Abstract
A high-performance liquid chromatography (HPLC) method for the determination of 4 nucleosides by one reference compound in Chinese cordyceps was developed. The sample of Chinese cordyceps was prepared by ultrasonic extraction with a 0.5% phosphoric acid aqueous solution. The separation of the sample was performed on a CORTECS T3 column (100 mm × 4.6 mm, 2.7 μm) by gradient elution with acetonitrile and water at a flow rate of 1.0 mL/min. The wavelengths were set as 258 nm (0-5 min), 252 nm (5-9.1 min), 258 nm (9.1-10.5 min), 249 nm (10.5-20 min) at which 4 nucleosides exhibited identical ultraviolet absorption. The accuracy, precision, reproducibility, and stability tests of the HPLC method were carried out, and the results indicated that the established HPLC method was suitable for the quantitative analysis of 4 nucleosides in Chinese cordyceps. This approach was compared to the traditional external standard method with 4 nucleoside reference compounds and the relative standard errors of the content determination results by the 2 methods were less than 3.5%. The HPLC method developed for quantitative measurement of 4 nucleosides in Chinese cordyceps is simple, lower cost, and time-saving, which provides a methodology alternative for quality control of Chinese cordyceps.
Introduction
Chinese cordyceps have been used as a traditional Chinese medicine and tonic food for a long time. It was first recorded as a herbal medicine for lung disease in “YueWangYaoZhen” at AD 710. 1 In Chinese, Chinese cordyceps is called “Dong chong xia cao,” which means “it is a worm in the winter, and becomes a grass in the summer.” Modern studies have proved that Chinese cordyceps processes many functions including immune-regulation, anticancer, antioxidant, and antifatigue.2,3 These bioactivities were related to the components such as nucleosides, polysaccharides, sterols, and so on.2–5 The nucleosides were considered the primary bioactive compounds in Chinese cordyceps, which had anticancer, antiviral, anti-inflammatory, and antitumor activities.5–7 Adenosine, inosine, guanosine, and uridine were the 4 major nucleosides, which had been used as the makers for quality evaluation of Chinese cordyceps.8–10
Currently, several high-performance liquid chromatography (HPLC) methods have been developed for the determination of nucleosides in Chinese cordyceps, including HPLC ultraviolet (UV), HPLC-MS and UPLC-UV.11–13 The reported methods usually used external standard methods (ESM), which required 4 or 5 markers for the determination of major nucleosides in Chinese cordyceps sample.8–10 In order to reduce the cost of reference compounds in herbal medicine analysis the quantitative analysis of multicomponents by single marker method (QAMS) was developed.14–16 The adenosine was used as a standard substance to establish the relative calibration factors (RCF) between itself and other nucleosides, and the simultaneous determination of nucleosides in the Chinese cordyceps sample was carried out by adenosine with the RCF. However, the reported QAMS method needed the RCF to determinate other nucleosides, which increased the operational complexity. Therefore, it is necessary to develop an HPLC method for the determination of the 4 major nucleosides in Chinese cordyceps by adenosine without RCF. The 4 major nucleosides might process the same UV absorption value at different UV wavelengths, which could be called as equal absorption wavelength (EAW) of the 4 nucleosides. Thus, developing an HPLC-UV method at EAW of 4 nucleosides could allow for the simultaneous determination of these 4 components by a single reference compound without RCF.
In the present study, an HPLC method for the determination of adenosine, inosine, guanosine, and uridine via 1 marker (adenosine) in Chinese cordyceps was proposed. The Chinese cordyceps sample was extracted with an ultrasonic bath, separated on a reverse phase column, and detected at EAW. The developed method was successfully applied to the analysis of 4 nucleosides in 9 batches of Chinese cordyceps samples. The developed method is simple, economical, and efficient, which can reduce the cost and time of Chinese cordyceps sample analysis.
Results and Discussion
Optimization of Extraction Conditions
The ultrasonic extraction and refluxing extraction were 2 common methods for extracting nucleoside from Chinese cordyceps. 11 Ultrasonic extraction, a simple and rapid extraction method, was selected in the current experiment. The transformation of nucleosides would be triggered by ultrasonic extraction with water.11,17 According to the previous study, a 0.5% phosphoric acid aqueous solution could inhibit the transformation of nucleosides. 8 Thus, the 0.5% phosphoric acid aqueous solution was used as the extraction solvent. In order to obtain effective ultrasonic extraction conditions, the different extraction times (5, 10, 20 min) and solvent volumes (10, 15, and 20 mL) were tested. The test range of extraction times and solvent volumes were based on the literary works.8,13,14,17 The results indicated that 0.2 g of Chinese cordyceps sample extracted by 10 mL 0.5% phosphoric acid aqueous solution for 5 min could give a satisfactory extraction efficiency.
Optimization of HPLC Conditions
Nucleosides are a kind of polar compound, which are often separated on special reversed-phase columns with 100% aqueous phase.18,19 In order to obtain the rapid and good separation of nucleosides, 2 core-shell reversed-phase columns (Poroshell 120 SB-Aq column and CORTECS T3 column) were tested, which could be used in 100% aqueous solution. The result showed that CORTECS T3 column provided better resolution of guanosine and inosine. Thus, the CORTECS T3 column was used in subsequent tests. Four different mobile phase systems (water-methanol, water-acetonitrile, 0.1% formic acid-acetonitrile, 5 mmol/L ammonium formate-acetonitrile) were compared. As a result, 0.1% formic acid-acetonitrile and water-acetonitrile present better separation of components than the other 2 mobile phases. The water-acetonitrile was chosen as the eluting solvent system based on easy preparation. Different column temperatures (30, 35, and 40 °C) were tested. The separations of nucleosides in 3 temperatures were similar and 35 °C was used in the current experiment.
The selection of EAW was the key factor in the present study. There were 3 conditions for the selection of EAW: (1) At least 2 analytes exhibited identical UV absorption at the same wavelength. (2) The UV response of analytes at EAW should be less than the maximum UV response of 4 analytes. (3) The EAW with a higher UV response of the analyte was preferred. The UV spectra of 4 nucleosides was shown in Figure 1. It was found that 3 nucleosides had highest similar UV response at A region, but this UV response was higher than the maximum UV response of uridine. In the B region (259 nm), guanosine and uridine had EAW points, as did adenosine (249, 270 nm) and inosine (243, 253 nm). Therefore, the B region (259 nm) was chosen as the testing EAW for uridine and guanosine, and 249, 243, 253, and 270 nm were used as the testing EAWs for adenosine and inosine by HPLC-UV. The EAW of uridine and guanosine were tested at 258, 259 , and 260 nm. Due to the fact that their chromatographic peak areas were identical at 258 nm, this wavelength was chosen as the EAW for guanosine and uridine. By comparing the chromatographic peak areas of adenosine (248, 249, 250; 269, 270, 271 nm) and inosine (242, 243, 244; 252, 253, 254 nm) with the peak areas of uridine and guanosine at 258 nm, it was found that 249 nm and 270 nm could be used as EAW of adenosine (249 nm is better), and 243 and 252 nm could be used as EAW of inosine (252 nm is better). Therefore, the EAWs of 4 nucleosides were established as follows: uridine (258 nm), guanosine (258 nm), inosine (252 nm), and adenosine (249 nm). In addition, the different bandwidths (1, 2, 4, 8 nm) of the detection wavelength were compared. The results showed that the relative standard deviation (RSD) of 4 nucleosides peak areas was the best when the bandwidth was set at 4 nm.
The ultraviolet (UV) spectrum of 4 nucleosides.
Method Validation
The validation of the current method was summarized in Tables 1 and 2. The current method showed good linearity in the tested range with correlation coefficients above 0.9996. The limit of detections (LODs) and limit of quantifications (LOQs) of 4 analytes were less than 0.21 and 0.83 μg/mL, respectively. The RSDs of intraday and interday precision were less than 0.93%. The RSDs of repeatability were less than 3.6%. The RSD of stability validation was less than 1.4% within 24 h. The recoveries of 4 analytes were 98.85 to 100.70% (RSD ≤ 1.6%).
The Linearity, LOD, and LOQ of Analytes.
Abbreviations: LOD, limit of detection; LOQ, limit of quantification.
Precision, Repeatability, Stability and Recovery (%) of Analytes.
Analysis of Chinese Cordyceps Samples
The developed HPLC method was successfully applied to the determination of 4 nucleosides in 9 batches of Chinese cordyceps samples. The typical chromatograms of reference compounds and sample are presented in Figure 2, and their contents are listed in Table 3. To evaluate the feasibility of the developed HPLC-UV with EAW for the determination of nucleosides in Chinese cordyceps samples, the contents of 4 nucleosides in 9 samples were determined by the ESM method with 4 nucleoside reference compounds and EAW method with adenosine, respectively. The RSDs of the results obtained by the 2 methods were less than 3.5%. These results indicated that the developed HPLC-UV EAW method could be used for quantitative analysis of the 4 nucleosides in Chinese cordyceps by adenosine. The contents of nucleosides in 9 Chinese cordyceps samples showed that uridine (0.0274%-0.0518%), inosine (0.0063%-0.0181%), guanosine (0.0056%-0.0074%), and adenosine (0.0125%-0.0355%) were the major nucleosides in Chinese cordyceps sample. These results were in accordance with the literature data. 10
Chromatograms of blank solution(A), reference compounds (B), and Chinese cordyceps samples (C): (1) uridine; (2) inosine; (3) guanosine; (4) adenosine.
The Contents (%) of 4 Nucleosides in Chinese Cordyceps Samples.
Note: *RSD was obtained from the tested contents by EAW method (n = 2) and ESM method (n = 2). Abbreviations: ESM, external standard method; RSD, relative standard deviation; EAW, equal absorption wavelength.
Comparison of the Developed Method and Reported One
For the determination of the 4 nucleosides in Chinese cordyceps, classic ESMs required 4 reference compounds.8–10 The newly developed HPLC process only required 1 reference compound, it was both reference compound-saving and cost-effective. Compared with the QAMS,14–16 the QAMS requires establishing the RCF for determining other components, increasing operational complexity. Furthermore, the RCFs for different detecting wavelengths were different,20,21 limiting its wide application. The new HPLC approach enabled the determinaiton of 4 analytes by adenosine at EAWs. It was simpler and easier to popularize.
In addition, the developed method was faster than the traditional HPLC method for the determination of the 4 nucleosides in Chinese cordyceps. The old HPLC method used ordinary C18 columns, resulting in a separation time of more than 30 min. 10 The rapid HPLC column (core-shell column) was employed in this experiment, and the separation of the 4 analytes took only 13 min.
Materials and Method
Chemicals and Reagents
Uridine (99.6%, Batch No. 110887-202104), inosine (99.2%, Batch No.140669-202007), and guanosine (93.6%, Batch No. 111977-201501) were obtained from National Institutes for Food and Drug Control. Adenosine (98.1%, Batch No. WPS-21001) was purchased from Shanghai Blue Yan Chemical Co. Ltd. Phosphoric acid (HPLC grade, Batch No G1803024) was obtained from Shanghai Aladdin Biochemical Technology Co. Ltd. Acetonitrile (HPLC grade, Batch No. 880501) was obtained from Krude company. The deionized water was purified by Milli-Q purification system.
Materials
Chinese cordyceps samples (s1-s9) were collected from Hubei, Guangdong, Gansu, Qinghai, Sichuan and Xizang province. The botanical origin of materials was identified by Dr Zhengming Qian. Voucher specimens were deposited at the Dongguan HEC Cordyceps R&D Co. Ltd, Guangdong, China.
Preparation of Reference Compound Solutions
The stock solution was prepared by dissolving uridine (10.45 mg), inosine (10.31 mg), guanosine (11.08 mg), and adenosine (10.25 mg) with 0.5% phosphoric acid aqueous solution in 25 mL volumetric flask. The tested reference compound solution was obtained by diluting the stock solution with 0.5% phosphoric acid aqueous solution.
Preparation of Sample Solution
The 0.2 g Chinese cordyceps powder was extracted with 10 mL of 0.5% phosphoric acid solution by ultrasonic extraction for 30 min. The sample solutions were cooled down to room temperature and the lost weight was compensated with 0.5% phosphoric acid solution. The sample solutions were filtered through a 0.2 μm filter before HPLC analysis.
UV Condition
The UV spectrums of the 4 nucleosides were obtained by scanning the 4 reference compound aqueous solutions (uridine: 20.84, inosine: 20.46, guanosine: 20.74, and adenosine: 20.11 µg/mL) from 200 to 350 nm using an Agilent Cary 60 UV spectrophotometer (Agilent Technologies). Water was used as a blank solution.
HPLC Condition
To obtain the optimal HPLC condition, 2 HPLC columns (Angilent Poroshell 120 SB-Aq column and Waters CORTECS T3 column), 4 different mobile phase systems (water-methanol, water-acetonitrile, 0.1% formic acid-acetonitrile, 5 mmol/L ammonium formate-acetonitrile), 3 different column temperatures (30, 35, and 40 °C) were tested. The EAWs were validated on HPLC by analyzing a mixed reference chemical solution containing the 4 nucleosides with the same concentration (20 µg/mL) at 1 nm intervals up and down.
The HPLC analysis was performed on an Agilent 1260 II Series HPLC system (Agilent Technologies). Waters CORTECS T3 Column (100 mm × 4.6 mm, 2.7 µm) with water (A) and acetonitrile (B) by gradient elution was used for separation. The gradient program: 0 to 9 min, 0% B; 9 to 9.5 min, 0 to 4% B; 9.5 to 15 min, 4% B; 15 to 16 min, 4% to 90% B; 16 to 19 min, 90% B; 19 to 20 min, 90% to 0% B. The flow rate was 1.0 mL/min. The column temperature was set at 35 °C. The detection wavelength was set as follows: 0 to 5 min (258, 4 nm), 5 to 9.1 min (252, 4 nm), 9.1 to 10.5 min (258, 4 nm), 10.5 to 20 min (249, 4 nm). The injection volume was 2 µL.
Method Validation
The method validation, including linearity, LOD, LOQ, precision, accuracy, repeatability, and stability tests, was carried out.
Linearity, LOD and LOQ. A series of concentrations of reference components solutions were prepared for evaluation of linearity. The uridine (2.0837-52.0933 μg/mL), inosine (2.0455-51.1376 μg/mL), guanosine (2.0742-51.8544 μg/mL), and adenosine (2.0111-50.2763 μg/mL) were analyzed by HPLC. The standard curve was constructed by plotting the peak areas versus concentrations of reference compounds. The LODs and LOQs were determined by reference components standards and recorded as the corresponding concentrations which gave the signal-to-noise (S/N) ratios approximately 3 and 10, respectively.
Precision, repeatability, and stability. The intraday and interday assays were used to assess the precision of the developed method. The intraday precision was determined by analyzing reference components solution in 6 times within 1 day. The interday precision was determined by analyzing the same reference components solution in 2 times per day for 3 days. The RSD was utilized as a measure of precision.
The repeatability of the developed method was evaluated by 6 replicates of the same Chinese cordyceps samples. The samples were extracted as “2.3” and analyzed as “2.4.” The RSD of the 6 results was used as a measurement of repeatability.
The sample solution stability was assessed by analyzing the sample solution 6 times within 24 h. Variation was evaluated by the RSD.
Recovery. A recovery test was used to evaluate the accuracy of the developed method. Known amounts of 4 nucleosides were added to approximately 0.1 g of Chinese cordyceps powder (sample s5), and then extracted and analyzed by the developed method. The sample s5 was analyzed 6 times. The recovery rates were calculated as 100% × (found amount – original amount)/spiked amount.
Conclusions
In the current study, an HPLC method was established for the quantification of 4 nucleosides in Chinese cordyceps. In comparison to the reported HPLC method, the new developed method was reference compound saving, rapid, and simple. The determination of 4 major nucleosides in Chinese Cordyceps was accomplished with 1 reference compound (without RCF) and took 15 min. The developed method is an improvement assay method for the quality evaluation of Chinese Cordyceps, which helps to lower the cost and enhance the efficiency of industrial Cordyceps analysis.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Key R&D Program of China, the Science and Technology Development Fund, Macau SAR, the GDST (DGSTB)-FDCT project (grant numbers: 2018YFC1706101, 0040/2021/AGJ, 2022A0505020031).
