ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2010, том 65, № 9, с. 966-971
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
ON-LINE CONCENTRATION BY FIELD-ENHANCED SAMPLE INJECTION WITH REVERSE MIGRATING MICELLES IN MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY FOR THE DETERMINATION OF TRITERPENOIDS IN TRADITIONAL CHINESE MEDICINE © 2010 Ting-Fu Jiang*, Zhi-Hua Lv*, Yuan-Hong Wang*, Mei-E Yue**
*Department of Pharmacology, Marine Drug and Food Institute, Ocean University of China
Qingdao 266003, P.R. China **College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology
266042, P.R. China Received 17.08.2009; in final form 05.01.2010
In this work, a simple, reproducible and sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of five triterpenoids, lupeol (1), 1p-hydroxy-lupeol (2), lup-3p,1a-diol (3), lup-1p, 3p,11a-triol (4) and 30-norlupan-3p,11a-diol-20-one (5) in traditional Chinese medicine of Salvia roborowskii Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI—RMM) was used for on-line concentration of triterpenoids. The optimum buffer contained 50 mM H3PO4, 160 mM SDS, 20% acetonitrile and 15% 2-propanol and pH of buffer was 2.0. The sample solution was diluted with 10 mM H3PO4 (pH 2.5, containing 10 mM SDS) and injected for 15 s with —8 kV after injection of 4 s water plug. The effects of concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection time ofwater plug, the injection voltage and injection time of sample on the separation and stacking efficiency were investigated. Under the optimum conditions, the analytes were well separated and by optimizing the stacking conditions, about 28-96-fold improvement in the detection sensitivity was obtained for triterpenoids. The contents of five triterpenoids in Salvia roborowskii Maxim were successfully determined with satisfactory repeatability and recovery.
Key words: micellar electrokinetic capillary chromatography, triterpenoids.
Salvia roborowskii Maxim, with a Chinese name "ye zhi ma", an annual or biennial herb distributed widely in Gansu province of China, has been used as a traditional folk medicine for the treatment of hepatitis and toothache [1]. The chemical constituents from this plant have been examined, and the isolation of fla-vonoids [2], aromatic acid [3], triterpenoids [4], sesquiterpene esters [5] have been reported. Terpenoids in Salvia roborowskii Maxim are biologically active compounds. Triterpenoids are major terpenoid in the extract of Salvia roborowskii Maxim. Most triterpenoids are important because of their biological activity [6, 7], and could be used as markers for chemical evaluation. So, identification and determination of triter-penoids will play an important role in the efficacy, the safety and therapeutic reproducibility of Salvia roborowskii Maxim.
The qualitative and quantitative analysis methods of triterpenoids include thin layer chromatography (TLC) [8, 9], gas chromatography (GC) [10], high-performance liquid chromatography (HPLC) [11-13] and (HPLC-MS) [14]. However, these methods suffer from materials and time consuming since large amounts of organic reagent and many operation steps are often required. Re-
cently, owing to its high resolving power, low solvent consumption and simple pretreatment, capillary electro-phoresis (CE) has been used as an attractive method for separating and monitoring triterpenoids and their related compounds [15-18].
To date, however, CE still does not hold the position as the most representative method among analytical separation techniques. One reason is that the sensitivity of this method is relatively low by comparison with HPLC, which is the most widely employed methods at present. So, the separating and monitoring Chinese traditional medicines by CE focus on normally the compounds with the conjugated system. Vigorous attempts (i.e., on-line solid-phase extractors, membrane pre-concentrators [19], powerful detectors [20], sample stacking [21-24], etc.) have been done to solve the detection problem and thus further extend the use of CE for the analysis of samples in trace amounts. In 1998, Quirino and Terabe developed an on-line concentration technique in MEKC, field-enhanced sample injection with reverse migrating micelles (FESI-RMM) [25]. Low-pH buffer was used to reduce the electro-osmotic flow. The sample is dissolved with the aid of micelles in solution and injected into the capillary using voltage. This concentration technique has
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pH 3.0
pH 2.5
pH 2.0
pH 1.5
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9 10 11
Fig. 1. Molecular structures of five triterpenoids 1, 2, 3, 4 and 5.
been shown to provide more than 100-fold increase in UV detector response with very high plate numbers. Sample stacking with a dynamic pH junction depends on the change in electrophoretic mobility as the charged an-alytes encounter the pH junction between the sample zone and the background eletrolyte (BGE) zone upon application of voltage. Due to the good result of the sample stacking, the FESI-RMM has been used as an attractive method for separating and monitoring Chinese traditional medicines [26, 27].
To the best of our knowledge, the determination of triterpenoids in Salvia roborowskii Maxim has not been reported. In this paper, we first developed a FESI-RMM method for the determination of five triterpenoids in the extract of Salvia roborowskii Maxim. The optimum separation and stacking conditions were achieved by systematically optimizing the concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection time of water plug and the injection voltage and injection time of sample.
EXPERIMENTAL
Apparatus and conditions. Experiments were carried out on an Agilent HP3D capillary electrophoresis system (Agilent, USA). The applied voltage was held constant at —25 kV. The column was an uncoated 50 ^m ID fused-silica capillary with a total length of 60 cm and an effective length of 51.5 cm (Yfongnian, Hebei Province, China). The temperature of the capillary cartridge during electrophoresis was maintained at 25°C and UV detection was done at 200 nm. Before each use, the capillary was rinsed with 1 M NaOH for 10 min, then with water for 10 min; it was then conditioned with running electrolyte for 10 min. Between runs, the capillary was rinsed with water and electrolyte for 5 min each. Samples were
Fig. 2. Effect of the pH on the migration time. Analytical 4, 160 mM SDS, 20% acetoni-
conditions: 50 mM H3PO,
trile and 15% 2-propanol. Voltage, —25 kV; temperature, 25°C; UV detection at 200 nm. The analytes were injected for 15 s at —8 kV after injection of 4 s water plug. The concentration of the analytes was 2.5 ^g/mL.
loaded by injected for 15 s with —8 kV after injection of4 s water plug.
Materials and reagents. Salvia roborowskii Maxim was collected from the Gansu provinces of China. Standards of five triterpenoids 1, 2, 3, 4 and 5 (were isolated from extracts of Salvia roborowskii Maxim, and their structures were confirmed by the *H, 13C NMR and MS data (molecular structures were shown in Fig. 1). All NMR data were identical to those of the corresponding known compounds previously reported in the literature [4]. All chemicals were of analytical-reagent grade from Beijing Chemical Factory (Beijing, P.R. China). Deionized water was used throughout. All solutions and samples were filtered through 0.45 ^m syringe filter.
The buffer solutions containing certain amounts of H3PO4, SDS and organic solvents adjusted to the desired pH with 0.1 M NaOH, were all filtered through a 0.45 ^m membrane filter and degassed prior to use. A standard solution of 2500 ^g/mLof each triterpenoids was prepared in methanol, filtered, and degassed by the same procedure as used for buffer solutions. The various concentration of the sample solutions were prepare by appropriate dilution from the stock solution with 10 mM H3PO4 (pH 2.5) when needed.
Sample preparation. The air-dried Salvia roborowskii Maxim (2.0 g) was powdered and extracted with 50 mL methanol in Soxhlet extractor for 2 h, then filtered through a filter paper and a 0.45 ^m membrane filter.
RESULTS AND DISCUSSION
Optimizing the separation conditions. To verify the effect of buffer pH on migration behavior, experiments
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Fig. 3. Effect of the SDS concentration on the migration time. Analytical conditions: H3PO4 buffer at pH 2.0. Other conditions were similar to Fig. 2. The concentration of the analytes was 2.5 ^g/mL.
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20% 15%
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4 6 8 10 Mirgation time, min
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Fig. 4. Effect of acetonitrile concentration on the migration time. Analytical conditions: H3PO4 buffer at pH 2.0. Other conditions were similar to Fig. 2. The concentration of the analytes was 2.5 ^g/mL.
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were performed using the running buffers containing 50 mM H3PO4, 160 mM SDS, 20% acetonitrile and 15% 2-propanol with different pH (1.5~3.0). Fig. 2 shows the effect of buffer pH on the migration time of five triterpe-noids. The results indicated that the migration time increased with the increase of buffer pH from 1.5 to 3.0. Taking account of both the resolution and analytical time, pH 2.0 was then selected as the preference pH for further optimization.
In FESI-RMM method, the effect of SDS concentration on the separation and stacking of analytes was important. Different SDS concentrations (80, 100, 120, 140, 160 and 180 mM) in the electrolytes were used to study the effect of SDS concentration. Fig. 3 shows the effect of buffer pH on the migration time of five triterpe-noids. With the SDS concentration increasing, the peak areas and peak heights of analytes increased and t
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