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Simultaneously determination of five components in Folium Syringae by ultrahigh performance liquid chromatography
Guan Qingxia Wang Yanhong Yang Zhixin Feng Yufei Li Yongji* College of Pharmacy, Heilongjiang University of Chinese medicine, Harbin City150040, China
Abstracts. A rapid and sensitive method was developed for the simultaneous determination of five bioactive components in folium Syringaeby ultra-performance liquid chromatography. The analytes were separated with a Waters BEH column (C18 1.7 ^m, 50 x 2.1 mm) and a gradient elutionsystem using water and methanol as the mobile phases. The detection wavelength was set at 280nm and flow rate was set at 0.3 mlmin-1. The column compartment was kept atthe temperature of 30° C. Theseparation was achieved within 25 minutes. The linear response range was from 0.500 ^g to 5.015^g for five components and the correlation coefficients were all higher than 0.9995. The method was validated with respect to precision, repeatability, accuracy, recovery, and was successfully applied to quality control for folium Syringae and the preparations.
Key words: ultra-high performance liquid chromatography; Folium Syringae;
determination
^Introduction
Folium Syringae is dry leaves of Syringa oblate Lindl. in Oleaceae plant [1]. The genus of deciduous shrub distributes widelyand is abundant resources in northeast of China, and it is the main courtyard greening tree species. The water decoction of folium Syringae is used to treat excellently diarrhea and eye disease in folk. Folium Syringae was found to possess therapeutict effects on antiviral[2],anti-inflammatory[3], antibacterial,antitussive, antioxidant and hypothermia[4,5], and also could enhance immune function[6]. It is well known that traditional Chinese medicine is a complex mixturecontaining hundreds of chemically different constituents with therapeutic effects. Therefore, itseems necessary to determine multi-component as muchas possible to ensure the quality of TCM.In the prior reseach , a total 9 compounds of the dry folium Syringae were separated andidentified from folium Syringae [7], and6 compounds of them the were obtained for the first time from folium Syringae.
The aims of the present study are to develop aappropriate analyticalmethod to simultaneously determinefive bioactive components with higher content in folium Syringae(Structure as shown infig.1), and be readily utilizedas a quality control for the medicinal herbs and preparations.
A
(+)pinoresinol-4"-O-P-D-glucopyranosidelariciresinol-4-O-P-D-glucopyranoside
(+)Epipinoresinol-4-O-y#-D-glucopyranoside3,4-dihydroxy-phenylglycol-glucopyranoside
syringopicroside
Fig. 1. Chemical structures of five compounds inFolium Syringae 2.Materials and methods 2.1.Materials
Waters ACQUITY UPLC I - Class (the United States), Uv detector (Waters, the United States),Empower workstation, electronic balance (JD500-3G, Dongguan yong xu electronics co., LTD.).
Five compounds of (+)pinoresinol-4"-O-P-D-glucopyranoside, lariciresinol-4-O-P-D-glucopyranoside, (+)Epipinoresinol-4-O-P-D-glucopyranoside,3,4-dihydroxy-phenylglycol-glucopyranoside and Syringopicroside were prepared in our laboratory(over 98% purity).methanol were purchased from Merck Company Inc. (Merck,Darmstadt, Germany) and acetonitrile purchased from Dikma company(Dikma co.china). ultrapure water purchasedfrom Watson (Watson,china). 2.2 Chromatographic conditions
A ACQUITY UPLC system was used (Waters, the United States) for quantitative determination. The chromatographic separation was performed on a waters BEH C18 analytical column (1.7^m 2.1x50mm) at 30°C of the column temperature.A gradient elution of A (water) and
HO
B (methanol) was used. The flowrate was set at 0.3mlmin-1 and the detection wavelength was set at 280nm.Gradient elution conditions was showed in table 1. table 1 Gradient elution conditions
t (min) 0 2 6 8 8.5 9.5 11 13 18 21 23 25
A (water) 97 95 85.5 77 75.4 73.8 73.8 70.3 60 30 10 10
B (methanol) 3 5 14.5 23 24.6 26.2 26.2 29.7 40 70 90 90
2.3 Preparation of standard solutions
Five references were accurately weighed, dissolved inmethanol and diluted to appropriate
concentration. The solutions wasfiltrated through a 0.22^m membrane filter before UPLC analysis.Table 2 showed concentration of standard solutions respectively. table 2 Concentration of standard solutions
Compounds Concentration(mg • mL-1)
3,4-dihydroxy-phenylglycol-glucopyranoside 0.500
lariciresinol-4-O-P-D-glucopyranoside 0.503
pinoresinol-4"-O-P-D-glucopyranoside 0.306
(+)Epipinoresinol-4-O-yS-D-glucopyranoside 0.402
Syringopicroside 1.003
2.4 Preparation of samples
A 2 g dry folium Syringae was accurately weighed and then extracted for 1 h with 20 times the amount of water, and the procedure was repeated two times.Decoction was concentrated andthen adopted at 70%alcohol-precipitation concentration for 12 hours. The filtrate was combined and reclaimed.The recovered solution was purified with HPD500 macroporous resin,and extracted with ethyl acetate. The effective parts was dissolvedin 10ml methanol and water (5:95) by ultrasonic dipersion 30 min.then filtrated through a 0.22^m membrane filte, After that obtained thesample solution.
2.5 Specific experiment
The reference substance solution and sample solution were injected into the liquid chromatograph and recorded the chromatograms, as showed in fig.2. Results indicate that the reference solution and sample solution retention time of main peak is consistent, and no other impurity component is interfered.___
(a) 1 V
(b) I'
(c) n-
(d)
III (e) --^-- I 1 1 1 1 [ I I I 1 I 1 1 ! I 1 1 ! 1 ! 1 III' 5 FIIÎTTTfTTTTITÎIf
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
400
9.00
в 00
1000 12 00 1400 1800 1800 20 00 22 00 2400 - ffSS« test7, ft1 b.2.«tt 1. ЯШ ACQUITYTUVChA, ЖЖ0М 2012-5-19 1:15:14 CST
Fig.2. the reference substance and samples of the UPLC chromatograms.(a)3,4-dihydroxy-phenylglycol-glucopyranoside;(b)lariciresinol-4-O-P-D-glucopyranoside;(c)pinoresinol-4"-O-P-D-glucopyranoside;(d) (+)Epipinoresinol-4-O-P-D- glucopyranoside;(e)Syringopicroside;(f)samples. 3.Results and discussion
3.1 Optimum condition of chromatography system
In our study,Mixture of methanol and water(containing 0.1% phosphoric acid solution and without 0.1% phosphoric acid solution )was used as mobile phase, there was no too big difference on the spectra, but retention time of the material was extended.Therefore, mixture of methanol and aqueous without 0.1% phosphoric acid solution was adopted. A method of the gradient elution condition was established for the simultaneous determination of five compounds in Folium Syringaein 25 minutes. The wavelength of 220 nm, 254 nm, 260 nm, 270 nm, 280 nm and 300 nm were chosen to recordchromatograms for fivecompounds to achieve maximum sensitivity. The best result wasachieved using wavelength of 280 nm.The flow rate was also investigated,and results showed flow rate of 0.3 mlmin-1was more appropriate. 3.2. Method validation 3.2.1 linear range
The linearity calibration curves for the five compoundswere assessed at at least five concentration levels, and triplicateinjections were applied at each concentration. Calibrationcurves were constructed by plotting the integrated chromatographicpeak areas (Y) versus the corresponding sample quantityof the injected standard solutions (X ^g). Least squaremethod regression was employed, and results were presentedin Table 3. High correlation coefficient values (R > 0.9997)were achieved in relatively wide concentration ranges for allthe analytes. Table 3 Result of regression analysis on calibration curves_
Compounds
3,4-dihydroxy-phenylglycol-glucopyranoside lariciresinol-4-O-P-D-glucopyranoside pinoresinol-4"-O-P-D-glucopyranoside (+)Epipinoresinol-4-O-S-D-glucopyranoside
Syringopicroside_
Regression Correlationcoefficient Linear
equation (R) range (^g)
y=555080x-4508 0.9997 0.500~2.500
y=562514x-3553.7 0.9998 0.503~2.515
y=519362x-2245.7 0.9998 0.306~1.530
y=565021x-11929 0.9997 0.402~2.010
y=525259x+57364 0.9998 1.003~5.015
3.2.2 Precision and repeatability
The precision were determined by the same analyzingsample and continuous injection 5 times bymentioned chromatographic conditions. Results showed that the RSD of peak area were 0.37%, 0.07%, 0.59%, 0.37% and 0.46%, respectively.To confirm the repeatability, five differentworking solutions was prepared and analyzed from the same sample,the resultof repeatability indicated that the RSD of peak area were0.44%, 0.30%, 0.77%, 0.34% and 0.44%, respectively.So the method is acceptable.
3.2.3Accuracy
The recoveries of the compounds were determined by themethod of standard addition. Suitable amounts of the five references were spiked into a sampleof folium Syringae, which were analyzed previously. The mixturewas extracted and analyzed by using the proposed procedure.Table 4 showed the results of accuracy. It indicated that the RSD was lessthan 5% respectively, and the method is thus workable.
Table 4 Accuracy test results_
Compounds
Added amount (mg)
Recorded amount (mg)
Recovery
(%)
Mean recover y (%) RSD (% )
100.3 1.33
99.5 1.68
101 1.84
99.9 1.92
98.8 1.27
3,4-dihydroxy-phenylglycol-
glucopyranoside
lariciresinol-4-O-ß-D-
glucopyranoside
pinoresinol-4"-O-ß-D-
glucopyranoside
(+)Epipinoresinol-4-O-ß-D
-glucopyranoside
Syringopicroside
1.750 1.509 1.530 1.407 7.021
1.771,1.
1.733,1.
1.453,1.
1.477,
1.571,1.
1.558,1.
1.394,1
1.392,1
6.957,7.
6.979,6
776,1.736, 764
518,1.504, 1.486 516,1.510, 568
375,1.425, 441
056,6.824, 888
101.2,101.5,99.2,
99.0,100.8
96.3,100.6,99.7,
97.9,98.5
102.7,99.1,98.7,
101.8,102.5
99.1,97.7,101.3,
98.9,102.4
98.9, 100.5, 97.2,
99.4,98.1
3.2.5 Content determination
The sample solution offolium Syringae was injected into the chromatograph according tothe mentioned experiment conditions.The study used external standard method to calculate the content of the five compounds. Table 5 showed the result of thetests of Content determination. Table 5 the results of thetests of Content determination(n=3)
Compounds Content( mg/g) Mean content( mg/g) RSD(%)
3,4-dihydroxy-phenylglycol- 94.7 95.5 94.8 95.00 0.46
glucopyranoside
lariciresinol-4-O-ß-D-glucopyranoside 86.7 86.7 86.6 86.67 0.07
pinoresinol-4"-O-ß-D-glucopyranoside 79.6 80.1 79.2 79.63 0.56
(+)Epipinoresinol-4-O-ß-D- 72.5 72.8 73.0 72.77 0.35
glucopyranoside
Syringopicroside 392.1 396.9 394.4 394.47 0.61
4.Conclusions
Folium Syringae is a great research value of medicinal herbs.Lariciresinol-4-O-P-D-glucopyranoside, 3,4-dihydroxy-phenylglycol-glucopyranoside, Syringopicroside and other componentswere the main active ingredient. Reviewing the relevantliterature,no data concerning theUPLC analysis of folium Syringaecontent were found.To controlthe quality effectively, this studied mainly five components were simultaneouslydetermined by UPLC.The chromatographic peak shape was symmetrical and good separation.The UPLC had the abilities of high degree of separation, precision, sensitivity and thecharacteristics of ultrahigh speed[8'9].This study saved test time and improved work efficiency,so the method can be applied for the quality evaluation of folium Syringa herbs and preparations.
References
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[8] TAMOSDZIUNAS V,PADARAUSKAS A.Comparison of LC and UPLC coupled to MS-MS for the determination of sulfonamides in egg and honey[J].Chromatographia,2008,67(l-2):783-788.
[9] Qirong Shi, Shikai Yan, Mingjin Liang, et al. Simultaneous determination of eight components in Radix Tinosporae byhigh-performance liquid chromatography coupled with diode arraydetector and electrospray tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 994-999.
Synthesis of Nanosized Metal-organic Frameworks (NMOFs) for Drug Delivery
Li Xiuyan Lv Shaowa Wang rui Li Weinan Li Yongji* (College of Pharmacy, Heilongjiang University of TCM, Harbin 150040, China)
Abstracts: Three types of nanoscale Metal-organic Frameworks IRMOF-1, IRMOF-3 and HKUST-1 were synthesized successfully at the room temperature and was characterized by powder X-ray diffraction(PXRD), SEM , TEM and Nitrogen adsorption instrument. Metal-organic frameworks (MOFs) have emerged as a promising platform for drug delivery, owing to their many characters like porosity, high surface areas, unsaturated metal coordination site, preparation simple and versatile structure etc.It is provided with high drug loadings and fixed drug release, so it has received considerable attention. But compared with other carrier materials,the study of the MOFs is only in the primary stage .
Key words:Metal-organic frameworks; MOFs; mesoporous material; drug delivery
Metal-organic Frameworks(MOFs) are a kind of hybrid materials that are built from metal ion connectors and polydentate bridging ligands which have been exploited on the bulk scale for a number of applications, including gas adsorption[1], catalysis[2], nonlinear optics[3] and drug delivery[4]. Moreover, the high structural flexibility of some porous MOFs [5] enables the adaptation of their porosity to the shape of the hosted molecule. Recently, nanoMOFs based on NPs of nontoxic porous iron(III) carboxylates were shown to exhibit important drug loadings and progressive release as well as interesting imaging properties [6]. Herein we report a general method for preparing nanoscale MOFs ( NMOFs ) using pouring rapidly and room-tempreture titrimetry and demonstrate the potential utility of NMOFs as drug carries.
We choose Cu and Zn as the metal connectors because they are necessary trace elements for the health of people. Benzenetricarboxylic acid (H3BTC) and terephthalic acid (H2BDC) are used as the
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