ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2014, том 69, № 1, с. 21-25
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
DETERMINATION OF MATRINE IN MEDICINE AND BIO-FLUIDS BASED ON INHIBITION OF LUMINOL-MYOGLOBIN CHEMILUMINESCENCE
© 2014 Judong You, Jiangman Liu, Donghua Chen, Zhenghua Song
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science Northwest University 710069, Xi'an, China Received 15.06.2011; in final form 05.05.2012
A green, rapid and sensitive flow injection procedure based on the inhibition of the chemiluminescence (CL) intensity from the luminol—myoglobin (Mb) system is proposed for the determination of matrine. The decrement of CL signal was linear with the logarithm of the matrine concentration over the range from 10 to 1000 ng/mL (R2 = 0.9978) offering a detection limit of 3.5 ng/mL. At a flow rate of 2.0 mL/min, one analysis cycle, including sampling and washing, could be accomplished in 0.5 min with a relative standard deviation (RSD) of less than 5.0%. The sensitive flow injection method was successfully applied to the determination of matrine in pharmaceutical injection and human serum, with recoveries from 94.1 to 113.4% and RSDs of less than 5.0%.
Keywords: matrine, myoglobin, chemiluminescence, luminol, flow injection.
DOI: 10.7868/S0044450214010095
As one of the major components found in Sophora roots, matrine has long been regarded as an anticancer herb in China. It is a typical quinolizidine alkaloid which was first isolated and identified in 1958 [1]. Matrine has a wide range of pharmacological actions such as anti-diarrhea [2], analgesic [3], anti-inflammatory [4] and immunoinhibition effects [5]. The main clinical applications are treatment of cancer (it could inhibit the growth of tumour cells directly and could also affect immune functions) [6, 7], viral hepatitis (it appears to inhibit the viral replication, reduce destruction of liver cells, and protect against fibrosis) [8], cardiac diseases (such as viral myocarditis) [9], and skin diseases [10]. Based on its good effect on curing skin diseases, a topical liniment was developed combining sophora's matrine with the anti-inflammatory flavonoid baicalin from scute for treatment of eczema, neurodermatitis, and psoriasis [11], and relaxant effects on aortic smooth muscles [12].
High performance liquid chromatography (HPLC) coupled with different detectors was widely used for determination of matrine [13-17]. Besides, gas chromatography (GC) [18], capillary electrophoresis (CE) [19-21], and electrogenerated chemiluminescence (ECL) [22] were also reported.
Chemiluminescence (CL) combined with flow injection (FI) technique, an attractive alternative for pharmaceutical analysis, is superior for its high sensitivity, rapidity, simplicity and feasibility [23]. It has been reported that myoglobin (Mb) could catalyze lu-
minol oxidation, while some ligands could inhibit the CL reaction [24]. In this paper, it was first found that matrine could greatly inhibit the CL intensity generated from the reaction between luminol and Mb. The decrement of CL signal was linear to the logarithm of the matrine concentration over the range from 10 to 1000 ng/mL with a detection limit of 3.5 ng/mL (3a). The sensitive FI-CL method was applied successfully to the determination of matrine in pharmaceutical injection and human serum samples, with recoveries from 94.1 to 113.4% and relative standard deviations (RSDs) of less than 5.0%.
EXPERIMENTAL
Reagents. All the reagents were of analytical grade, and water purified in a Milli—Q system (Millipore, Bedford, MA, USA) was used for the preparation of solutions. Matrine standard solution was obtained from Shaanxi Institute for Drug Control. Luminol (Fluka, Biochemika, Switzerland) was purchased from Xi'an Medicine Purchasing and Supply Station, China and used as supplied to prepare a 2.5 x 10-2 M stock standard solution by dissolving 0.44 g of luminol with 0.1 M sodium hydroxide to 100 mL in an amber glass calibrated flask. Horse heart Mb (Sigma) was used as received without further purification.
Apparatus. The FI—CL system used in this work is shown schematically in Fig. 1. A peristaltic pump of the IFFM—E Luminescence Analyzer (Xi'an Remax Elec-
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JUDONG YOU и др.
Pump
Fig. 1. Schematic diagram of the present FI—CL system for matrine.
tronic Science—Tech. Co. Ltd., Xi'an, China) was applied to deliver all streams at a flow rate of 2.0 mL/min on each line. PTFE tubing (1.0 mm i.d.) was used throughout the manifold for carrying reagents. A six-way valve with loop of100 ^L was used for sampling. The CL emission cell was a spiral quartz tube (1.0 mm i.d., 15 cm length) in order to produce a large surface area exposed to the adjacent photomultiplier tube (PMT) (model IP28, Hamamatsu, Japan). Extreme precautions were taken to ensure that the sample compartment and PMT were light tight. The CL signal produced in flow cell was detected without wavelength discrimination, and the PMT output was recorded by computer with an IFFE-E client system (Remax, China).
Procedures. The carrier (pure water) and the solutions (luminol, Mb and matrine) were propelled at a flow rate of 2.0 mL/min on each flow line. The pump was started in order to wash the whole flow system un-
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Fig. 2. Intensity of different CL systems in the flow injection system; a: luminol system; b: luminol—Mb—matrine system; c: luminol—Mb system. Luminol: 5.0 x 1(T5 M; NaOH: 0.025 M; Mb: 1.0 x 10-8 M; Matrine: 150 ng/mL.
til a stable baseline was recorded. 100 ^L of luminol was injected into the carrier stream by a six—way valve quantitatively, which was then merged with Mb and matrine stream. The mixed solution was delivered to the CL cell in an alkaline medium, and the peak height of the CL intensity was therefore detected by the PMT and luminometer. The concentration of the sample could be quantified based on the decrement of CL intensity, AI = Io — Is, where Io and Is were CL signals in the absence and in the presence of matrine, respectively.
RESULTS AND DISCUSSION
CL intensity—time profile. Before carrying out the FI—CL method, the kinetic curve was examined using 2.5 x 10-5 M luminol, 1.0 x 10-8 M Mb and 0.025 M sodium hydroxide. It was shown in Fig. 2 that the time (Tmax) for reaching maximum CL intensity (Imax) of luminol in the present of 1.0 x 10-8 M Mb shortened from 4.4 to 4.0 s, and the Imax increased rapidly from 71 to 255; in the present of 150 ng/mL matrine, the Imax of luminol—Mb decreased from 255 to 147 with the same Tmax of 4.0 s.
Effect of luminol, Mb and NaOH concentration.
Luminol concentrations from 5.0 x 10-7 to 5.0 x 10-4 M were tested. The CL signal increased steadily when luminol concentration was less than 2.5 x 10-5 M, over which the CL intensity tended to be stable. For a series of Mb solutions from 5.0 x 10-10 to 5.0 x x 10-7 M, the CL intensity arrived at the maximum with 1.0 x 10-8 M Mb and kept approximately constant over it. Therefore, 2.5 x 10-5 M of luminol and 1.0 x 10-8 M of Mb were used in this experiment. Due to the luminol reaction was more favorable under alkaline conditions and the CL intensity approached a maximum at 0.025 M NaOH, this con-
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DETERMINATION OF MATRINE IN MEDICINE AND BIO-FLUIDS BASED
Table 1. Results of matrine determination in pharmaceutical injection*
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Sample no. Added, ng/mL Found, ng/mL RSD, % Recovery, % Content, mg/mL
proposed method HPLC
0 50.1 2Л
1 30 0 79.8 48.5 0.6 l. 99.1 1.02 ± 0.01 1.08
2 50 0 96.4 50.! 1.2 I. 95.8 1.05 ± 0.01 1.06
3 70 0 128.4 71.. 1.2 H 111.7 1.13 ± 0.01 1.11
4 70 0 149.5 69.8 1.1 1.3 111.0 1.09 ± 0.01 1.06
5 100 0 163.9 103.7 1.1 0.9 94.1 1.06 ± 0.01 1.13
6 100 206.0 1.2 102.2 1.09 ± 0.01 1.05
* The average of five determinations.
centration was employed in the subsequent experiments.
Effect of flow rate and the length of mixing tubing.
The CL intensity increased with increasing flow rate, and the flow rate of 2.0 mL/min was chosen as a compromise between good precision and lower reagent consumption in this work. The length of the mixing tube was also adjusted to yield maximum light emission in the CL cell. It was observed that a 5.0 cm mixing tube afforded the best results with respect to sensitivity and was then selected as the optimum length of mixing tube.
Performance of the system for matrine measurements. Under the optimum conditions, a series of standard solutions of matrine was injected into the manifold depicted in Fig. 1. The decrease of CL intensity was found to be proportional to the logarithm of matrine concentration from 10 to 1000 ng/mL with the detection limit of 3.5 ng/mL (3a), and the regression equation was A/CL = 38.90LnCmatrine — 87.23, R2 = = 0.9978. The RSDs of five determinations were 2.02, 2.53 and 1.56% with matrine concentrations of 30, 300 and 1000 ng/mL, respectively. At a flow rate of 2.0 mL/min, the determination of analyte could be performed in 0.5 min, including sampling and washing, giving a throughput of 120 times per hour.
Interference studies. The interference of foreign substances was investigated by analyzing a standard solution of 100 ng/mL matrine to which increasing amounts of interfering species were added. And the
tolerable limit of interferents was taken as a relative error of less than 5.0%. Some ions and organic compounds commonly found in pharmaceutical or serum samples were considered as interferents. And the tolerable concentrations were as follows: over 50 ^g/mL for
I-, BrO-, amylum, sucrose, glucose, borate, maltose, methanol and ethanol; 10 ^g/mL for oxalate, urea, glutin, dextrin, tartrate and citrate; 1.0 ^g/mL for uric acid; 0.01 ^g/mL for Cu2+ and Fe2+/Fe3+. Compounds abundant in serum such as salt, lipid and proteins caused no obvious interference with the determination of matrine.
Possible mechanism of lumino
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