Ge Bai,Chuanguo Ma*,Xiaowei Chen*

College of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT Phytosterols,which are naturally occurring in plants,have excellent nutritional and health values on lowering both the blood cholesterol level and the risk of cardiovascular diseases.Edible oils are the main source of daily intake of phytosterols,whereas the properties of phytosterols may vary a lot depending on their sources.During the processing of edible oil including refining and frying,phytosterol's content fluctuates,which influences the properties of the final product.Phytosterols and their derivatives undergo physical migration between different phases and chemical conversion during the processing,which reduces the quality and the commercial value of edible oils.Therein,the loss of phytosterols is the major concern in the process of neutralization and deodorization.In addition,oxidation and thermal degradation of phytosterols occur simultaneously during frying,which also reduces the content of phytosterols.Nevertheless,the oil matrix has a promoting or an inhibitory effect on the thermal oxidation of phytosterols.Therefore,various efforts have been devoted to analyzing and improving the remaining contents of phytosterols in edible oil.Regardless of the processing method, temperature plays an important role in the loss of phytosterols.At present,the main analysis methods of phytosterols include gas chromatography and liquid chromatography,in which the pretreatment of different types of phytosterols is also a crucial step.This review focused on the following topics comprehensively:(i)the distribution of phytosterols in the oil-containing plants and edible oils during the refining processing;(ii)the pretreatment and analysis methods of various phytosterols(free phytosterols,phytosteryl fatty acid esters,phytosteryl glycosides and acylated phytosteryl glycosides);(iii)the variation of phytosterols in process of esterification and oxidation,storage and so on.The study also proposed that the investigation in the loss and safety of phytosterols during processing of the vegetable oils should be proceeded further in combination with efficient and accurate chromatography methods.

Keywords:Phytosterols Edible oil Distribution Gas chromatography High performance liquid chromatography Oil refining

1.Introduction

Phytosterols are widely distributed in plant sources,especially oil seeds like corn, soybean and rapeseed.They are well known for their various bioactive properties,such as reducing intestinal cholesterol absorption and potential contributions to the prevention of cancer,cardiovascular and metabolic diseases [1-3].Actually, phytosterols are triterpenes analogs of cholesterol;they have the structure of the four-ring steroid nucleus with a methyl or an ethyl group at the C-24 position or an additional double bond at the C-22 position on a side chain.Also, phytostanols are known as stanols with a saturated ring structure and considered to be a subgroup.Certain plant materials,such as wheat and rye, contain small amounts of saturated phytosterols,sitostanol and campestanol[4].It is worth mentioning that most of 4-methyl phytosterols have double bonds between C-5 and C-6 in the ring system and are called Δ5-phytosterols.However,one group of common desmethylsterols rich in certain plants has a double bond between C-7 and C-8,rather than C-5 and C-6,so they are called Δ7-phytosterols[5].Moreover, phytosterols can be classified into 4-demethylated phytosterols,4-methyl phytosterols and 4,4-dimethyl phytosterols,while most are 4-demethylated phytosterols,such as β-sitosterol,stigmasterol and campesterol,without methyl groups at the C-4 position of the sterol ring[6].Nowadays,in addition to the three main phytosterols mentioned above, more than 200 different sterols have been reported[7].

In the 1950s, researchers proposed that phytosterols could decrease the cholesterol level in humans by reducing the concentration of low-density lipoprotein cholesterol(LDL-C), as well as reduce the risk of cardiovascular diseases [8].Then, phytosterols, as functional components,may play a potential role in preventing breast cancer,colon cancer and cervical cancer[9].Subsequent extensive trials have shown that free phytosterols(FPs)or phytosteryl esters(PEs)with fatty acids in vegetable oils can also significantly reduce serum total cholesterol(TC)and LDL-C concentrations with an intake level of 2 g/d phytosterols(corresponding to a dose of 3.3 g/d phytosterol esters)[10].A significant effect of phytosterols on systolic and diastolic blood pressure was shown for a duration of ＜12 weeks[11].Taken together,phytosterols may be appropriate ingredients in functional food for disease prevention.

Edible oils are the main source of phytosterols for daily intake, whereas the properties of phytosterol may vary a lot depending on their sources.The structures of common phytosterols, FPs and their derivatives were shown in Fig.1.These derivatives [6], including phytosteryl hydroxycinnamic acids esters (HPEs), phytosteryl fatty acids esters (PEs), acylated phytosteryl glycosides esters(APGs)and phytosteryl glycosides esters(PGs),have different phytochemical effects and functional properties.Apart from the important role in maintaining adequate plant cell membrane,phytosterols are also precursors of many plant growth factors[12].FPs and,to a certain extent,PGs and APGs are integrated into cell membranes.Like cholesterol in mammalian cells,these components have critical roles in maintaining the structure and function of cell membranes.PEs are present in plant cells,mainly in a storage form similar to cholesterol esters in mammalian cells.Therefore,phytosterols are available in plant-based foods.However,the refining process of vegetable oil and the preparation process of oil-based products have adverse impacts on phytosterols.Several factors might lead to phytosterol loss,including chemical conversion and physical migration[13,14].Commonly,high-temperature treatments,such as deodorization,are a key factor influencing the stability of phytosterols.Apart from frying and baking,deodorization and hydrogenation at high temperatures also result in a content decrease of phytosterols in edible oils.Moreover,the chemical conversion of phytosterols seems to be associated with many health hazards.Currently,the content level of phytosterols was used as a marker of natural products such as edible oils and fats due to their unique components[15].Therefore,controlling the constituents and amount of phytosterols is essential for ensuring the high quality of edible oils,such as extra virgin olive oil.

Because of the variety difference of phytosterols in the common sources,the diverse and complex molecular structures of these compounds require multiple pretreatments before analysis.Although AOCS Official Method Ce 12-16 was proposed to determine the five FPs,many phytosterol conjugates cannot be determined in an intact manner yet.The variety in molecular conformation of phytosterol conjugates induce numerous analysis challenges.Therefore,the present review illustrated the phytosterols distribution in vegetable oils, also discussed novel analysis methods of phytosterols and their mechanisms,finally elaborated the physical and chemical loss of phytosterols during refining,frying and storage, aiming to provide the scientific basis for improving technologies in the refining of functionally edible oils.

2.Phytosterols distribution in edible oil

Phytosterols can be detected in various plants and edible oils,such as cereal,fruits and vegetable oils.Among these products, the highest content of total phytosterols was found in vegetable oil(150-1,231 mg/100 g),followed by legumes, nuts and cereals [16,17].Therefore, vegetable oils and foods made using these oils are the richest sources of phytosterols in normal diets[18].To evaluate the phytosterol contents of various oils, the contents of sitosterol,campesterol and stigmasterol,which are the vital components of phytosterols[18,19],should be considered thoroughly.As reported previously,the level of phytosterols in wheat germ oil was the highest(1,700-2,600 mg/100 g),followed by crude corn oil (780-1,390 mg/100 g), corn germ oil (1,070 mg/100 g), crude rapeseed oil (680-880 mg/100 g), crude soybean oil (300-440 mg/100 g),and palm oil(70-80 mg/100 g)[20].The difference of phytosterols in vegetable oils can be attributed to their species,origins,genetic makeup,environment,and other factors.The phytosterol composition in different edible oils was shown in Table 1.

3.Analysis of phytosterols

It is difficult to analyze the phytosterols in oils and fats because of complex components existing in the oil system.Thus,it is necessary to extract the target compounds or exclude the non-target ones.Traditional analysis methods were originally developed based on cholesterol analysis,including a series of purification and enrichment steps.Recently,remarkable progress has been made in terms of sample preparation and chromatographic technique improvements, and the analysis efficiency and accuracy have been greatly enhanced.

Fig.1.Chemical structure of free phytosterol,their conjugates and the sites of hydrolysis.

3.1.Phytosterols separation from edible oils

The types and polarity of phytosterols should be considered during extraction.The polarity of phytosterols varies greatly with their molecular configurations.Nonpolar lipids mainly contain FPs and PEs.Among various chromatographic separation technologies,column chromatography(CC),thin layer chromatography(TLC)and solid phase extraction(SPE)are commonly used to separate FPs and PEs[21-23],therein CC method is more suitable for samples with large quantity(＞200 mg).Better quantitative or qualitative results could be obtained by adopting capillary gas chromatography and liquid chromatography after sample pretreatment by CC and TLC.To purify and separate the initial sample,the elution solvent is generally selected according to the polarity of components.Lacoste et al.[24]selected acetone and ethanol as eluent and silica gel(particle size 70-230 meshes)as fillers to successfully collect PGs and APGs.Besides,lipids were separated or extracted via TLC[25,26].Spots on silica gel plates sprayed with fluorescent indicators(such as dichlorofluorescein)can become visible under ultraviolet(UV)lamps.However,the CC and TLC methods are complex and time consuming.Therefore,more and more attention has been paid to the development of more efficient methods.These techniques are finally replaced by SPE,which can achieve faster fractionation and requires less solvents.Conventional analysis methods of phytosterols still demand extensive sample preparation.

Different physical and chemical methods[27-29]have been reported to separate FPs and their derivatives according to their different molecular configurations.First of all,it is important to determine the target compounds, including FPs,PEs,PGs,APGs or HPEs,which have polarity difference.In refined vegetable oils, FPs and PEs are the main target compounds that can be extracted by nonpolar solvent hexane[30].However,the detected content of FPs or PEs might be slightly high due to the presence of residual PGs and APGs.

Owing to the difference in structures of hundreds types of phytosterols,abundant possible chemical combinations and polarity of the conjugates,there is not a single method available to analyze all molecular species of phytosterols simultaneously.Currently, the methods for the separation and purification of PEs cannot completely separate each PE in vegetable oils.With the innovation of analysistechnologies, gas chromatography (GC) and highperformance liquid chromatography(HPLC)are more specific for separation of the target compounds,and mass spectrometry(MS)is usually used for identifying characteristic fragment ions of phytosteryl/stanyl esters.For instance,the online LC-GC-based method has been applied for analyzing the compositions of the phytosterols in various nuts,especially FPs and PEs[34].

Table 1 Composition of phytosterols (mg/100 g) in different edible oils.

3.2.Hydrolysis and silylation of phytosterols

The total phytosterols could be extracted via hydrolysis and subsequently analyzed by chromatography.The ester linkages in PEs,HPEs and APGs can be hydrolyzed by saponification(KOH or NaOH in methanol),while the glycosidic linkages in PGs and APGs require acid hydrolysis,as shown in Fig.1.Unlike conventional methods, the novel method was capable of extracting and detecting the total phytosterols during saponification in foods using GC with flame ionization detector(FID)[35].However,acid or alkaline hydrolysis sometimes alters the structure of certain phytosterols [36,37], but enzymatic hydrolysis has been proposed to gently release FPs from conjugated compounds[38,39].

Phytosterols are involatile and relatively labile at high temperatures,resulting in unreliable analysis at high temperatures.Generally,FPs are silylated for the derivatization of-OH prior to GC analysis.Several silylation reagents are widely used,such as bis(trimethylsilyl)acetamide, bis(trimethylsilyl)ether,bis(trimethylsilyl)trifluoroacetamide(BSTFA),nitrogencontaining silyl ethers and pentafluorophenylsilyl ether.However,advances in the technologies of modern capillary columns have facilitated the analysis of non-silylated phytosterols[40,41].

3.3.Gas chromatography analysis

GC technology is widely used in the determination of phytosterols and becomes more accurate and efficient along with continuous development and updating.Crucial details of certain GC methods were listed in Table 2.GC is usually connected to FID for sensitive and responsive linearity and MS for structural identification.Meanwhile,it also requires authentic standards,such as 5α-cholestan-3-β-ol,to confirm the retention time and response factors by FID quantitation.If there is no commercial authentic standard of phytosterols,MS is adopted for further confirmation.Although not all samples are vegetable oil,the detection targets and the sample pretreatment methods are similar.

3.4.High performance liquid chromatography analysis

Owing to the advantages of low column temperature and nondestructive test conditions, HPLC seems to be appropriate for the analysis of thermally unstable compounds,such as phytosterols.The FPs and their derivatives could be separated and quantitatively analyzed by HPLC without the hydrolysis or silylation processes.However,the low hydrophilicity and structure difference of phytosterols make sample preparation and chromatographic analysis challenging [28].The separation of FPs homologues and unsaturated analogues is usually conducted by reversed-phase HPLC (RP-HPLC) methods.In RP-HPLC,separation is based on the hydrophobic interactions between the analyte and stationary phase,which are affected by the molecular size and number of double bonds;whereas for normal-phase HPLC (NP-HPLC), separation is mainly based on adsorption and polarity difference [20].UV,diode array detection(DAD),evaporative light scattering detection(ELSD)or refractive index(RI)were used in the detection and quantitation of phytosterols[42].HPLC-MS is exploited to accurately identify and quantify phytosterols,especially to measure the molecular weight of the various phytosterol derivatives.Zarrouk et al.[43]detected precursor ions and fragment ions of FPs for quantitation in virgin olive oils by using HPLC-APCI-tandem MS.It is worth mentioning that atmospheric pressure chemical ionization(APCI)is more suitable than electrospray ionization(ESI)in the identification of phytosterols by MS,which is mainly due to the weak polarity of phytosterols.Several selected HPLC methods reported in articles for the analysis of phytosterols in different samples were listed in Table 3.

4.Changes of phytosterols in edible oils

4.1.Changes of phytosterols during refining process

In the refining process of edible oil,some minor components,such as phospholipids,free fatty acids,chlorophyll and carotene,are generally removed,meanwhile the beneficial components(e.g.,phytosterols)are also lost.As the major components in edible oil,the content change of FPs and PEs is the main concern in the refining process.Based on detection,we have found that the content of phytosterols decreased gradually in each step of the oil refining process.As early as 1974,the content changes in total phytosterols during the refining process were observed [44].Anna Johansson and Hoffmann[45]studied the effect of refining process on FPs and PEs,in which the content of FPs and PEs in various refining processes was reduced from 3.1 to 1.8 mg/g, and from 3.4 to 1.6 mg/g, respectively.Crude corn oil is commonly subjected to the conventional refining process,and approximately 50%of total phytosterols was eliminated in the conventional refining process, while only 10%was lost in a moderate refinement[33].The loss of phytosterols largely depended on the refining conditions.Gutfinger and Letan[44]previously studied the effects ofthe local oil refining technologies on the phytosterol content,and the results showed that different refining technologies could cause different loss degrees of phytosterols.In general,the complete refinement of vegetable oils resulted in a 10%to 70%loss of phytosterols[56].Therefore,it is necessary to study the effect of each step on phytosterol loss during the refining process.

Table 2 Phytosterols detected by gas chromatography(GC).

Because the presence of phospholipids can affect the oxidation stability of edible oils,the first step of the refining process is degumming, which aims to remove phospholipids from crude oils.It is generally known that water degumming can effectively remove PGs and APGs to a non-detectable level due to the polarity of PGs and APGs[45,56].It is speculated that phytosterols were embedded with the phospholipids bilayer by hydration of phospholipids and their self-assembly into a lamellar liquidcrystalline mesophase[57].Verleyen et al.[58]reported a slight increment in the amount of FPs during acid degumming due to the acid-catalyzed hydrolysis of PEs.

The second step of the refining process is neutralization,in which free fatty acids can be neutralized in the degummed oils.Meanwhile soapstock is formed at the bottom of the oil phase by centrifugation.Phytosterols are probably adsorbed or wrapped in the soapstock.It was previously reported that certain amount of phytosterols(9%-21%)may be transferred to soapstock through physical adsorption [44,59,60].The unsaponifiable part of soapstock contains 70% phytosterols on average [44].However,the content of PEs remained constant in neutralization,most likely indicating that hydrolysis of PEs did not occur.Thus,the difference between the losses of FPs and PEs may be caused by their hydroxyls at C-3 position.

The third step of the refining process is bleaching to remove pigments in edible oils.Because the competitive adsorption is probably present between phytosterols and pigments,the interacting forces between phytosterols and adsorbent agents may be the major reason causing the loss of phytosterols [61].Besides, the pH value of adsorbent agents affects the formation of phytosterol by-products.The content of PEs decreased slightly after treatment with bleaching agent, and the reason may be due to the acidactivated bleaching agent having hydrolyzed PEs [58].The formation of steradienes and the hydrolysis of PEs may be caused by the acidity of bleaching agent[13,14,56,62].Moreover,the trace metal ions existing in the bleaching agent may induce thermal oxidation of phytosterols.It has been demonstrated that the metal ions in oils significantly induced oxidation of phytosterols at both low and high concentrations[63,64].In addition,it is important to be aware of the stability and by-product of phytosterols in edible oils.The phytosterol oxidation products are present in the crude oils.These oxidation products undergo complex chemical reactions during the refining process,particularly bleaching[65].

The aim of the fourth step,deodorization,is to remove free fatty acids and undesirable odors to improve thequality of edible oils.The content of FPs distilled from oil changes with the deodorization temperature(220-260°C)based on the vapor pressure.The high temperature would cause the chemical loss of phytosterols during deodorization.Ferrari, Johansson and Appelqvist [81,82] also observed that the content of PEs after refining was higher than the content of PEs in crude oil.In the physical refining process,it was found that the fraction of PEs increased considerably,which may be due to thermal promotion of the esterification reaction between FPs and FFAs[58].Ferrari et al.[81] found that the total phytosterols fraction of corn oil contained less stigmasterol and Δ7-stigmasterol than the PE fraction after refining.This may be explained by the fact that stigmasterol and Δ7-stigmasterol are more prone to esterification or transesterification[83].According to previous data,the effects of refining parameters on FPs and PEs are different during the refining process[83].Moreover, the content of steradienes, which is absent in crude oils,increased in every successive refining stage,in particular during deodorization[13].Scholars[62]found that steradienes were formed by heating-promoted dehydration during deodorization (Fig.2).Additionally, the Δ5-phytosterol was dehydrated to Δ3,5-steradiene through the 1,2-elimination reaction under high-temperature frying conditions[84].Furthermore,the presence of these compounds can be used to detect processed fats or their mixtures in fats and oils that have been designated as genuine and unrefined.

Table 3 Phytosterols detected by high-performance liquid chromatography(HPLC).

Overall,the physical migration of phytosterols seems to be the main reason for phytosterol loss during oil refining.It was concluded that the neutralization and deodorization processes resulted in the largest phytosterol losses.According to the previous data,the effects of refining parameters on FPs and PEs are different during the refining process[58].Nevertheless,few papers have described the mechanism of phytosterol loss during refining.

4.2.Changes of phytosterols during the frying process

Fig.2.Molecular conformational change of phytosterols during the frying processes.

Although the heating temperature during the frying process is not as high as that of the deodorization process,there is abundant air during the frying process to trigger the oxidation and thermal degradation of phytosterols simultaneously.At present, based on the common existence of phytosterol oxidation, there are more investigations on phytosterol oxidation during the frying process.Researchers have originally found plenty of cholesterol oxidation products in various foods,and then their negative effects have been thoroughly reviewed [85-87].Since then,elucidation of the mechanism for cholesterol oxidation has been a good example for the study of phytosterol oxidation[88].In the process of food production,many factors lead to chemical changes in the molecular structure of phytosterols,such as light,temperature,metal ions,lipid matrix, enzymes, and other trace components [86,89].Under the influence of these factors,FPs undergo oxidation reaction,and consequently the primary and secondary oxides are detected [85,86,90-99].Also, PEs are oxidized via the similar free radical mechanism as FPs[100].Moreover,the lipid matrix is one important factor influencing the oxidation of phytosterols and the formation of oxidative derivatives[101].In the case of an unsaturated lipid matrix,the oxidative coupling of the lipid matrix and phytosterols begins with the oxidation of unsaturated lipids and transits to the gradual oxidation of phytosterols [102].The phytosterol structure also seems to be one of the factors influencing the formation of phytosterol oxidation products.Furthermore,when phytosterols are combined with emulsions,these phytosterols are particularly easy to oxidize[103],because oxidation tends to occur at the interface compared with the oil phase or aqueous phase[99].

In the study of the oxidative properties of β-sitosterol,when compared with cholesterol both products exhibit thermal instability.When heated at 180°C for 2 h,75%of cholesterol and β-sitosterol are oxidized, and the oxidation rates and products are similar[104].The oxidation products of cholesterol comprise 7-ketonecholesterol,7-α-hydroxycholesterol,7-β-hydroxycholesterol,5,6-α-epoxycholesterol and 5,6-βepoxycholesterol, while the oxidation products of βsitosterol include 7-ketositosterol, 7-α-hydroxysitosterol,7-β-hydroxysitosterol, 5,6-α-epoxysitosterol, 5,6-βepoxysitosterol[102,105]and 3,5,6-triol[63](Fig.2).The C-7 position of phytosterols is more easily oxidized than the C-5 position.The longer the branched chain is, the more difficult it is to oxidize and degrade the compound[106].At the same time,unsaturated fatty acids can accelerate the oxidation of phytosterols.In the process of accelerating the oxidation of phytosterols by fatty acids and triglycerides,researchers have proposed two hypotheses.The first hypothesis is that both triglycerides and fatty acids can increase the solubility of cholesterol and βsitosterol,thereby attaining good dispersion.This improves the contact and permeation of oxygen,thus accelerating the oxidation of phytosterols.The second hypothesis is that the oxidation products of fatty acids can enhance the oxidation of cholesterol and β-sitosterol[105].At different temperatures and lipid unsaturation levels,once unsaturated lipids begin to be oxidized, phytosterols begin to be oxidized.The interaction between the lipid composition and temperature has an important effect on oxidation of phytosterols:phytosterols in unsaturated lipids are more stable than those in saturated lipids at high temperatures(＞140°C),whereas at low temperatures(＜140°C),the opposite occurs.It is assumed that unsaturated lipids are more easily oxidized at high temperatures,thus preventing phytosterols from degradation.In saturated lipids, high temperatures force the stronger lipid components to react with phytosterols.The difference in the content of oxidative phytosterol products was observed at low and high temperatures, indicating that the oxidation processes of phytosterols were different at high and low temperatures [101].However, fatty acid esters delay the oxidation of phytosterols and inhibit the formation of phytosterol oxides in the heating process,and unsaturated fatty acid esters play a more significant role [102].In addition, studies have shown that phytosterols act as antioxidants to scavenge free radicals in frying oil or similar conditions [90,107].The addition of βsitosterol could significantly reduce the formation of triacylglycerol polymers in trioleate and vegetable oil after heating at 180°C for 72 h due to the conversion of phytosterols to steradienes by the dehydration [84].

4.3.Changes of phytosterols during storage

Phytosterols are prone to oxidation, especially during long-term storage in thermal environment[108].The temperature of storage also plays a role in the oxidation stability of phytosterols during storage.The peroxide,thiobarbituric acid,and anisidine values and 7-ketone derivatives vary with the structure of phytosterols and the storage temperature, and each index showed an upward trend with prolongation of the storage time [108].Rudziñska et al.[103]reported that the oxidative rates of phytosterols and phytostanols were similar during storage of margarine enriched with these compounds at 4°C and 20°C.Phytostanols are considered to be less prone to oxidation and degradation due to the lack of double bonds in its structure;however,the presence of tertiary carbon makes these compounds prone to degradation,which might generate various oxidized derivatives[88].Although esterification can alleviate the oxidation of FPs, temperature and time are also important factors of the PEs stability during storage.Panpipate et al.[108]reported that all oxidative indices of sitosteryl esters tended to increase with increasing storage time at 25°C and 55°C up to 20 days.

Phytosterols not only are present in edible oils,but also are used to enrich or fortify various food products.The moisture content,temperature,and oxygen content mainly affect phytosterols degradation during storage.The degradation of phytosterols in foods with high water content is faster than that of phytosterols in edible oils.Thus,novel techniques have been applied to foods with high water content to improve oxidative stability,such as encapsulation[109].Additionally,a controlled atmosphere and a reduction in oxygen levels also inhibited the degradation of phytosterols[110].

5.Conclusions and perspectives

Phytosterols and their derivatives are able to reduce the concentration of LDL-C in human plasma significantly.For this reason, the underlying mechanisms for phytosterol loss and strategies for enriching food with phytosterols have become a hot topic of research recently.Edible vegetable oils are the main source of phytosterols.In the vegetable oil refining process,phytosterols and their derivatives undergo physical migration between different phases and chemical transformation,therefore reducing the value of oils.The phytosterol oxidation products occur in the undesirable reactions during the food processing.However,the generation of degradation products and the underlying mechanisms need to be further studied because of the inequality between contents of the phytosterol loss and contents of oxidation products.This situation drastically hampers the development of efficient refining methods,resulting in low phytosterols content in the edible oils in the current market.In addition,thermal oxidation products of phytosterols are associated with health hazards and safety issues.With regard to the analytical methods of phytosterols,researchers are mainly concerned with the direct methods including HPLC-MS and GC-MS,which are faster and more convenient.

Author Contributions

Ge Bai:Writing-original draft preparation, editing;Chuanguo Ma:Funding acquisition, supervision and conceptualization;Xiaowei Chen:Writing-review&editing.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(No.31972110)and the National Key R&D Program of China(No.2018YFD0401102).