Simultaneous Analysis of Hydrophobic Atractylenolides, Atractylon and Hydrophilic Sugars in Bai-Zhu Using a High-Performance Liquid Chromatography Column Tandem Technique

An analytical method was established using high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) with -C18 and -NH2 column tandem for the simultaneous determination of hydrophobic atractylenolide I, II, III, atractylone and hydrophilic compounds glucose, fructose and sucrose in the dried rhizome of Atractylodes macrocephala Koidz (a natural raw material for health foods, Bai-Zhu aka. in Chinese). The method combines the different separation capabilities of reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. It can provides a new choice for the simultaneous determination of hydrophilic and hydrophobic compounds in traditional Chinese medicines and health foods. It provided a reference method for the quality control of Bai-Zhu. The results showed that the linear correlation coefficients of the established column tandem chromatographic method were all greater than 0.9990, the relative standard deviation was 0.1–2.8%, and the average recovery was 96.7–103.1%. The contents of atractylenolide I, II, III, atractylone, fructose, glucose, and sucrose in 17 batches of Baizhu were 172.3–759.8 μg/g, 201.4–612.8 μg/g, 160.3–534.2 μg/g, 541.4–8723.1 μg/g, 6.9–89.7 mg/g, 0.7–7.9 mg/g, and 1.2–21.0 mg/g, respectively.


Introduction
Atractylodes macrocephala (Bai-Zhu aka. in Chinese) is the dried rhizome of Atractylodes macrocephala Koidz, which is widely used as a tonic herbal in eastern Asia.It belongs to the homologous resource of medicine and food in China [1][2][3][4].The components include hydrophobic lactones, volatile oils, and hydrophilic carbohydrates, etc. Hydrophobic atractylenolide I, II, III and atractylon are important active components in Bai-Zhu, which have anti-tumor, anti-inflammation, anti-depression, anti-hypertension bio-activites [5,6].The hydrophilic carbohydrate compounds in Bai-Zhu are also a kind of important component [7,8].They are two kinds of important components in the quality control of Bai-Zhu.
In China, the unique processing (aka.Pao-Zhi in traditional Chinese medicine) of raw herbal medicines is a technique used to fulfill the different requirements of therapy, resulting in pharmacological properties changing, and toxicity or side effects reducing.There are obvious different in clinical efficacy between the raw and processed state [9].Presently, the main processing methods of Bai-Zhu are steamed Bai-Zhu, stir-fried Bai-Zhu, soil-fried Bai-Zhu, soaked Bai-Zhu with rice swill, focal Bai-Zhu, bran-fried Bai-Zhu, and fried Bai-Zhu with honey bran [4].Among the methods, bran-fried Bai-Zhu is documented in the Chinese Pharmacopeia [3].Investigation on the changes occurring in the chemical components of Bai-Zhu before and after processing have been studied by utilizing HPLC, GC/MS, and other analytical techniques.Substantial changes in essential oil, as well as the sesquiterpenoids, e.g., atractylon, atractylnolides I, II, and III, etc., were observed.These changes can be explained as the result of evaporation, conversion, or degradation by heating, due to their physicochemical properties such as volatility and instability [10][11][12][13].
For the quality control of Bai-Zhu, as there is no quantitative identification of Bai-Zhu in the Chinese Pharmacopoeia [3]; research on the determination of the content of different chemical components in Bai-Zhu has been limited.Usually, analyses of atractylenolide I, II, III, atractylon and sugars in Bai-Zhu are completed using reversed-phase liquid chromatography (RP-HPLC) [14,15] and hydrophilic interaction liquid chromatography (HILIC) [16], independently [2].The two independent methods are time-consuming and laborious.Therefore, it is important to develop a simple and rapid analytical method for the simultaneous detection of hydrophobic atractylenolide, atractylon, and hydrophilic carbohydrates in Bai-Zhu.
In order to simultaneously analyze hydrophilic and hydrophobic components in a complex matrix in the field of chromatography research, two-dimensional HPLC (2D-HPLC) methods and a column tandem technique have been employed based on the excellent orthogonality of RP-HPLC and HILIC [17][18][19][20][21][22][23][24][25][26][27].Although the 2D-HPLC method allows for high separation resolution, the instrument system is highly complex.The column tandem technique can be employed on a simple instrument.The RP-LC column in tandem with an HILIC column has demonstrated that it is beneficial for the separation of hydrophilic and hydrophobic substances in complex samples, and has been successfully applied to the analysis of different targets in different sample matrices [20][21][22][23][24][25][26][27].Therefore, the series of the RP-LC column and HILIC column, whose mobile phase (an acetonitrile-H 2 O system) is also compatible, are easy to achieve, and can be used for the simultaneous analysis of hydrophobic components atractylenolide I, II, III, atractylone and hydrophilic sugars in Bai-Zhu.
In this paper, an analytical method was established via HPLC-DAD-ELSD with a C 18 -NH 2 column tandem for the simultaneous determination of hydrophobic components atractylenolide I, II, III, and atractylone, and hydrophilic compounds glucose, fructose and sucrose (Figure 1) in Bai-Zhu.The tandem of DAD and ELSD can simultaneously detect sugar with no UV absorption and atractylenolide I, II, III, and atractylone.It provides a reference method for the quality control of Bai-Zhu.
A Thermo Scientific Sorvall ST 16R high-speed centrifuge (Thermo Fisher Scientific, Shanghai, China) was used for sample pretreatment.An F-030SD ultrasonic device (Shenzhen Fuyang Technology Group Co., Ltd., Shenzhen, China) was used for sample extraction.

Preparation of Standard Solutions
To construct the calibration curves, 0.50 mL, 1.0 mL, 4.0 mL, and 8.0 mL of a mixed standard solution with concentrations of 1.0 mg/mL, 1.0 mg/mL, 1.0 mg/mL, 0.025 mg/mL, 0.025 mg/mL, 0.025 mg/mL, 0.025 mg/mL for fructose, glucose, sucrose, atractylon, and atractylenolide I, II, and III were diluted with methanol to 10.0 mL.A series of mixed standard working solutions were obtained.Some 20 µL of the solution was injected into the HPLC, respectively.Linear regression was calculated using a least-squares method.All the above solutions were stored in a 4 • C refrigerator.

Sample Preparation
The medicinal slices of raw Bai-Zhu and processed Bai-Zhu were crushed into powder (20 mesh).Briefly, 0.20 g of the powder was extracted in 5 mL of methanol in an ultrasonic bath for 45 min.After centrifuging at 5000 rpm for 5 min, the supernatant was filtered using a 0.22 µm filter membrane.The filtrate was injected into HPLC.

Selection of Detectors
Alongside a big difference in polarity, there are also differences in spectral characteristics between sugars and terpenoids.It is well known that sugars do not have UV absorption, and cannot be analyzed using DAD.Because atractylon, and atractylenolide I, II, and III have high volatility and low content, they are difficult to detect through ELSD, because of their sensitivity.Therefore, the selection and combination of detectors is important for the simultaneous analysis of sugars and atractylon and atractylenolide I, II, and III in Bai-Zhu.
For the quantitative analysis of atractylenolide I, II, III, and atractylenolide, the optimal UV detection wavelength was investigated.The UV absorption spectrum for each compound is shown in Figure 2. The characteristic absorption wavelength of atractylenolide I is 274 nm, and is the same for atractylenolide II, III and atractylon (218-219 nm).To obtain optimal sensitivity and anti-interference ability, 274 nm was selected as the optimal detection wavelength for the analysis of atractylenolide I, and 218 nm was selected as the optimal detection wavelength for atractylenolide II, III and atractylon in a number of reported methods [28][29][30][31].The optimal selection of the detection wavelength can improve the sensitivity.In this method, tandem DAD and ELSD was employed for the analysis of Bai-Zhu.As shown in Figure 3, the two kinds of compounds have very different responses in DAD and ELSD.It can be seen that both atractylenolide and atractylon have good responses on DAD, but there is no response on ELSD.On the contrary, sugars respond on ELSD and show no response on DAD.Therefore, the combination of DAD and ELSD is a good choice for the simultaneous detection of two kinds of compounds.In addition, the difference in responses according to the difference detector can also increase analytical selectivity due to incomplete separation.

Optimization of Separation Conditions
Using single-column mode for the separation, hydrophobic atractylenolide I, II, III, and atractylone can be separated on a C 18 column based on hydrophobic interaction (a revised phase separation mode) (Figure 4a ELSD), and hydrophilic sugars can be separated on a -NH 2 column based on hydrophilic interaction separation mode (Figure 4a PDA).The results were similar for Bai-Zhu sample solution (Figure 4b).
Sugars can not be separated completely on a -C 18 column, and atractylenolide I, II, III and atractylone can not be separated completely on a NH 2 column based on a H 2 O-ACN mobile phase system.
When a -C 18 column is combined with a -NH 2 column, two types of compounds can be separated completed.The chromatographic separation specification parameters, including retention factors (k), relative retention value (α), and resolutions ®, are as follows.In addition, two kinds of different connection, i.e., C 18 columns in front of NH 2 columns, and NH 2 columns in front of C 18 columns, on the simultaneous separation of atractylenolide I, atractylenolide II, atractylenolide III, atractylone, fructose, glucose, and sucrose were compared.The results show that there are no significant differences in separation efficiency and response intensity.

Optimization of Separation Conditions
Using single-column mode for the separation, hydrophobic atractylenolide I, II, III, and atractylone can be separated on a C18 column based on hydrophobic interaction (a revised phase separation mode) (Figure 4a ELSD), and hydrophilic sugars can be separated on a -NH2 column based on hydrophilic interaction separation mode (Figure 4a PDA).The results were similar for Bai-Zhu sample solution (Figure 4b).In addition, two kinds of different connection, i.e., C18 columns in front of NH2 columns, and NH2 columns in front of C18 columns, on the simultaneous separation of atractylenolide I, atractylenolide II, atractylenolide III, atractylone, fructose, glucose, and sucrose were compared.The results show that there are no significant differences in separation efficiency and response intensity.

Calibration Curves, LOD, and LOQ
Using the series of mixed standard working solutions, the linear regression of the method was completed.The LOD for seven compounds was 0.1-5.0μg/mL (S/N = 3), and the LOQ was 1.25-50.0μg/mL.The results are shown in Table 1.

Calibration Curves, LOD, and LOQ
Using the series of mixed standard working solutions, the linear regression of the method was completed.The LOD for seven compounds was 0.1-5.0µg/mL (S/N = 3), and the LOQ was 1.25-50.0µg/mL.The results are shown in Table 1.

Accuracy
Some 0.20 g of Bai-Zhu powder from the same batch with known contents was used, with six parallel portions, and a certain amount of reference solutions were added.The three spiked levels were 0.035, 0.35, and 0.625 mg/g for atractylenolide I, II, III, and atractylone in samples.The three spiked levels were 1.25, 12.5, 25.0 mg/g for glucose, fructose, and sucrose, respectively.Then, the extraction solution was obtained based on the preparation method described in Section 2.3, and analyzed using the HPLC method described in Section 2.4.The average recoveries of atractylenolide I, II, III, atractylone, glucose, fructose, and sucrose were in the range of 97.8-101.2%,98.7-102.2%,97.5-103.1%,97.1-100.7%,96.5-99.6%,97.1-101.2%,and 96.7-100.1%,respectively.Additionally, all RSDs were within acceptable ranges.The results are shown in Table 2.

Application
Briefly, 17 batches of Bai-Zhu sample were analyzed using the proposed C1 8 -NH 2 column tandem HPLC-DAD-ELSD method.The results were also compared with those obtained using single-column methods such as RP-HPLC-DAD and HILIC-ELSD.The results are listed in Table 3.
From the results, it can be seen that ( 1) there are no significant differences between results obtained with the C 18 -NH 2 column tandem HPLC-DAD-ELSD method and the RP-HPLC-DAD and HILIC-ELSD methods.(2) The unique processing of raw Bai-Zhu is one of the characteristics of traditional herbal medicines.Compared with raw materials, the content of atractylon in heat-prepared products decreased to different degrees (to almost ten times lower), and the contents of atractylenolide I, II, and III significantly increased.The reason for this is that atractylon is transformed under heating conditions, mainly into atractylenolides.Therefore, the heating process is very important for Bai-Zhu quality control [32][33][34].However, there are obviously differences in the contents of the same types of materials.Because the raw and heat-processed Bai-Zhu were purchased from different local pharmacies at different times, it is possible that the brands of the samples and the harvesting season differed.This shows that standardization of plant materials is necessarily.
(3) It has been found that the main low-molecular-weight sugar in Bai-Zhu is fructose.Moreover, oligosaccharides are abundant (Figure 4b).Therefore, the sugars in processed Bai-Zhu are more abundant than in raw materials due to the hydrolysis of oligosaccharides.In addition, the extract of Bai-Zhu has been shown to regulate disorders of glucose and lipid metabolism in mice with type 2 diabetes mellitus, enhancing insulin sensitivity, improving the level of inflammation, and alleviating liver injury and lipid deposition.The mechanism of these effects may be related to the up-regulation of the protein expression of GLP-1R [35].Because glucose intake is very dangerous for people with type 2 diabetes, monitoring

Figure 1 .
Figure 1.Chemical structure of the compounds.

Foods
Bai-Zhu is very important when Bai-Zhu is used for the treatment of type 2 diabetics.

Table 2 .
Recovery of compounds.