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Li-li Zhang1 Yong-liang Bai2 Shu-lan Shu1 Da-wei Qian1 Zhen Ou-yang2 Li Liu3 Jin-ao Duan1 1 Jiangsu
Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, China 2 College of Pharmacy, Jiangsu University, Zhenjiang, China 3 Sericultural Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
Received November 24, 2013 Revised February 24, 2014 Accepted March 1, 2014
Research Article
Simultaneous quantitation of nucleosides, nucleobases, amino acids, and alkaloids in mulberry leaf by ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry An ultra high performance liquid chromatography coupled with a triple quadrupole mass detection and electrospray ionization mass spectrometry method has been established for the simultaneous determination of the 14 nucleosides and nucleobases, 24 amino acids and two main alkaloids in mulberry leaf. In this method, a complicated mobile phase, the flow rate of which was 0.4 mL/min, was applied to the gradient elution, which provided a satisfied separation of the 40 compounds. The present method was validated, and sufficient reproducibility and accuracy were obtained for the quantitative measurement of the 40 compounds. The method was subsequently applied to ten mulberry leaves and the results showed that almost all of these samples were rich in nucleosides, nucleobases, amino acids, and alkaloids. The proposed method, which is convenient and economical, could serve as a prerequisite for the quality control of mulberry leaf herbs and be applied analogously to other Chinese medicines. Keywords: Alkaloids / Amino acids / Morus Alba / Nucleosides / Simultaneous quantitation DOI 10.1002/jssc.201301267
1 Introduction Morus Alba L., a Moraceous plant, is indigenous to China with a history of over 4000 years and is widely distributed in China, especially in Jiangsu and Zhejiang provinces. Its dried leaves, called mulberry leaf in China, have been commonly used as a traditional Chinese medicine for its efficacies of hypoglycemic [1], hypolipidemic [2], antihypertensive [3], anti-atherosclerotic [4], anti-inflammatory [5], antioxidant [1, 6, 7], and antitumor [8]. Phytochemical studies have revealed that mulberry leaf contains various constituents, including flavonoids [9, 10], alkaloids [11], polysaccharides [12], amino acids [13], nucleosides, microelements, and so on. Mulberry leaf, which is the only food of silkworms, has been commonly utilized as a food or food additive in recent decades due to their high nutritional value [14]. It is also well known that nucleosides [15–17] and amino acids [18–21] are involved in the regulation and modulation of various physiological processes in body and exhibit multiple bioactivities, Correspondence: Dr. Jin-ao Duan, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, P. R. China E-mail: [email protected] Fax: +86-25-85811116
Abbreviations: MRM, multiple reaction monitoring; TQ-MS, triple quadrupole mass spectrometry; UHPLC, ultra high performance liquid chromatography C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
such as antiplatelet aggregation, antiarrhythmic, antioxidant, and so on [22–29]. 1-Deoxynojirimycin, one of the alkaloids in mulberry leaf, is an ␣-glucosidase inhibitor [30], which is effective for diabetes [31]. Since mulberry leaf is increasingly being used in healthcare products and these benefit human health, it is very necessary to develop a fast, convenient, and efficient method to find the clear and definite amount of its nutritional constituents to make sure it is safe and improve their potential values. Ultra high performance liquid chromatography (UHPLC), which is widely used in medicines analysis, shows great advantages over traditional HPLC for its high power in separation and analysis speed. High sensitivity and high throughput can be obtained by using triple quadrupole mass detection and multiple reaction monitoring (MRM), which use one transition each from the precursor to the reporter ions for every compound, respectively. To our knowledge, unfortunately, there are just quantitative methods [32–34] for each particular class of the compounds, but no published analytical methods are available for the quantification of nucleosides, nucleobases, amino acids, and alkaloids together. Thus, the UHPLC–triple quadrupole MS (TQ-MS) method, with which the 40 analytes were completely separated and detected, was established and applied to ten mulberry leaf samples. The aim of this study is to report a development, validation, and application of a rapid and sensitive Colour Online: See the article online to view Figs. 2 and 3 in colour. www.jss-journal.com
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Figure 1. Chemical structures of the 40 identified nucleosides, nucleobases, amino acids, and alkaloids.
UHPLC–TQ-MS method for the detection and quantitation of underivatized nucleosides, nucleobases, amino acids, and alkaloids together. The proposed method can be used for the quality control of mulberry leaf and other traditional Chinese medicines.
2 Materials and methods 2.1 Chemicals and reagents Acetonitrile was of HPLC grade (Tedia, USA), formic acid was of HPLC grade (Merck, Germany), and deionized water (H2 O) was purified by a superpurification system
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(Eped Technology Development, Nanjing, China). Other reagent solutions, such as ammonium formate (Lingfeng Chemical Reagent, Shanghai, China) and ammonium acetate (Sinopharm Chemical Reagent), were of analytical grade. Chemical standards of adenine (1), guanine (2), xanthine (3), thymine (10), inosine (13), arginine (15), histidine (16), lysine (17), tyrosine (20), valine (21), proline (22), glutamate (24), serine (27), threonine (28), alanine (29), tryptophan (31), phenylalanine (32), leucine (34), isoleucine (35), aspartic acid (37), 1-deoxynojirimycin (DNJ) (39), and fagomine (40) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Reference compounds of cytidine-5 -monophosphate (4), 2 -deoxyguanosine (5),
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Figure 1. Continued
2 -deoxyuridine (8), 2 -deoxyinosine (9), cytidine (11), 2 -deoxyadenosine-5 monophosphate (12), thymidine (14), ornithine (18), hydroxyproline (19), citrulline (23), glutamine (25), asparagine (26), glycine (30), ␥-aminobutyric acid (36), and cysteine (38) were obtained from Sigma (St. Louis, MO). Chemical standards of 2 -deoxyadenosine (6) and 2 -deoxycytidine (7) were obtained from Aladdin Chemical, Shanghai, China. A reference of methionine (33) was purchased from Sinopharm Chemical Reagent, Beijing, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
China. The purity of each compound was >98%, determined by HPLC analysis. The chemical structures of these reference compounds are shown in Fig. 1. 2.2 Plant materials Seven mulberry leaves (samples 1–7) were purchased from seven Pharmacies of Traditional Medicine in Nanjing. Others (samples 8–10), were collected on November 1, 2012 from a www.jss-journal.com
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Table 1. The information of samples
2.3 Preparation of standard solutions
Sample
Origin
Batch number
1 2 3 4 5 6 7 8 9 10
Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Zhenjiang, Jiangsu Zhenjiang, Jiangsu Zhenjiang, Jiangsu
120813 130130 130303 130111 130211 130101 120804002 12110101 12110102 12110103
mulberry garden in Zhenjiang, China. Their botanical origins were identified by the corresponding author. After collection, the leaves were dried at 55⬚C. The information of these samples is summarized in Table 1.
A mixed standard stock solution containing the reference compounds 1–40 was prepared in methanol/water (9:1, v/v). Working standard solutions for calibration curves were prepared by diluting the mixed standard stock solution with 10% methanol at different concentrations, and the concentration ranges for the 40 analytes were as follows: 1, 0.55–27.50 g/mL; 2, 0.60–30.00 g/mL; 3, 0.25–12.50 g/mL; 4, 0.54– 27.00 g/mL; 5, 0.65–32.50 g/mL; 6, 0.56–28.00 g/mL; 7, 0.55–27.25 g/mL; 8, 0.77–38.25 g/mL; and 9, 0.58– 28.75 g/mL; 10, 0.60–30.00 g/mL; 11, 0.53–26.50 g/mL; 12, 0.60–30.00 g/mL; 13, 0.66–32.75 g/mL; 14, 0.64– 32.00 g/mL; 15, 0.55–27.50 g/mL; 16, 0.52–25.75 g/mL; 17, 0.60–30.00 g/mL; 18, 0.60–29.75 g/mL; 19, 0.74– 37.00 g/mL; 20, 0.50–25.00 g/mL; 21, 0.52–26.00 g/mL; 22, 0.67–33.25 g/mL; 23, 0.51–25.25 g/mL; 24, 0.51– 25.50 g/mL; 25, 0.63–31.50 g/mL; 26, 0.80–40.00 g/mL; 27, 0.57–28.50 g/mL; 28, 0.62–31.00 g/mL; 29, 0.63– 31.50 g/mL; 30, 0.65–32.25 g/mL; 31, 0.65–32.50 g/mL; 32, 0.51–25.50 g/mL; 33, 0.57–28.50 g/mL; 34, 0.50– 24.75 g/mL; 35, 0.53–26.25 g/mL; 36, 0.49–24.50 g/mL;
Figure 2. Effects of solvent, extraction temperature, solvent volume, and extraction time on the extraction efficiency of investigated nucleosides, amino acids, and alkaloids from the mulberry leaf sample. When one of the parameters was determined, the others were set at the default (solvent, water; extraction temperature, 50⬚C; solvent volume, 50 mL; extraction time, 50 min).
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Table 2. Precursor/product ion pairs and parameters for MRM of compounds used in this study
No.
Compound
tR (min)
[M+H]+ (m/z)
Product ion (m/z)
Cone voltage (V)
Collision energy (eV)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Adenine Guanine Xanthine Cytidine-5 -monophosphate 2 -deoxyguanosine 2 -deoxyadenosine 2 -deoxycytidine 2 -deoxyuridine 2 -deoxyinosine Thymine Cytidine 2 -deoxy-ade-5 -monophosphate Inosine Thymidine Arginine Histidine Lysine Ornithine Hydroxyproline Tyrosine Valine Proline Citrulline Glutamate Glutamine Asparagine Serine Threonine Alanine Glycine Tryptophan Phenylalanine Methionine Leucine Isoleucine GABA Aspartic acid Cysteine DNJ Fagomine
1.93 4.82 3.32 16.95 3.45 1.68 2.99 3.01 1.66 1.22 3.95 7.24 2.81 1.21 16.98 17.12 10.89 17.17 8.76 6.95 6.16 6.25 16.91 10.95 10.76 11.51 10.96 9.43 8.62 9.84 4.85 4.51 5.63 4.48 4.92 5.02 11.69 6.14 11.25 8.17
136.0 152.0 153.0 324.0 268.1 252.0 228.0 229.0 253.0 127.0 244.0 332.0 269.0 243.0 175.2 156.1 147.0 133.0 132.0 182.1 118.0 116.0 176.0 147.9 147.0 132.9 106.0 120.0 90.0 76.0 205.1 166.1 150.1 132.1 132.1 103.9 134.0 122.0 163.3 147.9
136.0 152.0 153.0 111.9 152.0 135.9 111.9 113.0 136.9 127.0 111.9 135.9 136.9 126.9 70.0 110.0 83.9 69.9 67.9 136.0 72.1 70.0 69.9 83.9 83.9 73.9 60.0 74.0 44.0 30.0 146.0 120.1 104.0 86.1 86.1 87.0 88.0 75.8 68.7 112.0
30 30 30 16 10 16 28 8 22 30 28 20 10 10 22 22 14 14 18 16 12 20 16 12 8 12 14 38 16 12 16 18 14 16 16 16 14 14 4 22
20 20 20 14 12 14 10 10 12 15 10 16 14 10 18 14 14 14 16 16 10 10 20 14 16 14 8 20 10 6 18 14 10 10 10 10 10 17 18 14
37, 0.64–31.75 g/mL; 38, 0.66–33.00 g/mL; 39, 1.45– 145.00 g/mL; 40, 0.12–12.00 g/mL. The standard solutions were filtered through a 0.22 m membrane filter prior to injection. All solutions were stored in a refrigerator at 4⬚C before analysis.
and then the same solvent was added to compensate for the weight lost during the extraction. After centrifugation (13 000 rpm, 10 min) and filtering (0.22 m membrane filter), the supernatant was stored at the sample plate whose temperature was set at 4⬚C before injection into the UHPLC system for analysis.
2.4 Preparation of sample solutions The dried leaves were pulverized to homogeneous powders (40 mesh). The dried powder (1.0 g), which was weighed accurately, was put into a 100 mL conical flask with a stopper, and 50 mL water was added. After accurate weighing, ultrasonication (80 kHz) was performed at 50⬚C for 50 min, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Chromatographic conditions and instrumentation Analysis was performed on a Waters Acquity UHPLC system (Waters, Milford, MA), consisting of a quaternary pump solvent management system, an online degasser, an www.jss-journal.com
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Figure 3. Chromatogram of the 40 compounds analyzed in this study.
autosampler, and a triple quadrupole mass detector. An Acquity UPLC BEH Amide (100 mm × 2.1 mm, 1.7 m) column was applied for all analyses. The raw data were acquired and processed with MassLynx 4.1 software. The mobile phase was composed of A (5 mM ammonium formate and ammonium acetate solution, 0.2% formic acid) and B (acetonitrile with 1 mM ammonium formate, ammonium acetate solution, and 0.2% formic acid) with a gradient elution: 0– 3 min, 10% A; 3–9 min, 10–18% A; 9–15 min, 18–20% A, 15–16 min, 20–46% A, 16–18 min, 46% A. The flow rate of the mobile phase was 0.4 mL/min, and the column temperature was maintained at 35⬚C. High-purity nitrogen was used as the nebulizer and auxiliary gas; argon was utilized as the collision gas. The triple quadrupole (TQ) mass spectrometer was operated in positive ion mode with a capillary voltage of 3 kV, a sampling cone voltage of 30 V, a cone gas flow of 20 L/h, a desolvation gas flow of 1000 L/h, a desolvation temperature of 350⬚C, a source temperature of 120⬚C, a collision energy of 6 V, and full-scan spectra from 100 to 1000 Da.
2.6 Validation of the method For calibration, the linearity was obtained by plotting the peak areas versus the corresponding concentrations of each analyte. The lowest concentration of working solution for calibration use was diluted with water to a series of appropriate concentrations. They were analyzed until the S/N for each compound was about 3 for the LOD and 10 for the LOQ. The precision was evaluated by analyzing the standard solutions containing the 40 standard compounds six times. Then, the RSD of the peak area for each of the marker compounds was calculated. To confirm the repeatability, six different sample solutions prepared from the same sample (sample 9) were analyzed and variations were expressed by RSD. To evaluate C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the stability of the solution, one of the sample solutions mentioned above was stored at 25⬚C and analyzed at 0, 2, 4, 8, 12, and 24 h, respectively. A recovery test was used to evaluate the accuracy of this method. It was performed by adding corresponding marker compounds with high (120%), medium (100%), and low (80%) levels into accurately weighed samples (sample 9, 0.5 g). The spiked samples were then extracted, processed, and quantified in accordance with the methods mentioned above. The detected amounts (actual) were calculated by subtracting the total amount of each compound before spiking from the total amount after spiking. The ratio of the detected amount (actual) to spiked amount (theoretical) was used to calculate the recovery percentage.
2.7 Identification and quantification The identification of the nucleosides, nucleobases, amino acids, and alkaloids was carried out by comparing the UHPLC retention time of target peaks with those of the standards by UHPLC–TQ-MS in positive ion mode. Quantification was performed on the basis of linear calibration plots of the peak areas versus the concentration.
3 Results and discussion 3.1 Optimization of extraction procedure In this study, extraction variables such as extraction solvent (water, 10, 20, and 30% aqueous methanol), solvent volume (10, 20, 30, 40, 50, and 60 mL), extraction temperature (20, 30, 40, 50, 60, 70⬚C), and extraction time (10, 20, 30, 40, 50, and 60 min) were investigated for sample 9 (1.0 g, 40 mesh) by sonicating to obtain optimal extraction conditions. When www.jss-journal.com
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Table 3. Calibration curves, LOD, LOQ, precision, repeatability, and stability of the 40 analytes
NO.a)
Calibration curves
r2
Linear range g/mL
LOD
LOQ
Precision RSD,%, n = 6
Repeatablity
Stability
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
y = 56706x + 4185.7 y = 12041x – 2477.4 y = 17580x – 1103 y = 6321.6x + 1600 y = 33732x – 1634 y = 123054x + 2303.4 y = 148482x – 33562 y = 3744.8x – 221.9 y = 8969.6x – 2653.8 y = 30601x – 6103.5 y = 3326x – 142.1 y = 8057.2x + 500 y = 46313x – 6166.2 y = 8995.6x – 405.7 y = 33694x + 10764 y = 96645x + 28000 y = 22071x – 1712 y = 19718x + 3000 y = 20010x – 1940 y = 13766x – 6047.1 y = 118887x – 31410 y = 172631x – 8252 y = 3006.7x + 280 y = 16326x – 8764.8 y = 23880x – 3959 y = 12303x – 8406.5 y = 10987x + 4071.9 y = 585.52x – 433.86 y = 12795x + 11.807 y = 4411.1x + 339.45 y = 25987x – 7387 y = 163984x – 61671 y = 35932x – 2972 y = 93166x – 423.76 y = 137944x – 6779 y = 102825x + 14772 y = 5295.9x + 0 y = 1482.3x – 910.75 y = 682x – 46.17 y = 190000x + 11796
0.9994 0.9969 0.9920 0.9946 0.9972 0.9987 0.9889 0.9976 0.9949 0.9987 0.9935 0.9942 0.9990 0.9982 0.9947 0.9986 0.9951 0.9997 0.9944 0.9930 0.9949 0.9982 0.9912 0.9935 0.9951 0.9958 0.9939 0.9918 0.9981 0.9976 0.9957 0.9986 0.9920 0.9988 0.9979 0.9981 0.9943 0.9984 0.9900 0.9902
0.55–27.50 0.60–30.00 0.25–12.50 0.54–27.00 0.65–32.50 0.56–28.00 0.55–27.25 0.77–38.25 0.58–28.75 0.60–30.00 0.53–26.50 0.60–30.00 0.66–32.75 0.64–32.00 0.55–27.50 0.52–25.75 0.60–30.00 0.60–29.75 0.74–37.00 0.50–25.00 0.52–26.00 0.67–33.25 0.51–25.25 0.51–25.50 0.63–31.50 0.80–40.00 0.57–28.50 0.62–31.00 0.63–31.50 0.65–32.25 0.65–32.50 0.51–25.50 0.57–28.50 0.50–24.75 0.53–26.25 0.49–24.50 0.64–31.75 0.66–33.00 0.145–145.00 0.12–12.00
0.015 0.036 0.068 0.034 0.004 0.004 0.005 0.012 0.014 0.037 0.069 0.026 0.005 0.005 0.003 0.004 0.023 0.010 0.006 0.041 0.016 0.010 0.022 0.044 0.050 0.088 0.045 0.119 0.045 0.041 0.013 0.009 0.004 0.016 0.017 0.003 0.131 0.076 0.003 0.002
0.050 0.115 0.226 0.112 0.012 0.012 0.015 0.038 0.048 0.120 0.230 0.086 0.017 0.016 0.011 0.014 0.078 0.031 0.021 0.135 0.054 0.035 0.072 0.150 0.166 0.296 0.150 0.413 0.150 0.129 0.046 0.030 0.014 0.055 0.058 0.009 0.438 0.254 0.007 0.004
0.6 1.4 1.5 2.9 0.7 0.8 0.7 1.1 1.4 0.3 1.7 0.8 1.9 0.4 2.5 2.5 0.5 2.3 0.4 0.8 0.9 0.4 2.5 0.7 1.1 0.9 0.6 2.3 0.4 2.8 1.2 0.3 0.7 0.5 1.1 0.6 2.8 1.9 1.7 1.3
1.2 0.2 0.7 0.9 0.9 1.4 1.6 0.6 0.8 0.7 1.4 1.1 2.2 2.2 2.0 2.2 0.7 2.4 2.4 0.8 0.4 0.3 2.8 1.2 1.1 0.7 0.6 1.9 0.4 1.6 1.1 1.0 2.6 0.7 1.1 0.6 2.2 1.1 2.1 2.9
0.2 1.7 0.9 1.8 2.1 2.8 2.9 2.2 2.5 2.2 0.8 0.8 1.9 2.7 2.2 0.8 1.8 1.6 2.3 1.6 0.6 1.2 1.6 1.8 0.5 0.6 0.4 1.2 0.6 2.0 2.0 2.3 2.0 0.5 1.8 1.0 2.2 2.2 3.8 3.1
a) The 40 analytes were the same as in Fig. 1. (The part of recovery has been deleted).
one of the parameters was determined, the others were set at the default (solvent, water; solvent volume, 50 mL; extraction temperature, 50⬚C; extraction time, 50 min). The results are shown in Fig. 2, which indicates that the best solvent was found to be water, which could obtain the highest extraction efficiency for the 40 constituents analyzed among these investigated solvents. Furthermore, it was found that the amounts of the compounds analyzed in mulberry leaf increased with the extraction time extension and reached the maximum at 50 min; thereafter, a further increase of extraction time did not result in a significant increase in the amount.
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On the contrary, it could lead to dramatic decreases in yields of lysine, asparagine, and DNJ. For example, compared with the amounts detected after an extraction of 50 min, the detected amount decreased about 0.47 mg/g for DNJ when the extraction time was extended to 60 min. In addition, the volume of solvent was chosen as 50 mL, and the extraction temperature was chosen as 50⬚C, which was sufficient for sample extraction. Therefore, the final extraction conditions were as follows: each sample was extracted by sonication with 50 mL of water for 50 min at 50⬚C, which was adequate and appropriate for the analysis.
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Table 4. Recovery of the 40 analytes in mulberry leaves
NO.a)
Recovery,%, n = 3 Low level (80%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Medium level (100%)
High level (120%)
Mean
RSD
Mean
RSD
Mean
RSD
97.5 102.7 97.4 97.7 98.6 97.9 96.8 97.1 103.4 95.7 102.1 96.7 98.3 97.3 101.6 102.4 98.2 103.5 97.7 101.8 98.0 97.6 95.9 103.7 102.9 98.5 97.6 98.2 101.9 103.5 97.8 102.6 98.4 97.3 101.9 102.6 104.1 95.9 97.7 101.8
2.7 3.5 4.6 2.9 2.0 2.3 1.7 2.8 3.6 4.8 2.9 3.6 4.7 4.1 4.5 3.4 3.0 2.1 3.5 2.3 2.2 3.8 4.3 3.8 2.7 3.0 3.5 3.9 2.4 4.4 3.3 2.5 2.7 3.8 3.1 3.7 4.1 4.0 2.2 1.9
102.5 102.4 103.5 97.6 102.4 99.1 98.8 101.9 102.3 97.6 102.7 98.5 103.1 103.4 102.4 103.6 101.8 101.2 102.5 103.7 98.3 102.4 96.7 104.5 97.4 103.9 98.3 102.4 96.8 102.2 104.2 98.5 101.9 98.8 103.7 103.5 103.9 101.7 102.6 103.1
3.2 2.8 2.9 2.5 2.3 2.2 2.4 2.6 3.1 2.5 3.4 3.7 2.6 2.3 2.5 2.6 2.1 3.4 2.8 3.4 1.7 3.2 3.7 2.2 3.8 3.7 2.3 2.7 4.1 1.9 2.0 3.3 2.3 3.6 2.4 2.5 4.1 2.6 3.7 2.8
102.8 98.4 101.6 103.4 102.1 102.4 97.6 101.9 97.5 102.5 103.5 97.1 103.9 102.5 102.8 104.6 103.5 98.7 103.1 104.2 103.6 103.2 97.3 102.1 98.3 103.7 102.4 96.8 102.6 98.0 101.9 104.2 97.2 102.8 103.2 97.4 95.8 97.0 102.3 98.0
1.9 2.6 2.5 1.8 1.5 1.4 2.0 1.7 2.2 3.1 2.7 1.9 3.8 3.6 2.9 2.3 3.7 1.5 3.9 2.5 1.9 4.7 2.9 2.6 1.9 2.4 2.7 4.5 2.3 3.6 2.8 1.7 1.9 4.0 3.9 2.7 3.3 2.9 1.5 1.5
a) The 40 analytes were the same as in Fig. 1. (New added).
3.2 Optimization of MS conditions In this study, to select a proper transition for the MS/MS detection of the analytes, all the compounds were examined separately in direct infusion mode by full-scan MS method in both positive and negative ionization modes. It was found that compared to the negative ion mode, the tested compounds had not only higher sensitivity but also clearer mass spectra in the positive ion mode, which made it easier to detect them of lower content in mulberry leaves, and easier to confirm C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
molecular ions or quasi-molecular ions in the identification of each peak. In MRM mode, the peak could appear only when the parent and daughter ions were both detected by choosing the appropriate parent and daughter ions, and then the influence of nucleosides could be minimized. After comparing with the peak of [M+Na]+ , [M+K]+ , and [M+H-ribose]+ , [M+H]+ was selected as both parent and daughter ions for the 40 compounds to obtain a good peak separation with their corresponding compounds. All the MRM transitions and parameters applied in the study are listed in Table 2. www.jss-journal.com
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Table 5. Contents (mg/g) of nucleosides, nucleobases, amino acids, and alkaloids in mulberry leaves
NO.a) 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1 2 3 4 5 6 7 8 9 10
0.130 0.136 0.166 0.124 0.202 0.172 0.159 0.147 0.080 0.172
0.123 0.200 0.121 0.142 0.137 0.121 0.133 0.083 0.095 0.083
0.034 0.025 0.032 0.033 0.042 0.038 0.040 0.030 0.017 0.022
0.024 0.054 0.039 0.107 0.036 0.036 0.036 0.054 0.073 0.054
0.028 0.025 0.033 0.049 0.014 0.009 0.031 0.015 0.036 0.064
0.060 0.077 0.070 0.091 0.068 0.067 0.073 0.207 0.107 0.206
0.040 0.071 0.067 0.099 0.053 0.051 0.070 0.066 0.241 0.062
0.038 0.032 0.042 0.059 0.023 0.018 0.040 0.043 0.044 0.088
0.012 0.020 0.015 0.032 0.016 0.017 0.018 0.035 0.052 0.034
0.044 0.025 0.036 0.024 0.036 0.038 0.050 0.064 0.022 0.071
0.003 0.002 0.005 0.001 0.002 0.001 0.003 0.007 0.001 0.012
0.019 0.052 0.032 0.024 0.072 0.065 0.038 0.026 0.011 0.043
0.024 0.051 0.034 0.089 0.033 0.036 0.039 0.056 0.067 0.056
0.008 0.006 0.017 0.106 0.037 0.033 0.128 0.237 0.042 0.045
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.232 0.028 0.219
0.034 0.040 0.068 0.071 0.050 0.049 0.051 0.207 0.927 0.226
0.008 0.008 0.009 0.013 0.010 0.008 0.009 0.032 0.012 0.036
0.022 0.022 0.023 0.022 0.023 0.022 0.022 0.089 0.023 0.089
0.029 0.050 0.047 0.073 0.045 0.041 0.036 0.118 0.033 0.182
0.701 0.551 0.740 0.671 0.645 0.591 0.705 0.928 0.430 0.744
NO. 21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
1 2 3 4 5 6 7 8 9 10
0.110 0.060 0.398 0.156 0.246 0.122 0.253 0.306 0.807 1.311
0.142 0.161 0.138 0.085 0.118 0.132 0.081 0.774 0.028 0.675
0.138 0.166 0.223 0.212 0.182 0.140 0.185 0.551 0.597 0.667
0.034 0.036 0.066 0.074 0.050 0.049 0.051 0.173 0.061 0.183
0.105 0.055 0.294 0.104 0.249 0.172 0.521 0.970 2.520 1.491
0.015 0.027 0.040 0.050 0.037 0.029 0.032 0.115 0.237 0.164
0.102 0.074 0.099 0.143 0.131 0.108 0.143 0.159 0.239 0.168
0.050 0.076 0.124 0.133 0.140 0.101 0.113 0.319 0.149 0.408
0.008 0.010 0.016 0.016 0.016 0.014 0.013 0.030 0.020 0.046
0.031 0.035 0.053 0.092 0.043 0.038 0.058 0.232 0.212 0.322
0.020 0.031 0.032 0.047 0.033 0.031 0.034 0.081 0.040 0.118
0.022 0.028 0.027 0.035 0.025 0.026 0.023 0.081 0.021 0.084
0.025 0.048 0.048 0.084 0.066 0.052 0.045 0.125 0.166 0.211
0.023 0.039 0.036 0.061 0.039 0.036 0.029 0.093 0.043 0.123
0.063 0.044 0.205 0.073 0.194 0.149 0.217 0.594 0.289 0.566
0.067 0.140 0.077 0.121 0.207 0.221 0.197 0.033 0.135 0.060
0.014 0.014 0.017 0.015 0.016 0.015 0.015 0.059 0.019 0.066
0.538 1.223 0.754 0.370 2.630 1.924 1.708 0.212 1.095 0.407
0.005 0.006 0.007 0.007 0.009 0.006 0.015 0.002 0.022 0.000
0.030 0.049 0.054 0.073 0.070 0.055 0.053 0.174 0.209 0.273
a) The 10 samples and 40 analytes were the same as in Table 1 and Fig. 1.
3.3 Optimization of the UHPLC chromatographic conditions In our preliminary test, two columns, an Acquity BEH C18 (100 mm × 2.1 mm, 1.7 m) and an Acquity BEH Amide (100 mm × 2.1 mm, 1.7 m), were compared. The result showed that the latter has a stronger retention ability as well as better resolution for these hydrophilic components of mulberry leaf with the same mobile phase; thus, the Acquity Amide (100 mm × 2.1 mm, 1.7 m) column was chosen for this analysis. As for the mobile phase, methanol is a polar protic solvent, while acetonitrile is a polar aprotic solvent, which has been proven the best organic solvent for hydrophilic interaction liquid chromatography producing narrower peaks in a short analysis time so that acetonitrile was selected as the organic phase. It was reported that ammonium acetate aqueous solution or ammonium formate aqueous solution can improve the separation of nucleosides, nucleobases, and amino acids for HPLC analysis [35–40]. So different mobile phase additives, such as ammonium formate and ammonium acetate, were compared. The results showed that ammonium formate and ammonium acetate used together as a salt additive of mobile phase could provide much improved sensitivity and peak shapes of these compounds. However, it was found that for the acquity BEH amide column, tailings were observed for most of the target peaks when only ammonium acetate and ammonium formate was used as a mobile phase modifier. Hence, different concen-
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tration of formic acid (0.05, 0.1, 0.15, 0.2%) was added and compared. The result showed that the solution at 0.2% was better than those at 0.05, 0.1, and 0.15% for improving the peak shape. As a result, a mixed solution including acetonitrile solution (1 mM ammonium formate and ammonium acetate, 0.2% formic acid) and aqueous solution (5 mM ammonium formate and ammonium acetate, 0.2% formic acid) was chosen as the preferred mobile phase, and gradient elution was applied. The injection volume was set at 1 L while the flow rate was set at 0.4 mL/min and the column temperature was kept at 35⬚C. The typical chromatograms of the 40 analytes are presented in Fig. 3.
3.4 UHPLC method validation The proposed UHPLC method was validated by determining the linearity, LOD, LOQ, precision, repeatability, stability, and recovery. The results demonstrated that all calibration curves exhibited excellent linear regressions with the determination coefficients (r2 ) ranging from 0.9889 to 0.9997, and the calibration ranges adequately covered variations in the amounts of the compounds investigated in the samples. The overall LODs and LOQs were
J. Sep. Sci. 2014, 37, 1265–1275
Li-li Zhang1 Yong-liang Bai2 Shu-lan Shu1 Da-wei Qian1 Zhen Ou-yang2 Li Liu3 Jin-ao Duan1 1 Jiangsu
Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, China 2 College of Pharmacy, Jiangsu University, Zhenjiang, China 3 Sericultural Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China
Received November 24, 2013 Revised February 24, 2014 Accepted March 1, 2014
Research Article
Simultaneous quantitation of nucleosides, nucleobases, amino acids, and alkaloids in mulberry leaf by ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry An ultra high performance liquid chromatography coupled with a triple quadrupole mass detection and electrospray ionization mass spectrometry method has been established for the simultaneous determination of the 14 nucleosides and nucleobases, 24 amino acids and two main alkaloids in mulberry leaf. In this method, a complicated mobile phase, the flow rate of which was 0.4 mL/min, was applied to the gradient elution, which provided a satisfied separation of the 40 compounds. The present method was validated, and sufficient reproducibility and accuracy were obtained for the quantitative measurement of the 40 compounds. The method was subsequently applied to ten mulberry leaves and the results showed that almost all of these samples were rich in nucleosides, nucleobases, amino acids, and alkaloids. The proposed method, which is convenient and economical, could serve as a prerequisite for the quality control of mulberry leaf herbs and be applied analogously to other Chinese medicines. Keywords: Alkaloids / Amino acids / Morus Alba / Nucleosides / Simultaneous quantitation DOI 10.1002/jssc.201301267
1 Introduction Morus Alba L., a Moraceous plant, is indigenous to China with a history of over 4000 years and is widely distributed in China, especially in Jiangsu and Zhejiang provinces. Its dried leaves, called mulberry leaf in China, have been commonly used as a traditional Chinese medicine for its efficacies of hypoglycemic [1], hypolipidemic [2], antihypertensive [3], anti-atherosclerotic [4], anti-inflammatory [5], antioxidant [1, 6, 7], and antitumor [8]. Phytochemical studies have revealed that mulberry leaf contains various constituents, including flavonoids [9, 10], alkaloids [11], polysaccharides [12], amino acids [13], nucleosides, microelements, and so on. Mulberry leaf, which is the only food of silkworms, has been commonly utilized as a food or food additive in recent decades due to their high nutritional value [14]. It is also well known that nucleosides [15–17] and amino acids [18–21] are involved in the regulation and modulation of various physiological processes in body and exhibit multiple bioactivities, Correspondence: Dr. Jin-ao Duan, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, P. R. China E-mail: [email protected] Fax: +86-25-85811116
Abbreviations: MRM, multiple reaction monitoring; TQ-MS, triple quadrupole mass spectrometry; UHPLC, ultra high performance liquid chromatography C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
such as antiplatelet aggregation, antiarrhythmic, antioxidant, and so on [22–29]. 1-Deoxynojirimycin, one of the alkaloids in mulberry leaf, is an ␣-glucosidase inhibitor [30], which is effective for diabetes [31]. Since mulberry leaf is increasingly being used in healthcare products and these benefit human health, it is very necessary to develop a fast, convenient, and efficient method to find the clear and definite amount of its nutritional constituents to make sure it is safe and improve their potential values. Ultra high performance liquid chromatography (UHPLC), which is widely used in medicines analysis, shows great advantages over traditional HPLC for its high power in separation and analysis speed. High sensitivity and high throughput can be obtained by using triple quadrupole mass detection and multiple reaction monitoring (MRM), which use one transition each from the precursor to the reporter ions for every compound, respectively. To our knowledge, unfortunately, there are just quantitative methods [32–34] for each particular class of the compounds, but no published analytical methods are available for the quantification of nucleosides, nucleobases, amino acids, and alkaloids together. Thus, the UHPLC–triple quadrupole MS (TQ-MS) method, with which the 40 analytes were completely separated and detected, was established and applied to ten mulberry leaf samples. The aim of this study is to report a development, validation, and application of a rapid and sensitive Colour Online: See the article online to view Figs. 2 and 3 in colour. www.jss-journal.com
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Figure 1. Chemical structures of the 40 identified nucleosides, nucleobases, amino acids, and alkaloids.
UHPLC–TQ-MS method for the detection and quantitation of underivatized nucleosides, nucleobases, amino acids, and alkaloids together. The proposed method can be used for the quality control of mulberry leaf and other traditional Chinese medicines.
2 Materials and methods 2.1 Chemicals and reagents Acetonitrile was of HPLC grade (Tedia, USA), formic acid was of HPLC grade (Merck, Germany), and deionized water (H2 O) was purified by a superpurification system
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(Eped Technology Development, Nanjing, China). Other reagent solutions, such as ammonium formate (Lingfeng Chemical Reagent, Shanghai, China) and ammonium acetate (Sinopharm Chemical Reagent), were of analytical grade. Chemical standards of adenine (1), guanine (2), xanthine (3), thymine (10), inosine (13), arginine (15), histidine (16), lysine (17), tyrosine (20), valine (21), proline (22), glutamate (24), serine (27), threonine (28), alanine (29), tryptophan (31), phenylalanine (32), leucine (34), isoleucine (35), aspartic acid (37), 1-deoxynojirimycin (DNJ) (39), and fagomine (40) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Reference compounds of cytidine-5 -monophosphate (4), 2 -deoxyguanosine (5),
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Figure 1. Continued
2 -deoxyuridine (8), 2 -deoxyinosine (9), cytidine (11), 2 -deoxyadenosine-5 monophosphate (12), thymidine (14), ornithine (18), hydroxyproline (19), citrulline (23), glutamine (25), asparagine (26), glycine (30), ␥-aminobutyric acid (36), and cysteine (38) were obtained from Sigma (St. Louis, MO). Chemical standards of 2 -deoxyadenosine (6) and 2 -deoxycytidine (7) were obtained from Aladdin Chemical, Shanghai, China. A reference of methionine (33) was purchased from Sinopharm Chemical Reagent, Beijing, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
China. The purity of each compound was >98%, determined by HPLC analysis. The chemical structures of these reference compounds are shown in Fig. 1. 2.2 Plant materials Seven mulberry leaves (samples 1–7) were purchased from seven Pharmacies of Traditional Medicine in Nanjing. Others (samples 8–10), were collected on November 1, 2012 from a www.jss-journal.com
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Table 1. The information of samples
2.3 Preparation of standard solutions
Sample
Origin
Batch number
1 2 3 4 5 6 7 8 9 10
Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Bozhou, Anhui Zhenjiang, Jiangsu Zhenjiang, Jiangsu Zhenjiang, Jiangsu
120813 130130 130303 130111 130211 130101 120804002 12110101 12110102 12110103
mulberry garden in Zhenjiang, China. Their botanical origins were identified by the corresponding author. After collection, the leaves were dried at 55⬚C. The information of these samples is summarized in Table 1.
A mixed standard stock solution containing the reference compounds 1–40 was prepared in methanol/water (9:1, v/v). Working standard solutions for calibration curves were prepared by diluting the mixed standard stock solution with 10% methanol at different concentrations, and the concentration ranges for the 40 analytes were as follows: 1, 0.55–27.50 g/mL; 2, 0.60–30.00 g/mL; 3, 0.25–12.50 g/mL; 4, 0.54– 27.00 g/mL; 5, 0.65–32.50 g/mL; 6, 0.56–28.00 g/mL; 7, 0.55–27.25 g/mL; 8, 0.77–38.25 g/mL; and 9, 0.58– 28.75 g/mL; 10, 0.60–30.00 g/mL; 11, 0.53–26.50 g/mL; 12, 0.60–30.00 g/mL; 13, 0.66–32.75 g/mL; 14, 0.64– 32.00 g/mL; 15, 0.55–27.50 g/mL; 16, 0.52–25.75 g/mL; 17, 0.60–30.00 g/mL; 18, 0.60–29.75 g/mL; 19, 0.74– 37.00 g/mL; 20, 0.50–25.00 g/mL; 21, 0.52–26.00 g/mL; 22, 0.67–33.25 g/mL; 23, 0.51–25.25 g/mL; 24, 0.51– 25.50 g/mL; 25, 0.63–31.50 g/mL; 26, 0.80–40.00 g/mL; 27, 0.57–28.50 g/mL; 28, 0.62–31.00 g/mL; 29, 0.63– 31.50 g/mL; 30, 0.65–32.25 g/mL; 31, 0.65–32.50 g/mL; 32, 0.51–25.50 g/mL; 33, 0.57–28.50 g/mL; 34, 0.50– 24.75 g/mL; 35, 0.53–26.25 g/mL; 36, 0.49–24.50 g/mL;
Figure 2. Effects of solvent, extraction temperature, solvent volume, and extraction time on the extraction efficiency of investigated nucleosides, amino acids, and alkaloids from the mulberry leaf sample. When one of the parameters was determined, the others were set at the default (solvent, water; extraction temperature, 50⬚C; solvent volume, 50 mL; extraction time, 50 min).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 2. Precursor/product ion pairs and parameters for MRM of compounds used in this study
No.
Compound
tR (min)
[M+H]+ (m/z)
Product ion (m/z)
Cone voltage (V)
Collision energy (eV)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Adenine Guanine Xanthine Cytidine-5 -monophosphate 2 -deoxyguanosine 2 -deoxyadenosine 2 -deoxycytidine 2 -deoxyuridine 2 -deoxyinosine Thymine Cytidine 2 -deoxy-ade-5 -monophosphate Inosine Thymidine Arginine Histidine Lysine Ornithine Hydroxyproline Tyrosine Valine Proline Citrulline Glutamate Glutamine Asparagine Serine Threonine Alanine Glycine Tryptophan Phenylalanine Methionine Leucine Isoleucine GABA Aspartic acid Cysteine DNJ Fagomine
1.93 4.82 3.32 16.95 3.45 1.68 2.99 3.01 1.66 1.22 3.95 7.24 2.81 1.21 16.98 17.12 10.89 17.17 8.76 6.95 6.16 6.25 16.91 10.95 10.76 11.51 10.96 9.43 8.62 9.84 4.85 4.51 5.63 4.48 4.92 5.02 11.69 6.14 11.25 8.17
136.0 152.0 153.0 324.0 268.1 252.0 228.0 229.0 253.0 127.0 244.0 332.0 269.0 243.0 175.2 156.1 147.0 133.0 132.0 182.1 118.0 116.0 176.0 147.9 147.0 132.9 106.0 120.0 90.0 76.0 205.1 166.1 150.1 132.1 132.1 103.9 134.0 122.0 163.3 147.9
136.0 152.0 153.0 111.9 152.0 135.9 111.9 113.0 136.9 127.0 111.9 135.9 136.9 126.9 70.0 110.0 83.9 69.9 67.9 136.0 72.1 70.0 69.9 83.9 83.9 73.9 60.0 74.0 44.0 30.0 146.0 120.1 104.0 86.1 86.1 87.0 88.0 75.8 68.7 112.0
30 30 30 16 10 16 28 8 22 30 28 20 10 10 22 22 14 14 18 16 12 20 16 12 8 12 14 38 16 12 16 18 14 16 16 16 14 14 4 22
20 20 20 14 12 14 10 10 12 15 10 16 14 10 18 14 14 14 16 16 10 10 20 14 16 14 8 20 10 6 18 14 10 10 10 10 10 17 18 14
37, 0.64–31.75 g/mL; 38, 0.66–33.00 g/mL; 39, 1.45– 145.00 g/mL; 40, 0.12–12.00 g/mL. The standard solutions were filtered through a 0.22 m membrane filter prior to injection. All solutions were stored in a refrigerator at 4⬚C before analysis.
and then the same solvent was added to compensate for the weight lost during the extraction. After centrifugation (13 000 rpm, 10 min) and filtering (0.22 m membrane filter), the supernatant was stored at the sample plate whose temperature was set at 4⬚C before injection into the UHPLC system for analysis.
2.4 Preparation of sample solutions The dried leaves were pulverized to homogeneous powders (40 mesh). The dried powder (1.0 g), which was weighed accurately, was put into a 100 mL conical flask with a stopper, and 50 mL water was added. After accurate weighing, ultrasonication (80 kHz) was performed at 50⬚C for 50 min, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5 Chromatographic conditions and instrumentation Analysis was performed on a Waters Acquity UHPLC system (Waters, Milford, MA), consisting of a quaternary pump solvent management system, an online degasser, an www.jss-journal.com
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Figure 3. Chromatogram of the 40 compounds analyzed in this study.
autosampler, and a triple quadrupole mass detector. An Acquity UPLC BEH Amide (100 mm × 2.1 mm, 1.7 m) column was applied for all analyses. The raw data were acquired and processed with MassLynx 4.1 software. The mobile phase was composed of A (5 mM ammonium formate and ammonium acetate solution, 0.2% formic acid) and B (acetonitrile with 1 mM ammonium formate, ammonium acetate solution, and 0.2% formic acid) with a gradient elution: 0– 3 min, 10% A; 3–9 min, 10–18% A; 9–15 min, 18–20% A, 15–16 min, 20–46% A, 16–18 min, 46% A. The flow rate of the mobile phase was 0.4 mL/min, and the column temperature was maintained at 35⬚C. High-purity nitrogen was used as the nebulizer and auxiliary gas; argon was utilized as the collision gas. The triple quadrupole (TQ) mass spectrometer was operated in positive ion mode with a capillary voltage of 3 kV, a sampling cone voltage of 30 V, a cone gas flow of 20 L/h, a desolvation gas flow of 1000 L/h, a desolvation temperature of 350⬚C, a source temperature of 120⬚C, a collision energy of 6 V, and full-scan spectra from 100 to 1000 Da.
2.6 Validation of the method For calibration, the linearity was obtained by plotting the peak areas versus the corresponding concentrations of each analyte. The lowest concentration of working solution for calibration use was diluted with water to a series of appropriate concentrations. They were analyzed until the S/N for each compound was about 3 for the LOD and 10 for the LOQ. The precision was evaluated by analyzing the standard solutions containing the 40 standard compounds six times. Then, the RSD of the peak area for each of the marker compounds was calculated. To confirm the repeatability, six different sample solutions prepared from the same sample (sample 9) were analyzed and variations were expressed by RSD. To evaluate C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the stability of the solution, one of the sample solutions mentioned above was stored at 25⬚C and analyzed at 0, 2, 4, 8, 12, and 24 h, respectively. A recovery test was used to evaluate the accuracy of this method. It was performed by adding corresponding marker compounds with high (120%), medium (100%), and low (80%) levels into accurately weighed samples (sample 9, 0.5 g). The spiked samples were then extracted, processed, and quantified in accordance with the methods mentioned above. The detected amounts (actual) were calculated by subtracting the total amount of each compound before spiking from the total amount after spiking. The ratio of the detected amount (actual) to spiked amount (theoretical) was used to calculate the recovery percentage.
2.7 Identification and quantification The identification of the nucleosides, nucleobases, amino acids, and alkaloids was carried out by comparing the UHPLC retention time of target peaks with those of the standards by UHPLC–TQ-MS in positive ion mode. Quantification was performed on the basis of linear calibration plots of the peak areas versus the concentration.
3 Results and discussion 3.1 Optimization of extraction procedure In this study, extraction variables such as extraction solvent (water, 10, 20, and 30% aqueous methanol), solvent volume (10, 20, 30, 40, 50, and 60 mL), extraction temperature (20, 30, 40, 50, 60, 70⬚C), and extraction time (10, 20, 30, 40, 50, and 60 min) were investigated for sample 9 (1.0 g, 40 mesh) by sonicating to obtain optimal extraction conditions. When www.jss-journal.com
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Table 3. Calibration curves, LOD, LOQ, precision, repeatability, and stability of the 40 analytes
NO.a)
Calibration curves
r2
Linear range g/mL
LOD
LOQ
Precision RSD,%, n = 6
Repeatablity
Stability
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
y = 56706x + 4185.7 y = 12041x – 2477.4 y = 17580x – 1103 y = 6321.6x + 1600 y = 33732x – 1634 y = 123054x + 2303.4 y = 148482x – 33562 y = 3744.8x – 221.9 y = 8969.6x – 2653.8 y = 30601x – 6103.5 y = 3326x – 142.1 y = 8057.2x + 500 y = 46313x – 6166.2 y = 8995.6x – 405.7 y = 33694x + 10764 y = 96645x + 28000 y = 22071x – 1712 y = 19718x + 3000 y = 20010x – 1940 y = 13766x – 6047.1 y = 118887x – 31410 y = 172631x – 8252 y = 3006.7x + 280 y = 16326x – 8764.8 y = 23880x – 3959 y = 12303x – 8406.5 y = 10987x + 4071.9 y = 585.52x – 433.86 y = 12795x + 11.807 y = 4411.1x + 339.45 y = 25987x – 7387 y = 163984x – 61671 y = 35932x – 2972 y = 93166x – 423.76 y = 137944x – 6779 y = 102825x + 14772 y = 5295.9x + 0 y = 1482.3x – 910.75 y = 682x – 46.17 y = 190000x + 11796
0.9994 0.9969 0.9920 0.9946 0.9972 0.9987 0.9889 0.9976 0.9949 0.9987 0.9935 0.9942 0.9990 0.9982 0.9947 0.9986 0.9951 0.9997 0.9944 0.9930 0.9949 0.9982 0.9912 0.9935 0.9951 0.9958 0.9939 0.9918 0.9981 0.9976 0.9957 0.9986 0.9920 0.9988 0.9979 0.9981 0.9943 0.9984 0.9900 0.9902
0.55–27.50 0.60–30.00 0.25–12.50 0.54–27.00 0.65–32.50 0.56–28.00 0.55–27.25 0.77–38.25 0.58–28.75 0.60–30.00 0.53–26.50 0.60–30.00 0.66–32.75 0.64–32.00 0.55–27.50 0.52–25.75 0.60–30.00 0.60–29.75 0.74–37.00 0.50–25.00 0.52–26.00 0.67–33.25 0.51–25.25 0.51–25.50 0.63–31.50 0.80–40.00 0.57–28.50 0.62–31.00 0.63–31.50 0.65–32.25 0.65–32.50 0.51–25.50 0.57–28.50 0.50–24.75 0.53–26.25 0.49–24.50 0.64–31.75 0.66–33.00 0.145–145.00 0.12–12.00
0.015 0.036 0.068 0.034 0.004 0.004 0.005 0.012 0.014 0.037 0.069 0.026 0.005 0.005 0.003 0.004 0.023 0.010 0.006 0.041 0.016 0.010 0.022 0.044 0.050 0.088 0.045 0.119 0.045 0.041 0.013 0.009 0.004 0.016 0.017 0.003 0.131 0.076 0.003 0.002
0.050 0.115 0.226 0.112 0.012 0.012 0.015 0.038 0.048 0.120 0.230 0.086 0.017 0.016 0.011 0.014 0.078 0.031 0.021 0.135 0.054 0.035 0.072 0.150 0.166 0.296 0.150 0.413 0.150 0.129 0.046 0.030 0.014 0.055 0.058 0.009 0.438 0.254 0.007 0.004
0.6 1.4 1.5 2.9 0.7 0.8 0.7 1.1 1.4 0.3 1.7 0.8 1.9 0.4 2.5 2.5 0.5 2.3 0.4 0.8 0.9 0.4 2.5 0.7 1.1 0.9 0.6 2.3 0.4 2.8 1.2 0.3 0.7 0.5 1.1 0.6 2.8 1.9 1.7 1.3
1.2 0.2 0.7 0.9 0.9 1.4 1.6 0.6 0.8 0.7 1.4 1.1 2.2 2.2 2.0 2.2 0.7 2.4 2.4 0.8 0.4 0.3 2.8 1.2 1.1 0.7 0.6 1.9 0.4 1.6 1.1 1.0 2.6 0.7 1.1 0.6 2.2 1.1 2.1 2.9
0.2 1.7 0.9 1.8 2.1 2.8 2.9 2.2 2.5 2.2 0.8 0.8 1.9 2.7 2.2 0.8 1.8 1.6 2.3 1.6 0.6 1.2 1.6 1.8 0.5 0.6 0.4 1.2 0.6 2.0 2.0 2.3 2.0 0.5 1.8 1.0 2.2 2.2 3.8 3.1
a) The 40 analytes were the same as in Fig. 1. (The part of recovery has been deleted).
one of the parameters was determined, the others were set at the default (solvent, water; solvent volume, 50 mL; extraction temperature, 50⬚C; extraction time, 50 min). The results are shown in Fig. 2, which indicates that the best solvent was found to be water, which could obtain the highest extraction efficiency for the 40 constituents analyzed among these investigated solvents. Furthermore, it was found that the amounts of the compounds analyzed in mulberry leaf increased with the extraction time extension and reached the maximum at 50 min; thereafter, a further increase of extraction time did not result in a significant increase in the amount.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
On the contrary, it could lead to dramatic decreases in yields of lysine, asparagine, and DNJ. For example, compared with the amounts detected after an extraction of 50 min, the detected amount decreased about 0.47 mg/g for DNJ when the extraction time was extended to 60 min. In addition, the volume of solvent was chosen as 50 mL, and the extraction temperature was chosen as 50⬚C, which was sufficient for sample extraction. Therefore, the final extraction conditions were as follows: each sample was extracted by sonication with 50 mL of water for 50 min at 50⬚C, which was adequate and appropriate for the analysis.
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Table 4. Recovery of the 40 analytes in mulberry leaves
NO.a)
Recovery,%, n = 3 Low level (80%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Medium level (100%)
High level (120%)
Mean
RSD
Mean
RSD
Mean
RSD
97.5 102.7 97.4 97.7 98.6 97.9 96.8 97.1 103.4 95.7 102.1 96.7 98.3 97.3 101.6 102.4 98.2 103.5 97.7 101.8 98.0 97.6 95.9 103.7 102.9 98.5 97.6 98.2 101.9 103.5 97.8 102.6 98.4 97.3 101.9 102.6 104.1 95.9 97.7 101.8
2.7 3.5 4.6 2.9 2.0 2.3 1.7 2.8 3.6 4.8 2.9 3.6 4.7 4.1 4.5 3.4 3.0 2.1 3.5 2.3 2.2 3.8 4.3 3.8 2.7 3.0 3.5 3.9 2.4 4.4 3.3 2.5 2.7 3.8 3.1 3.7 4.1 4.0 2.2 1.9
102.5 102.4 103.5 97.6 102.4 99.1 98.8 101.9 102.3 97.6 102.7 98.5 103.1 103.4 102.4 103.6 101.8 101.2 102.5 103.7 98.3 102.4 96.7 104.5 97.4 103.9 98.3 102.4 96.8 102.2 104.2 98.5 101.9 98.8 103.7 103.5 103.9 101.7 102.6 103.1
3.2 2.8 2.9 2.5 2.3 2.2 2.4 2.6 3.1 2.5 3.4 3.7 2.6 2.3 2.5 2.6 2.1 3.4 2.8 3.4 1.7 3.2 3.7 2.2 3.8 3.7 2.3 2.7 4.1 1.9 2.0 3.3 2.3 3.6 2.4 2.5 4.1 2.6 3.7 2.8
102.8 98.4 101.6 103.4 102.1 102.4 97.6 101.9 97.5 102.5 103.5 97.1 103.9 102.5 102.8 104.6 103.5 98.7 103.1 104.2 103.6 103.2 97.3 102.1 98.3 103.7 102.4 96.8 102.6 98.0 101.9 104.2 97.2 102.8 103.2 97.4 95.8 97.0 102.3 98.0
1.9 2.6 2.5 1.8 1.5 1.4 2.0 1.7 2.2 3.1 2.7 1.9 3.8 3.6 2.9 2.3 3.7 1.5 3.9 2.5 1.9 4.7 2.9 2.6 1.9 2.4 2.7 4.5 2.3 3.6 2.8 1.7 1.9 4.0 3.9 2.7 3.3 2.9 1.5 1.5
a) The 40 analytes were the same as in Fig. 1. (New added).
3.2 Optimization of MS conditions In this study, to select a proper transition for the MS/MS detection of the analytes, all the compounds were examined separately in direct infusion mode by full-scan MS method in both positive and negative ionization modes. It was found that compared to the negative ion mode, the tested compounds had not only higher sensitivity but also clearer mass spectra in the positive ion mode, which made it easier to detect them of lower content in mulberry leaves, and easier to confirm C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
molecular ions or quasi-molecular ions in the identification of each peak. In MRM mode, the peak could appear only when the parent and daughter ions were both detected by choosing the appropriate parent and daughter ions, and then the influence of nucleosides could be minimized. After comparing with the peak of [M+Na]+ , [M+K]+ , and [M+H-ribose]+ , [M+H]+ was selected as both parent and daughter ions for the 40 compounds to obtain a good peak separation with their corresponding compounds. All the MRM transitions and parameters applied in the study are listed in Table 2. www.jss-journal.com
Liquid Chromatography
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Table 5. Contents (mg/g) of nucleosides, nucleobases, amino acids, and alkaloids in mulberry leaves
NO.a) 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1 2 3 4 5 6 7 8 9 10
0.130 0.136 0.166 0.124 0.202 0.172 0.159 0.147 0.080 0.172
0.123 0.200 0.121 0.142 0.137 0.121 0.133 0.083 0.095 0.083
0.034 0.025 0.032 0.033 0.042 0.038 0.040 0.030 0.017 0.022
0.024 0.054 0.039 0.107 0.036 0.036 0.036 0.054 0.073 0.054
0.028 0.025 0.033 0.049 0.014 0.009 0.031 0.015 0.036 0.064
0.060 0.077 0.070 0.091 0.068 0.067 0.073 0.207 0.107 0.206
0.040 0.071 0.067 0.099 0.053 0.051 0.070 0.066 0.241 0.062
0.038 0.032 0.042 0.059 0.023 0.018 0.040 0.043 0.044 0.088
0.012 0.020 0.015 0.032 0.016 0.017 0.018 0.035 0.052 0.034
0.044 0.025 0.036 0.024 0.036 0.038 0.050 0.064 0.022 0.071
0.003 0.002 0.005 0.001 0.002 0.001 0.003 0.007 0.001 0.012
0.019 0.052 0.032 0.024 0.072 0.065 0.038 0.026 0.011 0.043
0.024 0.051 0.034 0.089 0.033 0.036 0.039 0.056 0.067 0.056
0.008 0.006 0.017 0.106 0.037 0.033 0.128 0.237 0.042 0.045
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.232 0.028 0.219
0.034 0.040 0.068 0.071 0.050 0.049 0.051 0.207 0.927 0.226
0.008 0.008 0.009 0.013 0.010 0.008 0.009 0.032 0.012 0.036
0.022 0.022 0.023 0.022 0.023 0.022 0.022 0.089 0.023 0.089
0.029 0.050 0.047 0.073 0.045 0.041 0.036 0.118 0.033 0.182
0.701 0.551 0.740 0.671 0.645 0.591 0.705 0.928 0.430 0.744
NO. 21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
1 2 3 4 5 6 7 8 9 10
0.110 0.060 0.398 0.156 0.246 0.122 0.253 0.306 0.807 1.311
0.142 0.161 0.138 0.085 0.118 0.132 0.081 0.774 0.028 0.675
0.138 0.166 0.223 0.212 0.182 0.140 0.185 0.551 0.597 0.667
0.034 0.036 0.066 0.074 0.050 0.049 0.051 0.173 0.061 0.183
0.105 0.055 0.294 0.104 0.249 0.172 0.521 0.970 2.520 1.491
0.015 0.027 0.040 0.050 0.037 0.029 0.032 0.115 0.237 0.164
0.102 0.074 0.099 0.143 0.131 0.108 0.143 0.159 0.239 0.168
0.050 0.076 0.124 0.133 0.140 0.101 0.113 0.319 0.149 0.408
0.008 0.010 0.016 0.016 0.016 0.014 0.013 0.030 0.020 0.046
0.031 0.035 0.053 0.092 0.043 0.038 0.058 0.232 0.212 0.322
0.020 0.031 0.032 0.047 0.033 0.031 0.034 0.081 0.040 0.118
0.022 0.028 0.027 0.035 0.025 0.026 0.023 0.081 0.021 0.084
0.025 0.048 0.048 0.084 0.066 0.052 0.045 0.125 0.166 0.211
0.023 0.039 0.036 0.061 0.039 0.036 0.029 0.093 0.043 0.123
0.063 0.044 0.205 0.073 0.194 0.149 0.217 0.594 0.289 0.566
0.067 0.140 0.077 0.121 0.207 0.221 0.197 0.033 0.135 0.060
0.014 0.014 0.017 0.015 0.016 0.015 0.015 0.059 0.019 0.066
0.538 1.223 0.754 0.370 2.630 1.924 1.708 0.212 1.095 0.407
0.005 0.006 0.007 0.007 0.009 0.006 0.015 0.002 0.022 0.000
0.030 0.049 0.054 0.073 0.070 0.055 0.053 0.174 0.209 0.273
a) The 10 samples and 40 analytes were the same as in Table 1 and Fig. 1.
3.3 Optimization of the UHPLC chromatographic conditions In our preliminary test, two columns, an Acquity BEH C18 (100 mm × 2.1 mm, 1.7 m) and an Acquity BEH Amide (100 mm × 2.1 mm, 1.7 m), were compared. The result showed that the latter has a stronger retention ability as well as better resolution for these hydrophilic components of mulberry leaf with the same mobile phase; thus, the Acquity Amide (100 mm × 2.1 mm, 1.7 m) column was chosen for this analysis. As for the mobile phase, methanol is a polar protic solvent, while acetonitrile is a polar aprotic solvent, which has been proven the best organic solvent for hydrophilic interaction liquid chromatography producing narrower peaks in a short analysis time so that acetonitrile was selected as the organic phase. It was reported that ammonium acetate aqueous solution or ammonium formate aqueous solution can improve the separation of nucleosides, nucleobases, and amino acids for HPLC analysis [35–40]. So different mobile phase additives, such as ammonium formate and ammonium acetate, were compared. The results showed that ammonium formate and ammonium acetate used together as a salt additive of mobile phase could provide much improved sensitivity and peak shapes of these compounds. However, it was found that for the acquity BEH amide column, tailings were observed for most of the target peaks when only ammonium acetate and ammonium formate was used as a mobile phase modifier. Hence, different concen-
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tration of formic acid (0.05, 0.1, 0.15, 0.2%) was added and compared. The result showed that the solution at 0.2% was better than those at 0.05, 0.1, and 0.15% for improving the peak shape. As a result, a mixed solution including acetonitrile solution (1 mM ammonium formate and ammonium acetate, 0.2% formic acid) and aqueous solution (5 mM ammonium formate and ammonium acetate, 0.2% formic acid) was chosen as the preferred mobile phase, and gradient elution was applied. The injection volume was set at 1 L while the flow rate was set at 0.4 mL/min and the column temperature was kept at 35⬚C. The typical chromatograms of the 40 analytes are presented in Fig. 3.
3.4 UHPLC method validation The proposed UHPLC method was validated by determining the linearity, LOD, LOQ, precision, repeatability, stability, and recovery. The results demonstrated that all calibration curves exhibited excellent linear regressions with the determination coefficients (r2 ) ranging from 0.9889 to 0.9997, and the calibration ranges adequately covered variations in the amounts of the compounds investigated in the samples. The overall LODs and LOQs were
