Simultaneous Determination of 17 Nucleotides, Nucleosides and Nucleobases in Pinellia Ternata by High Performance Liquid Chromatography

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1 Simultaneous Determination of 17 Nucleotides, Nucleosides and Nucleobases in Pinellia Ternata by High Performance Liquid Chromatography Hua-Li Zuo, MD, Feng-Qing Yang PhD *, Zhi-Ning Xia PhD Abstract Pinellia ternate is an important medicinal herb in various compound traditional Chinese medicine preparations. In the present study, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of 17 nucleotides, nucleosides and nucleobases in P. ternate. An Agilent Zorbax SB-Aq column ( mm, 5 μm) was used for the separation. Different mobile phases include room temperature ionic liquids ([BMIm]Cl - and [BMIm]PF 6 ), phosphate (KH 2 PO 4 -Na 2 HPO 4 ) and ammonium acetate were compared for the separation of 17 investigated compounds. The results indicated that ionic liquid solution only show limit improvement on the separation of target compounds, while the best resolution and shape of peaks can be obtained by using mixture of phosphate and ammonium acetate solution as mobile phase. Therefore, gradient elution of 1.5 mmol/l phosphate and 1 mmol/l ammonium acetate water solution (ph 4.90) (A) - methanol (B) was used as mobile phase. Then the developed method was fully validated and successfully applied for the determination of those 17 investigated analytes in P. ternate from different collecting places of China. The results indicated that P. ternate contains abundance of uridine, guanosine and adenine. Furthermore, the developed method can be applied for the determination of nucleosides and their related compounds in other medicinal herbs as well as biological samples. Keywords Pinellia ternate, nucleosides and their related compounds, high performance liquid chromatography (HPLC). more than 2000 years and is officially listed in Chinese pharmacopeia (2010 edition) [1]. Furthermore, P. ternate is an important herb in a famous compound preparation of TCM namely Huoxiang Zhengqi preparations, which have spasmolysis, analgesic, antiemetic, anti-type I allergic reaction and anti-bacterial effects, as well as the effect of enhancement the peristalsis and absorbability of gastrointestinal [2, 3]. Actually, the P. ternate were frequently used for drying dampness and resolving phlegm, dissolving lumps and resolving masses, and preventing or arresting vomiting, etc [4]. Moreover, the Compendium of Materia Medica ( Ben Cao Gang Mu in Chinese, composed by Li Shi Zhen in Ming dynasty) recorded that P. ternate was the best choice for treating dyscatabrosis or carcinoma of esophagus in modern medicine [5], and the modern research illustrated that the β-sitosterol, organic acids and alkaloids, etc. were mainly related to its antineoplastic activity [6-8]. However, nucleosides and their related compounds, which can affect on the immune system or show antibacterial and antiviral effect [9], and have potential therapeutically effects on treating tumors by direct acting on the tumor cell or through the regulation of immune system [10-13], have not been carefully investigated in P. ternate. There only a limit number of nucleosides such as adenosine, guanosine, thymine and hypoxanthine in P. ternate have been determined [14]. Therefore, in order to investigated the nucleosides profile in P. ternate, a high performance liquid chromatographic (HPLC) method was developed in the present study for the simultaneous determination of 17 nucleotides, nucleosides and nucleobases in P. ternate from different producing areas of China. P I. INTRODUCTION INELLIA ternate, belong to the Araceae family, was the dried tuber of Pinellia ternata (Thunb.) Breit mainly produced in Sichuan, Gansu, Hubei, Anhui, Jiangsu and Zhejiang provinces in China. It is one of the most famous traditional Chinese medicines (TCMs), which has been used for Received 1st March 2013, Accepted 9th May Project No. CQDXWL supported by the Fundamental Research Funds for the Central Universities. School of Chemistry and Chemical Engineering, Chongqing University, Chongqing , China *Correspondence to Feng-Qing Yang ( fengqingyang@cqu.edu.cn). 9 II. MATERIALS AND INSTRUMENTS A. Materials Twelve P. ternate samples were collected from different producing places of China, and detail information of the samples was shown in Table I. The identification of P. ternate was confirmed by the corresponding author. Furthermore, the samples were dried in an oven (DHG-9023A, Shanghai Pudong Rong-feng Scientific Instrument Co., Ltd. Shanghai, China) toughly, and were smashed into powder by a shredders (Hangzhou Huier Instrument Co., Ltd. Jiangsu, China), then

2 sieved through a 200 mesh sieve (Zhejiang Shangyu daoxuzhangxing sieve factory, China) before extraction. TABLE I THE LIST OF PINELLIA TERNATE SAMPLES Sample code Origin Sample code Origin Chongqing Hunan Province Y Gansu Province Hunan Province Gansu Province Shanxi Province Guizhou Province Shanxi & Sichuan Province Y Hunan province Y Sichuan Province Y Hunan Province Sichun Province Cytosine, uracil, cytidine, hypoxanthine, guanine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine, uridine monophosphate (UMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), Thymidine monophosphate (TMP) and cytosine monophosphate (CMP) were purchased from Sigma (St. Louis, MO, USA). Methanol for liquid chromatography was purchased from Innochem (Beijing InnoChem Science & Technology Co., Ltd., Beijing, China). The KH 2 PO 4, Na 2 HPO 4, ammonium acetate and acetate acid were AR grade obtained from Chengdu Kelong Chemical Works (Chengdu, China). Room temperature ionic liquids (RTILs) ([BMIm]Cl -, 1-butyl-3-methylimidazolium chloride, C 8 H 15 N 2 Cl, purity > 99%, [BMIm]PF 6, 1 - butyl methylimidazolium hexafluorophosphate, C 8 H 15 N 2 PF 6, purity > 99%) were purchased from Lanzhou Institute of Chemical Physics (Lanzhou, China). B. Instruments The analysis of nucleosides was performed on an Agilent 1260 Series (Agilent Technologies, USA) liquid chromatograph system, equipped with a vacuum degasser, a binary pump, an autosampler, and a diode array detector (DAD), connected to an Agilent ChemStation software. An Agilent Zorbax SB-Aq column ( mm, 5 μm) and a Zorbax SB-Aq guard column ( mm, 5 μm) were used for the separation of the analytes. A Delta 320 ph meter (Mettler-Toledo Instruments, Shanghai, China) was used for the measurement of ph of aqueous mobile phase. Ultra-pure water was prepared using AKWL-IV-16 water purification system (Chengdu Tang s Kangning Science and Technology Development Co., Ltd., Chengdu, China), The KQ-100B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd. China) was applied for the extraction of samples (Frequency 40 KHz, power 100 W, bath dimensions mm). A centrifuge (GL-16A, Shanghai Fulgor Analysis Apparatus Co. Ltd., China) and membrane filters (Tianjin Automatic Science Instrument Co. Ltd, Tianjin, China) were used for sample preparation after extraction. A. Sample preparation III. RESULTS AND DISCUSSIONS Boiling water extraction (BWE) was adopted from the previous report [15]. The powder of P. ternate (0.5 g) were mixed with 10 ml boiling ( ) ultra-pure water in a glass tube with a stopper, accurately weighted and placed onto a ultrasonic cleaner and treated at 75 for 20 min. Made up the lost weight with water after the extract cooled down to room temperature (25 ), then centrifuged at 5000 rpm for 10 min. The supernatant was filtered through a 0.45 μm filter. 10 μl aliquot was injected into the HPLC system for analysis. B. Optimization of LC conditions for the separation of nucleosides and their related compounds The nucleosides and their related compounds were components which share the character of high polarity and have poor retention properties on reversed-phase column even using the high proportion of aqueous mobile phases [16]. Actually, some mobile phase additives in high proportion of aqueous mobile phases may improve the separation, thus the Agilent Zorbax SB-Aq column was used in the present study for its much higher tolerance on high aqueous mobile phases than normal SB-C 18 columns, and the RTILs (hydrophilic ones, [BMIm]Cl - & hydrophobic ones, [BMIm]PF 6 ), phosphate (KH 2 PO 4 -Na 2 HPO 4 ) and ammonium acetate were considered as the mobile phase additives. 1) RTILs water solution as mobile phase There are several reports [17-19] indicated that the RTILs could be added into the mobile phase during LC analyses to improve the separation of polar compounds, such as alkaloids, nucleosides and amine compounds. He et al. [20] firstly applied the RTILs in HPLC for the separation of ephedrines, illustrated that RTILs could suppress the deleterious effects of free silanols on the surface of silica-based stationary phases at low concentration. The results were confirmed by the following researches done by Kaliszan et al. and Zheng et al. [21-23]. Different concentration of [BMIm]PF 6 (0-1.0 mmol/l) and [BMIm]Cl - (0-0.5 mmol/l) in water solution were tested for the separation of 17 analytes in the present study, and the best results were shown in Fig. 1a and 1b, respectively. Unfortunately, the RTILs only have little improvement on the separation of target analytes. Furthermore, the peak shapes as well as the baseline were poor with the RTILs as additive. 2) Phosphate buffer (KH 2 PO 4 -Na 2 HPO 4 ) as mobile phase Different concentration of phosphate aqueous solutions ( mmol/l) was investigated for the separation of 17 investigated analytes. The results showed that the concentrations of phosphate buffer had significant effect on the retention time of uracil, GMP and AMP. And the best separation conditions for 17 nucleosides could be obtained under 1.5 mmol/l phosphate buffer (ph 5.76) (Fig. 2a). Under the optimum conditions, resolution (Rs) of adjacent peaks meet the demand of quantity analyses (Rs >1.5) except the peak of TMP (7) and uracil (11) (Rs=1.2), adenosine (16) and adenine 10

3 (17) (Rs=1.1). Then the ph, and gradient elution were adjusted in order to get a better resolution of TMP and uracil, but with little improvement. 3) Ammonium acetate aqueous solution as mobile phase As the ammonium acetate buffer usually applied in the separation of nucleosides compounds [24, 25], so ammonium acetate was also considered in this work. Based on our previous work [26], the 1 mmol/l ammonium acetate was adopted, though the best separation was obtained under the 1 mmol/l ammonium acetate, ph 4.63 (adjust by ammonium acid, the column temperature was 30 ), the peak of AMP (11) and guanosine (14) broadened serious (Fig. 2b), and the separation of some analytes such as GMP (4) and cytidine (6) were poor than the results when using phosphate as mobile phase additive. C. Method validation and the determination of nucleosides in P. ternate Mixture of 17 reference nucleosides was prepared by their stock solutions and diluted to appropriate concentrations for the construction of calibration curves. Nine concentrations of the 17 analytes solution were analyzed in duplicate, and then the calibration curves were constructed by plotting the relative peak areas versus the concentrations of each analyte. The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. The regression data, LODs and LOQs were summarized in Table II. Fig. 1 LC chromatograms for the mixture of 17 nucleosides with RTILs. (a) 0.4 mmol/l [BMIm]BF 6 (A)-methanol (B); column temperature 30 ; flow rate: 1.0 ml/min. Gradient: 0-20 min, 0%-5% B; min, 5%-15% B; min, 15%-20% B; min, 20% B; min, 20%- 0%B. (b) 0.25 mmol/l [BMIm]Cl - (A)-methanol (B); column temperature 30 ; flow rate: 1.0 ml/min. Gradient: 0-20 min, 0%-5% B; min, 5%-15% B; min, 15%-20% B; min, 20% B; min, 20%- 0%B. 4) Mixed phosphate and ammonium acetate aqueous solution as mobile phase From the results shown above, the phosphate and ammonium acetate had their respective effects on the separation of 17 investigated compounds. Therefore, mixed of phosphate and ammonium acetate aqueous solution was used in the present test. Finally, water solution (A) contains 1 mmol/l ammonium acetate and 1.5 mmol/l (the optimized concentration) phosphate -methanol (B) was employed, the optimum separation conditions were obtained under ph 4.90 (adjust by acetic acid), column temperature at 25, flow rate of 0.9 ml/min with the gradient condition: 0-20 min, 0%-2% B; min, 2%-20% B; min, 20% B; min, 20%-0% B; min, 0%B (Fig. 2c). Fig. 2 LC chromatograms for the mixture of 17 nucleosides with different mobile phase additives. (a) 1.5 mmol/l phosphate water solution (A)-methanol (B), ph 5.76; column temperature 30 ; flow rate: 1.0 ml/min. Gradient: 0-40 min, 0-5% B; min, 5%-12% B; min, 12%-0% B; min, 0% B. (b) 1 mmol/l ammonium acetate water solution (A)-methanol (B), ph 4.63; column temperature 30 ; flow rate: 1.0 ml/min. Gradient: 0-20 min, 1-5% B; min, 5%-20% B; min, 20% B; min, 20%-1% B, 42-47, 1%B. (c) water solution contains 1.5 mmol/l phosphate and 1 mmol/l ammonium acetate (A)-methanol (B), ph 4.90; column temperature 25 ; flow rate: 0.9 ml/min. Gradient: 0-20 min, 0%-2% B; min, 2%-20% B; min, 20% B; min, 20%-0% B; min, 0%B. 1, CMP; 2, UMP; 3, Cytosine; 4, GMP; 5, Uracil; 6, Cytidine; 7, TMP; 8,Uridine;9,Thymine; 10, Hypoxanthine; 11, AMP; 12, Inosine; 13, Guanine; 14,Guanosine; 15, Thymidine; 16, Adenosine; 17, Adenine 11

4 TABLE II LINEAR REGRESSION DATA, LOD AND LOQ OF INVESTIGATED COMPOUNDS Analytes Linear regression data Calibration curves Linear range (μg/ml) R 2 LOD (μg/ml) LOQ (μg/ml) CMP y= UMP y= x Cytosine y= x GMP y=8.6570x Uracil y= x Cytidine y= x TMP y=5.6862x Uridine y= x Thymine y= x Hypoxanthine y= x AMP y= x Inosine y= x Guanine y= x Guanosine y= x Thymidine y= x Adenosine y= x Adenine y=33.674x Intra-day and inter-day variations were used to determine the precision of the developed method. For intra-day variability test, the mixed standards solutions were analyzed for six replicates within one day, while for inter-day variability test, the solutions were examined in duplicates for consecutive three days. Variations were expressed by RSDs. Furthermore, the accuracy was evaluated by the recovery of the method. Known amounts of individual standards were added into certain amount (0.40g, 0.50 g, 0.60 g) of P. ternate (Batch code Y ) material, extracted and analyzed under the conditions mentioned above. The results were summarized in Table III, which indicated that the developed method can be used for the determination of 17 investigated nucleotides, nucleosides and nucleobases in real samples. TABLE III THE INTRA- AND INTER- DAY PRECISION, RECOVERY OF THE INVESTED COMPOUNDS Precision (RSD, %) Recovery (%, n=6) Analytes Intraday Interday (n=6) (n=6) Mean (RSD, %) CMP UMP Cytosine GMP Uracil Cytidine TMP Uridine Thymine Hypoxanthine AMP Inosine Guanine Guanosine Thymidine Adenosine Adenine Under the optimum conditions, the P. ternate samples were analyzed. The LC chromatograms for mixtures of standards and typical P. ternate were shown in Fig. 3. By using the calibration curve for each analyte, the contents of 17 investigated compounds in P. ternate samples were determined, and the results were summarized in Table IV, which indicated that P. ternate contains abundance of uridine, guanosine and adenine. Furthermore, the contents of nucleotides, nucleosides and nucleobases are variant in P. ternate samples from different collecting places. Further studies on the relationship between the chemical compositions of P. ternate and its pharmacological activities should be done to reveal the role of nucleosides. IV. CONCLUSIONS An LC method was developed in the present study for the simultaneous determination of 17 nucleosides, including six nucleosides (adenosine, cytidine, guanosine, uridine, inosine and thymidine), six nucleobases (adenine, cytosine, guanine, uracil, hypoxanthine and thymine) and five nucleotides (AMP, CMP, GMP, UMP, and TMP). Furthermore, the developed method was applied for the determination of nucleosides and their related compounds in P. ternate samples from different locations of China. On the other hand, the developed LC method can also be used for the analysis of nucleosides and their related compounds in other herbal medicines or biological fluids. Fig. 3 Chromatograms of mixture of 17 nucleosides (a) and the P. ternate sample (b) (Y ). 1, CMP; 2, UMP; 3, Cytosine; 4, GMP; 5, Uracil; 6, Cytidine; 7, TMP; 8,Uridine;9,Thymine; 10, Hypoxanthine; 11, AMP; 12, Inosine; 13, Guanine; 14,Guanosine; 15, Thymidine; 16, Adenosine; 17, Adenine There is no conflict of interest. V. ACKNOWLEDGMENTS 12

5 TABLE IV THE CONTENTS OF 17 NUCLEOSIDES IN P. TERNATE (mg/g) Analytes Y Y Y Y CMP OL ND ND ND OL ND ND ND UMP Cytosine GMP ND ND Uracil Cytidine TMP ND ND ND Uridine Thymine Hypoxanthine AMP ND Inosine Guanine Guanosine Thymidine Adenosine Adenine Note: OL means it lower the linear range, ND means it is not detected. 13

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