Determination of glibenclamide in some traditional Chinese medicine preparations by liquid chromatography

Abstract: A liquid chromatography-electrospray ion trap mass spectrometry method was established to identify the unrecognized chemical hypoglycemic agent glibenclamide in pure Chinese medicine hypoglycemic drugs. Method: A ZorbaxEclipseXDB-C8 column was used, with acetonitrile-water-formic acid as the active phase. According to the chromatographic storage time and multi-level mass spectrometry information of the detected compound, and compared with the reference substance, the chemical hypoglycemic drugs illegally incorporated in the Chinese medicine preparation Qualitative identification of drugs.

In recent years, the development of traditional Chinese medicine preparations has been relatively rapid, but the addition of chemical drugs to pure Chinese medicine preparations has seriously violated national regulations and threatened the health of patients. For example, adding "sildenafil" to the "pure traditional Chinese medicine" for the treatment of male sexual dysfunction; adding sibutramine to the weight loss medicine or thyroid hormones that can cause symptoms of hyperthyroidism. Therefore, it is necessary to establish a rapid, sensitive and reliable analysis and detection method to verify the true body of pure Chinese medicine preparations. Liquid chromatography-mass spectrometry (LC / MS) method is widely used in the qualitative detection of drugs due to its high specificity. This article establishes the LC / MS method for detecting whether pure Chinese medicine hypoglycemic drugs are illegally mixed with human chemistry Glyburide, a hypoglycemic drug. Glyburide is a second-generation sulfonylurea drug. If the dosage is not properly controlled, hypoglycemia can easily occur.

1 Instruments and reagents

LCQ type liquid chromatography (mass spectrometer, including electrospray ion source (ESI) and LCQ data processing system of Finnigan Company, USA, equipped with syringe pump and 250μL sampler for direct injection; HPLC system of Shimadzu Corporation, Japan) Including LC-10AD pump, Rheodyne 7125 injection valve).

Glibenclamide reference substance (purity 99.5%, Shenyang Pharmaceutical University Pharmacy Department); 6 pure Chinese medicine hypoglycemic drugs (provided by Liaoning Provincial Institute for Drug Control, 6 batches for different manufacturers, 200203O2, 20020601, 010401, 20020621 and 20010313); methanol, acetonitrile (chromatographically pure, Tianjin Kangkede Technology Co., Ltd.), formic acid (analytical pure, Tianjin Bodi Chemical Co., Ltd.), the water is double-distilled water.

2 Experimental methods

2.1 Chromatographic conditions Chromatography column: ZorbaxEclipseXDB-C8 column (5μm particle size, 150 × 4.6mm, Agilent); active phase: acetonitrile-water-formic acid (70: 30: 1); flow rate: O.5mL / min; column temperature: Room temperature; injection volume 5OμL.

2.2 Mass spectrometry conditions Electrospray ion source, positive ion detection, ion source ejection voltage: 4.50kV; capillary temperature: 200 ° C; capillary voltage: 6.25V; sheath gas flow rate: 1.05L / min; auxiliary gas flow rate: O.15L / min ; Detection methods: first-level full-scan method, second-level full-scan method, third-level full-scan method and selective ion monitoring method (SIM).

2.3 Preparation of glibenclamide standard solution Precisely weigh a certain amount of reference substance into a volumetric flask, add human active phase to dissolve and dilute to the mark, so that the concentration is 400 μg / mL.

2.4 Preparation of the test solution Take 10 capsules of different batches of the test capsules, take out the contents and mix them respectively, put the sample equivalent to the weight of 1 capsule into a 10mL glass test tube with a stopper, add 3mL of methanol, vortex 2min, ultrasound 20min, centrifugation 10min, take supernatant for analysis and determination.

3 Experimental results

3.1 Chromatography and mass spectrometry behavior of glibenclamide The LC / MS analysis of the glibenclamide reference substance was carried out by positive ion detection, and its storage time was 6.4 min; the results of the multi-stage mass spectrometry analysis were generated in the first-stage full-scan mass spectrometry The excimer ion [MH] peak is m / z494, [M Na] peak is m / z516, [2M H] peak is m / z987, [2M Ma] peak is m / zlO09, analyzed by two-stage full-scan mass spectrometry In the molecule, the amide bond to the left of the guanidine group in the molecule is broken.

Generate m / z369 fragment ions, and further perform a three-stage full-scan mass spectrometry analysis on m / z369, which mainly generates the fragment ions m / z169 generated by the left amide bond, and summarizes its cleavage pathways, which provides the identification of compounds in actual samples. Reliable basis.

3.2 Test results of the tested samples LC / MS analysis of the methanol extracts of 6 pure traditional Chinese medicine preparations under the same chromatographic and mass spectrometry conditions, all compounds with the same chromatographic behavior as the glibenclamide reference substance were detected, and the corresponding The same quasi-molecular ion m / z494 as glibenclamide was detected in the first-level full-scan mass spectrometry; to further determine the structure of the detected compound, it was subjected to multi-level mass spectrometry analysis. The full-scan mass spectrometry is exactly the same as the glibenclamide reference substance, thus it can be confirmed that the compound detected in the six tested Chinese medicine hypoglycemic drugs is the chemical glibenclamide.

4 Discussion

The detection methods of glibenclamide in the literature include ultraviolet detection method, fluorescence detection method and mass spectrometry detection method. Traditional Chinese medicine preparations usually contain many kinds of Chinese medicinal materials, each of which contains many kinds of components. When using ultraviolet detection method or fluorescence detection method to detect traditional Chinese medicine extracts, due to the low selectivity, there are many interference components In some cases, the chromatographic separation of the test object puts forward higher requirements, so it may produce false positive results. In this paper, LC / MS technology is used to combine the high separation capacity of high-performance liquid chromatography with the high selectivity of mass spectrometry analysis, and the multi-level mass spectrometry analysis of the chemical hypoglycemic agent glibenclamide illegally incorporated in pure Chinese medicine preparations. Its structural confirmation was carried out, which enhanced the credibility of the test results. The method has high sensitivity and strong selectivity, and has practical significance for the quality control of traditional Chinese medicine preparations.

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