Determination of Nucleotides, Nucleosides, and Their Transformation Products in Cordyceps by Ion-Pairing Reversed-Phase Liquid Chromatography–Mass Spectrometry

Keywords: Ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS), Nucleotides, Nucleosides, Transformation, Cordyceps

Abstract

An ion-pairing reversed-phase liquid chromatography–mass spectrometry (IP-RP-LC-MS) method was developed for the determination of nucleotides, nucleosides, and their transformation products in Cordyceps. Pentadecafluorooctanoic acid (PDFOA, 0.25 mM) was used as a volatile ion-pairing agent, and an Agilent ZORBAX SB-Aq column was used for the separation of three nucleotides-uridine-5′-monophosphate (UMP, 0.638–10.200 μg/mL), adenosine-5′-monophosphate (AMP, 0.24–7.80 μg/mL), and guanosine-5′-monophosphate (GMP, 0.42–13.50 μg/mL)-seven nucleosides, including adenosine (0.55–8.85 μg/mL), guanosine (0.42–6.75 μg/mL), uridine (0.33–10.50 μg/mL), inosine (0.21–6.60 μg/mL), cytidine (0.48–15.30 μg/mL), thymidine (0.20–6.30 μg/mL), and cordycepin (0.09–1.50 μg/mL), as well as six nucleobases: adenine (0.22–6.90 μg/mL), guanine (0.26–4.20 μg/mL), uracil (0.38–12.15 μg/mL), hypoxanthine (0.13–4.20 μg/mL), cytosine (0.39–12.45 μg/mL), and thymine (0.26–8.25 μg/mL), with 5-chlorocytosine arabinoside as the internal standard. The limits of detection (LOD) and quantification (LOQ) for the 16 analytes were 0.01–0.16 μg/mL and 0.04–0.41 μg/mL, respectively. The contents of the 16 compounds in natural and cultured Cordyceps were determined and compared after validation of the developed IP-RP-LC-MS method. Transformations of nucleotides and nucleosides in Cordyceps were evaluated based on quantification in three extracts: boiling water extraction (BWE), 24 h ambient temperature water immersion (ATWE), and 56 h ATWE. Transformation pathways UMP→uridine→uracil and GMP→guanosine→guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. AMP→adenosine→inosine→hypoxanthine was proposed in natural C. sinensis, while AMP→adenosine→adenine occurred in cultured C. militaris. No transformation was found in commercial cultured C. sinensis.

1. Introduction

Nucleosides play crucial roles in physiological regulation via purinergic and pyrimidine receptors. Nucleotides, beyond being nucleic acid precursors, enhance immune response, influence fatty acid metabolism, contribute to iron absorption, and aid in gastrointestinal repair. Cordyceps, a renowned traditional Chinese medicine, contains nucleosides regarded as its bioactive ingredients. Nucleosides may be degraded from nucleic acids or nucleotides, but their origins in Cordyceps have not been thoroughly investigated. Accurate determination of these compounds is essential for understanding their roles in Cordyceps.

Previous analytical methods include enzymatic assays, reversed-phase liquid chromatography (RP-LC), ion-exchange chromatography (IEC), capillary electrophoresis (CE), and capillary electrophoresis-mass spectrometry (CE-MS). However, these methods have limitations such as poor selectivity, low sensitivity, or incompatibility with MS detection. Ion-pairing reversed-phase liquid chromatography (IP-RP-LC) is commonly used for nucleotide separation, but traditional tetraalkylammonium salts as ion-pairing reagents cause high background and MS source pollution. Volatile salts, such as perfluorinated carboxylic acids like PDFOA, offer improved sensitivity and less source contamination. However, most previous studies focused on nucleotides or a few nucleosides, not the full profile of nucleotides, nucleosides, and nucleobases.

This study developed an IP-RP-LC-MS method using PDFOA for comprehensive qualitative and quantitative determination of nucleotides, nucleosides, and nucleobases in Cordyceps, and investigated their transformation under various extraction conditions.

2. Materials and Methods
2.1. Chemicals and Materials

Sixteen reference compounds (cytosine, uracil, cytidine, hypoxanthine, guanine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine, cordycepin, UMP, AMP, GMP) and 5-chlorocytosine arabinoside (internal standard) were purchased from Sigma. Acetonitrile (Merck), Milli-Q water, and various ion-pairing reagents (HFBA, NFPA, PFPA, PDFOA) were used. Natural Cordyceps sinensis samples were obtained from Qinghai, Sichuan, and Tibet; cultured C. sinensis and C. militaris samples were sourced from various regions and certified by relevant authorities. Voucher specimens are deposited at the Institute of Chinese Medical Sciences, University of Macau.

2.2. Sample Preparation
2.2.1. Boiling Water Extraction (BWE)

0.2 g Cordyceps powder was mixed with 10 mL boiling Milli-Q water, refluxed at 100°C for 30 min, cooled, and made up to original weight. After centrifugation (12,000 rpm, 5 min), the supernatant was filtered (0.45 μm) and diluted with internal standard solution (9:1). A 10 μL aliquot was injected into the HPLC.

2.2.2. Ambient Temperature Water Extraction (ATWE)

0.2 g Cordyceps powder was mixed with 10 mL water at 25°C, kept at room temperature for 24 or 56 h, then refluxed at 100°C for 5 min. The extract was processed as above for HPLC analysis.

2.3. IP-RP-LC-MS Analysis

Separation was performed on an Agilent Series 1200 system with a ZORBAX SB-Aq column (5 μm, 4.6 × 250 mm) and guard column at 50°C. The mobile phase was 0.25 mM PDFOA in water (A) and acetonitrile (B) with a gradient: 0–40 min, 0–5% B; 40–50 min, 5–12% B; 50–55 min, 12–0% B; 55–60 min, 0% B; flow rate 0.5 mL/min. Peaks were detected at 260 nm. MS analysis used positive ESI mode on an LC/MSD Trap system, with MS/MS or SIM for quantification.

2.4. Calibration Curves

Stock solutions of 16 standards were diluted for calibration. At least five concentrations per analyte were analyzed in duplicate, and calibration curves were constructed by plotting relative peak areas versus concentrations.

2.5. Limits of Detection and Quantification

LODs and LOQs were determined at signal-to-noise ratios of 3 and 10, respectively, by injecting serial dilutions of standards.

2.6. Precision and Accuracy

Intra- and inter-day precision were assessed by analyzing mixed standards in replicates within a day and across three days. Recovery was evaluated by spiking known amounts of standards into Cordyceps powder and calculating the percentage recovered after extraction.

3. Results and Discussion
3.1. Optimization of HPLC-ESI-MS/MS Conditions

PDFOA was selected as the ion-pairing reagent for its low required concentration and minimal MS suppression. Optimal separation was achieved at 0.25 mM PDFOA and 50°C. ESI-MS parameters were optimized for sensitivity. SRM or SIM was used for quantification, with time-programmed detection to enhance specificity.

3.2. Validation of the Method

All analytes showed good linearity (R² > 0.9917) over their test ranges. LODs and LOQs were below 0.16 and 0.41 μg/mL, respectively. Intra- and inter-day RSDs were less than 5.7% and 8.1%. Recoveries ranged from 81.5% to 120.2%, indicating high accuracy and precision.

3.3. Identification of Compounds in Cordyceps by LC-MS/MS

The method provided good separation and identification of nucleotides, nucleosides, and nucleobases in Cordyceps. Peaks were assigned based on retention time, UV, and MS/MS spectra compared to standards. The method overcame challenges such as poor retention and peak tailing of polar compounds by using PDFOA and PEEK tubing.

3.4. Quantification in Natural and Cultured Cordyceps

Sixteen compounds were quantified in various Cordyceps samples. Generally, natural C. sinensis contained higher levels of nucleotides than cultured samples, except for some exceptions. The method allowed for detailed comparison across sample types.

3.5. Effects of Sample Preparation on Transformation of Nucleosides

Comparing BWE and ATWE (24 h and 56 h) extracts revealed significant changes in nucleotide, nucleoside, and nucleobase content, indicating transformation during extraction. In natural C. sinensis, GMP and UMP decreased as guanosine and uridine increased, suggesting degradation. AMP decreased with a corresponding increase in inosine, possibly via oxidative deamination of adenosine. Further extraction (ATWE2) led to decreases in nucleosides and increases in their bases. In cultured C. militaris, similar patterns were observed, but with AMP decreasing and adenosine increasing. In commercial cultured C. sinensis, little change was observed, likely due to high-temperature processing deactivating enzymes.

4. Conclusions

An IP-RP-LC-MS method using PDFOA (0.25 mM) as a volatile ion-pairing agent was developed for the simultaneous determination of 16 nucleotides, nucleosides, and nucleobases in natural and cultured C. sinensis and C. militaris. The method was validated for sensitivity, precision, and accuracy. It was successfully applied to quantify these compounds and investigate their transformation during different extraction processes, revealing significant effects of sample preparation on compound content and transformation pathways.