobile phase (94% ethanol) at a flow rate of 1.0 mL min−1. The detection wavelength was set at 283 nm. The calibration curve of ergosterol showed good linearity (R = 0.9999) within the test range (4.21–25.27 μg mL−1). The RSD of precision was less than 2.0% and the recovery was 100.4% (RSD = 3.23%). The developed method was successfully applied to quantitative analysis of ergosterol in six edible fungi and the contents of ergosterol were in the range of 1.68–4.02 mg g−1. Only 11.5 mL ethanol water solution was used in the sample extraction and LC separation in the newly developed method, and no toxic organic solvents were used. The total analysis time was less than 15.5 min, about 12–14 min for sample extraction and 1.5 min for LC analysis. This method was environmentally friendly and time-saving, which is helpful to improve the quality evaluation of edible fungi.


1 Introduction

Liquid chromatography (LC) is a powerful analytical technique, which is widely applied in the analysis of components in food, including nutritional ingredients and contaminants.1,2 In general, LC analysis of components in food includes sample extraction and LC separation. Conventional sample extraction techniques, such as ultrasonic extraction (UE) and reflux extraction (RE), have been widely used in food sample preparation with satisfactory extraction efficiency for analytes.3 However, they are usually time-consuming and require a large amount of organic solvent. In some cases, the sample extracts need to be further purified or concentrated before LC analysis.4,5 Most of the traditional LC methods were operated on 5 μm particle packed column, which usually took a long time and consumed much organic mobile phase.6–8 It is desirable to develop new LC methods for the analysis of components in food, which are solvent saving and faster compared with the time-consuming traditional methods.

In recent years, green chemistry has attracted more and more attention and obtained acceptance. Many environmentally friendly extraction methods were developed, such as pressurized liquid extraction (PLE),9 supercritical fluid extraction (SFE)10 and matrix solid-phase dispersion extraction (MSPD),11 which can reduce the harmful solvent consumption. However, specialized and expensive instruments are necessary for PLE and SFE, which limit the application of these techniques in many labs. MSPD is a simple, rapid and low solvent consuming extraction technology, which is easy to be carried out in most labs. It also could combine sample extraction and purification together. MSPD has been used for the extraction of investigated compounds from foods, such as nucleosides,12 flavonoids,11 saponins13 and so on. On the other hand, the LC separation method was very important for decreasing the consumption of harmful solvent and analysis time. Sub-2 μm and Poroshell LC columns were applied as two effective separation technologies, which were rapid and solvent saving.14–16 The sub-2 μm LC column would generate high back pressure and must be operated on an ultrahigh pressure liquid chromatography (UHPLC) system. The UHPLC apparatus is not so easily available in most labs due to it high cost. The Poroshell LC column could provide a similar speed and efficiency to the sub-2 μm particle LC column, while maintaining low back pressure and thus can be used on conventional LC systems, which makes it a good choice for developing green and rapid LC separation methods.15,17 In addition, the solvents, such as methanol and acetonitrile, used in sample extraction or LC separation are usually harmful to the environment. In the literature, ethanol as a clean solvent has been used in the extraction and LC separation of samples.18–20 Therefore, the combination of MSPD and LC separation using a Poroshell column and clean solvent would offer a green and rapid LC method for component analysis in food.

Ergosterol is one of the major bioactive components in edible fungi, which possess various bioactivities such as anti-viral, anti-arrhythmia, anti-oxidative, anti-inflammatory and anti-tumor activities.21–23 It has been employed as the quality marker for edible fungi in the Chinese Pharmacopoeia (2015 edition).3 In the literature, many LC methods have been developed for the determination of ergosterol in edible fungi, including UE combined with LC-UV,3 PLE combined with LC-UV24 and RE combined with LC-UV.25 However, most of the reported approaches usually used harmful solvent and took a long analysis time. Therefore, it is necessary to develop a green and rapid LC method for the determination of ergosterol in edible fungi.

In the current study, a green LC method for the rapid determination of ergosterol in edible fungi was established. The sample was purified and extracted by MSPD and rapidly analyzed by LC using a Poroshell column, and an ethanol aqueous solution was used as the extraction solvent and mobile phase.

2 Materials and methods

2.1 Chemicals and reagents

Ergosterol (96.2%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Ethanol (HPLC grade) was purchased from Fisher (Missouri, USA). Ethanol (analytical grade) was provided by Xilong Scientific Co., Ltd (Sichun, China). The deionized water used for the experiments was purified by using a Milli-Q purification system (Millipore, USA).

Diatomaceous earth was purchased from Sigma (Merck, Germany). ODS (particle size: 50 μm, carbon loading: 14%) was purchased from Shenzhen Kemis Technology Co., Ltd. (Guangdong, China). The solid phase extraction column (12 mL) and sieve plate were purchased from Agilent Technologies (California, USA).

2.2 Fungi materials

Fermented Cordyceps (Cs-4) sample (S1) was purchased from Jiangxi Province, Fermented Cordyceps [CS-C-Q80] samples (S2) were purchased from Guangdong Province, and Cordyceps militaris samples (S3) were collected from Guangdong Province. Cordyceps sinensis samples were collected from Hubei (S4), Xizang (S5), Qinghai (S6) Province. The information of samples is also listed in Table 1.
Table 1 The contents of ergosterol in edible fungi samplesa
No. Name Collection place Contents (mg g−1)
Current method Chinese Pharmacopoeia method
a Data are expressed as mean ± SD (n = 2).
S1 Fermented Cordyceps (Cs-4) Jiangxi 3.62 ± 0.11 3.65 ± 0.07
S2 Fermented Cordyceps [CS-C-Q80] Guangdong 3.19 ±0.05 3.16 ±0.02
S3 Cordyceps militaris Guangdong 4.02 ± 0.19 3.95 ± 0.08
S4 Cordyceps sinensis Hubei 2.67 ± 0.01 2.61 ± 0.02
S5 Cordyceps sinensis Xizang 2.31 ± 0.21 2.25 ± 0.02
S6 Cordyceps sinensis Qinghai 1.68 ± 0.08 1.73 ± 0.11


2.3 Preparation of standard stock solutions and working solutions

The ergosterol stock solution at a concentration of 0.42 mg mL−1 was prepared with ethanol, and the stock solution was further diluted with ethanol to obtain a series of working standard solutions.

2.4 MSPD extraction conditions

The edible fungi sample was triturated with a pulverizer and passed through a 110 mesh sieve. The powder of the sample (0.50 g) was dispersed with diatomaceous earth (2.50 g). The homogeneous mixture (approximately 0.10 g) was accurately weighed and transferred into a SPE column, which was filled with 0.25 g ODS and a piece of sieve plate. The sample mixture was firstly washed with 5.0 mL 70% ethanol, and then the analytes were eluted with 5.0 mL of 100% ethanol solution. The analyte elution solution was collected by using a volumetric flask and the volume adjusted to 5.0 mL. The sample elution solvent was filtered through a 0.45 μm membrane prior to LC analysis.

2.5 UE extraction conditions

According to the Chinese Pharmacopoeia, 0.50 g of sample was put into an Erlenmeyer flask equipped with a stopper, in which 30.0 mL methanol was added. The flask was weighed. The extraction was performed in an ultrasonic bath for 1 hour, and after cooling, the flask was weighed again, and the loss weight was made up with methanol. The sample solution was filtered through a 0.45 μm membrane prior to LC analysis.

2.6 LC instruments and conditions

An Agilent 1260 LC system (Agilent Technologies, USA) equipped with a degasser, quaternary pump, autosampler, column oven, and diode array detector.
2.6.1 Poroshell column LC conditions. An Agilent Poroshell 120 SB-C18 column (4.6 mm × 30 mm, 2.7 μm) was used for sample separation. The column temperature was set at 35 °C. The mobile phase was 94% ethanol with isocratic elution. The flow rate was 1.0 mL min−1. The detection wavelength was 283 nm. The injection volume was 2 μL.
2.6.2 Traditional 5 μm column LC conditions. According to the Chinese Pharmacopoeia, an Agilent Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm) was utilized for sample separation. The column temperature was set at 25 °C. The mobile phase was 98% methanol with isocratic elution. The flow rate was 1.0 mL min−1. The detection wavelength was 283 nm. The injection volume was 10 μL.

2.7 Method validation

The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, recovery, and stability.
2.7.1 Linearity. Six ergosterol solutions were prepared in the concentrations of 4.21, 8.42, 12.64, 16.85, 21.06 and 25.27 μg mL−1 for the construction of calibration curves. The calibration curve was obtained by plotting the mean peak areas versus the concentrations of standards. The limits of detection (LOD) and quantification (LOQ) were individually determined at signal-to-noise ratios (S/N) of approximately 3 and 10, respectively. Briefly, the standard solutions were injected into the LC to ensure that the signal-to-noise ratios (S/N), which were automatically calculated by Openlab software in the Agilent 1260 LC system, were close to 3 and 10. Then, the LOD and LOQ were recorded as the corresponding analyte concentrations at which the right signal-to-noise ratios (S/N) were obtained.
2.7.2 Precision. Intra- and inter-day tests were used to assess the precision of the developed method. The intra-day precision was determined by analyzing ergosterol solution in six replicates within one day. The inter-day precision was determined by analyzing the ergosterol solution twice per day for three days. The relative standard deviation (RSD, %) was used as a measure of precision.
2.7.3 Recovery. The recovery was used to evaluate the accuracy of the developed assay. A known amount of ergosterol was added into sample S1. The mixture was extracted and analyzed using the method mentioned above. Six replicates were performed for the test. The percentage recovery was evaluated by calculating the ratio of detected amount to added amount.
2.7.4 Stability. The stability of sample S1 solution was tested at 4 °C. The sample solution was analyzed every 2 hours within 12 hours. The variation was expressed as RSD.

3 Results and discussion

3.1 Optimization of MSPD conditions

In order to obtain a satisfactory extraction yield and purification effect, the conditions of MSPD extraction were optimized. In this study, the MSPD column was packed in two layers to achieve a good extraction and purification effect, which included an extraction layer and a purification layer. The adsorbent was packed at the bottom of the column as the purification layer and a mixture of the sample and dispersant was packed on the top of the adsorbent as the extraction layer. Diatomaceous earth, which has good dispersion, was used as the dispersant. ODS, which has good retention of ergosterol, was selected as the adsorbent. Furthermore, the ratio of diatomaceous earth and sample, the amount of ODS, the type and volume of washing solvent, and the volume of elution solvent were studied. Sample S1 was used in all tests. Four ratios of sample and dispersant (1[thin space (1/6-em)]:[thin space (1/6-em)]5, 1[thin space (1/6-em)]:[thin space (1/6-em)]10, 1[thin space (1/6-em)]:[thin space (1/6-em)]20, and 1[thin space (1/6-em)]:[thin space (1/6-em)]40) were tested. The results of ergosterol content in the four different ratios were similar. The ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]5 was chosen in this experiment. Different amounts of ODS (0.25 g, 0.5 g, and 1.0 g) used were also compared. The result showed that there was no obvious difference of extraction efficiency. Thus 0.25 g ODS was packed as the purification layer.

In order to reduce the use of harmful solvent, ethanol aqueous solution was used as washing solvent. The polarity of washing solvent was a critical factor for removing impurities and retaining ergosterol from the samples. Four concentrations of ethanol (50%, 60%, 70%, and 80%) were used to wash the impurities from the samples. The results (impurity peak in Fig. 1B) showed that 70% and 80% ethanol solutions had better washing ability than 50% and 60% ethanol solution. In the 80% ethanol solution washing test, the ergosterol was also eluted along with impurities (ergosterol peak in Fig. 1A). So 70% ethanol solution was chosen as the washing solvent. Furthermore, the washing volume of 70% ethanol solution was investigated. The washing solution was collected in six tubes (1 mL of solution per tube). The LC analysis results of the six tubes of washing solution are presented in Fig. 2A. It was found that 5.0 mL of 70% ethanol solution can remove the impurities.


image file: d0ay00714e-f1.tif
Fig. 1 Chromatograms of different concentrations of ethanol washing solution (A) and 100% ethanol elution solution after being washed with different concentrations of ethanol (B).

image file: d0ay00714e-f2.tif
Fig. 2 Chromatograms of 70% ethanol washing solution (A) and 100% ethanol elution solution after being washed with 70% ethanol (B).

Ethanol was used to extract and elute ergosterol. The elute solution was also collected in six tubes (1 mL of solution per tube). The LC analysis results of the six tubes of elution solution are presented in Fig. 2B. This result indicated that the ergosterol was completely extracted by 5.0 mL ethanol solution.

3.2 Optimization of LC conditions

Ergosterol, which was usually separated on a reverse phase column with a high ratio of methanol as the mobile phase, was a weakly polar component. In the literature, a traditional 5 μm C18 column was usually used for ergosterol analysis.7,20 In order to develop rapid LC methods, a Poroshell 120 SB-C18 (4.6 × 30 mm, 2.7 μm) column was used, which could rapidly separate analytes on a conventional LC system. The ethanol aqueous solution was utilized as the mobile phase to avoid harmful organic solvent involved. Different concentrations of ethanol aqueous solutions (90%, 94%, and 98%) were tested for the separation of ergosterol in sample S1. It was found that the separation time increased in the 90% ethanol aqueous mobile phase test while the peak resolution decreased in the 98% ethanol aqueous mobile phase test. Thus, 94% ethanol aqueous solution was used in this study, to obtain a short separation time and good resolution. Meanwhile, different column temperatures (25, 30, 35 and 40 °C) were compared, and the results showed that the higher column temperatures could reduce the separation time, but too high temperature would decrease the resolution. Eventually, 35 °C was selected. According to the UV maximum absorption of ergosterol, 283 nm was selected as the detection wavelength.

3.3 Method validation

The calibration curve of ergosterol was Y = 7.331X − 0.0373, which showed good linear regression (R = 0.9999) within the tested range (4.21–25.27 μg mL−1). The LOD and LOQ of ergosterol were 0.04 μg mL−1 and 0.13 μg mL−1. The intra- and inter-day variations of ergosterol were 0.7% and 0.8% respectively. The average recovery was 100.4% (RSD = 3.23%). The RSD of stability validation was 2.29%. The detailed data of method validation are presented in the ESI. As the results showed that the developed LC method was sensitive and accurate, this method is suitable for the determination of ergosterol in edible fungi.

3.4 Analysis of ergosterol in edible fungi samples

The newly developed LC method was applied to determinate the ergosterol content in edible fungi samples. The representative chromatograms are shown in Fig. 3 and the content results are summarized in Table 1. The content of ergosterol in edible fungi samples was in the range from 1.68 to 4.02 mg g−1, which was similar to previous reported.3,26,27 In order to validate the newly developed LC method, the edible fungi samples were analyzed by the Chinese Pharmacopoeia (2015 edition) LC method and newly developed LC method respectively. The contents of ergosterol obtained by the current developed method were comparable to those obtained by the Chinese Pharmacopoeia (2015 edition) LC method. This result indicated that the developed LC method complied with the Chinese Pharmacopoeia LC method.
image file: d0ay00714e-f3.tif
Fig. 3 Chromatograms of the blank sample, reference compound and edible fungi samples.

3.5 Comparison with reported LC methods

In recent years, there were many reports about the quantitative analysis of ergosterol in food. The information of reported methods is listed in Table 2. Most reported methods used traditional extraction methods (RE or UE) and performed LC analysis on a conventional LC system using a 5 μm particle column, the organic solvent consumption of these methods was in the range of 55.0–105.0 mL, the sample consumption was in the range of 0.25–1.00 g and the total analysis time (sample extraction and LC separation time) reached 85.0 min. For example, the Chinese Pharmacopoeia method (method 5) was carried out by UE combined with LC analysis. The UE took 0.50 g sample, 30.0 mL methanol and 60.0 min. The LC separation consumed more than 21.0 mL methanol mobile phase and took 21.0 min (Fig. 4). A few rapid analytical methods (methods 3 and 4), which used a fast extraction equipment and rapid LC system, were developed for reducing the analysis time and organic solvent consumption. However, these rapid analytical methods usually depended on expensive instruments, which were limited to apply in some labs. In the current experiment, the analysis of ergosterol was carried out by using MSPD and conventional LC systems. In MSPD, the sample was extracted and purified by using an ethanol aqueous solution, which required 19 mg sample, 10 mL ethanol aqueous solution and about 14 min extraction time. In Poroshell column separation, the sample was separated by using an ethanol aqueous mobile phase within 1.5 min. Therefore, the developed LC method is green, rapid and sample saving, which is helpful to improve the determination of ergosterol in edible fungi.
Table 2 The reported LC method for the determination of ergosterola
No. Sample extraction LC separation Total solvent consumption (mL) Total time (min) References
Methods Samples (g) Solvents Time (min) Column Mobile phase Time (min)
a LC separation time was the end time of chromatogram in literature; “MAE” means microwave assisted extraction.
1 RE 0.25 44.0 mL ethanol 45.0 C18 column (250 mm × 4.6 mm, 5 μm) 40.0 mL methanol/water 40.0 84.0 85.0 20
2 UE 1.00 55.0 mL chloroform/methanol mixture (2[thin space (1/6-em)]:[thin space (1/6-em)]1. v/v) 20.0 C30 column (250 mm × 4.6 mm, 5 μm) 50.0 mL acetone/acetonitrile/hexane/water 50.0 105.0 70.0 7
3 MAE 0.10 1.0 mL methanol and 12.0 mL pentane 5.7 C18 column (100 mm× 2.1 mm, 1.7 μm) 2.5 mL acetonitrile/methanol/water 5.0 15.5 10.7 28
4 MAE 20.00 50.0 mL n-hexane/dichloromethane (1[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v) 20.0 C18 column (150 mm × 2.0 mm, 2.2 μm) 6.0 mL methanol/water 10.0 56.0 30.0 29
5 UE 0.50 30.0 mL methanol 60.0 C18 column (250 × 4.6 mm, 5 μm) 25.0 mL methanol/water 25.0 55.0 85.0
6 Current method 0.02 5.0 mL 70% ethanol and 5.0 mL ethanol 14.0 Poroshell C18 column (30 × 4.6 mm, 2.7 μm) 1.5 mL ethanol/water 1.5 11.5 15.5



image file: d0ay00714e-f4.tif
Fig. 4 Chromatograms of the sample obtained by the current method and Chinese Pharmacopoeia method.

4 Conclusion

In the current study, a LC method for the determination of ergosterol in edible fungi combining MSPD with Poroshell column separation was established and applied to six batches of samples. The results indicated that the newly developed LC method was sensitive and accurate, which was suitable for ergosterol analysis in edible fungi samples. Compared with the reported LC method, it was eco-friendly, rapid and sample saving, and was an improved analytical method for assaying ergosterol from edible fungi.

Ethical approval

This article does not contain any studies with animals.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was funded by the National Key R&D Program of China (2018YFC1706101) and Dongguan Academician Workstation Project (DGYSZ-2018-03).

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ay00714e

This journal is © The Royal Society of Chemistry 2020