Analytical Method And Strategy For Discovery, Identification And Determination Of Target Multiple Compounds In Complex Matrix Samples

It has always been difficult to discover, identify and determine target multiple compounds in complex matrix samples. For pharmaceutical analysis, the complex matrix sample analysis mainly includes the determination of components or illegal adulterants in herbal medicines and their prescriptions, and the analysis of drugs and their metabolites or endogenous metabolites in biological samples. On the one hand, the composition and the matrix of the samples are very complex:a herbal medicine may contain thousands of components, and a biological sample can contain a large variety of endogenous substances; On the other hand, a lot of target compounds are micro and even trace, and many of their structures are unknown. Therefore, it has been a hotspot in recent years that how to rapidly and sensitively discover, confirm and identify known and unknown target compounds from the complex matrix and further quantify them accurately.In this thesis, several rutine complex matrix samples were selected to develope feasible and effective analytical methods and strategies for discovery, identification and determination of target mutiple compounds.The first part of this thesis chose metabolomic analytical method for micro biological samples as the research target. Biological samples in metabolomics study are always micro amount, but it need to comprehensive cover different abundance and properties of endogenous metabolites simultaneously. Therefore a new biosample pretreatment method was developed:100μl of plasma or tissue homogenate was added internal standards and 3 ml of methanol/methyl tert-butyl ether (1:1, v/v). After vortexed, centrifuged and removed protein precipitation, the solution was added 3.5 ml of methyl tert-butyl ether and 1.2 ml of water, and then vortexed for extraction. Lipids were extracted to the organic supernatant and polar metabolites were extracted to the aqueous phase. After centrifugation and separartion, the organic phase and the aqueous phase were dried under nitrogen stream and then re-dissolved respectively. This method could extract lipids and polar metabolites from the same micro amount sample (100μl). On the basis of this, a targeted analytical method for 106 polar metabolites was developed using a HILIC column which has a strong retention to polar compounds and triple quadrupole mass spectrometer. And combined with the established lipidomic profiling method and free fatty acids analytical method, the endogenous metabolites could be determined by different types of LC-MS according to their polarity and aboundance after pretreatment, forming a novel targeted metabolomic profiling platform which could simultaneously extract and quantify 808 metabolites covering the core network of lipid, energy, amino acid and nucleotide metabolism from the same micro-amount biological sample.Using this platform, cardioprotective effect of Ginkgo biloba L. extract and ginkgolide on myocardial ischemia rats was studied from the perspective of metabolomics. It revealed that Ginkgo biloba L. extract and ginkgolide could significantly regulate the disorders of lipid metabolism, amino acid metabolism and energy metabolism caused by myocardial ischemia. It was found 5 fatty acids,1 sphingolipid,4 phospholipids,9 glycerides,8 amino acids in the plasma and 2 sphingolipids,12 phospholipids,1 glyceride,7 polar metabolites in the myocardium were biomarkers for the cardioprotective effect of Ginkgo biloba L. extract; 5 fatty acids,10 sphingolipids,2 phospholipids,9 glycerides,7 polar metabolites in the plasma and 3 sphingolipids,6 phospholipids,1 glyceride,11 polar metabolites in the myocardium were biomarkers for the cardioprotective effect of ginkgolide. These regulation effects were closely related to their antioxidant, PAF antagonist and hypolipidemic properties of flavonol glycosides and terpene lactones.In order to further study the material foundation of the cardioprotective effect of Ginkgo biloba L. extract and ginkgolide, their components and metabolites were also studied. An analytical method for determination of components and metabolites using liquid chromatography coupled with high resolution mass spectrometry was established, which could simultaneously collect high resolution mass spectral data and multi-stages mass spectral data of compounds in the extract and biological samples. Then after analyzing the extract and urine, feces, plasma, myocardium of the admistrated rats, combined with the mass spectral tree similarity filter technique, it was found and identified 6 categories of 48 components in the Ginkgo biloba L. extract,3 prototypes with 1 methylated metabolite in the ginkgolide administrated rats, and 18 prototypes with 21 methylated, glucuronidated and sulfated metabolites in the Ginkgo biloba L. extract administrated rats.The second part of this thesis chose health food and herbal medicine as the study objects. At present, there are various kinds of illegal adulterants and their new analogues in health food. The detection, confirmation, and quantification of multiple illegal adulterants in health foods and herbal medicines by using a single analytical method are a challenge. This section reported on a new strategy to meet this challenge by employing high-performance liquid chromatography coupled with high-resolution mass spectrometry and a mass spectral tree similarity filter technique. First, illegal adulterant standards were used to establish an HPLC-HRMS method:samples were ultrasonic-extractated by methanol and separated on a Shiseido Capcell Core C18 column (50 × 2.1 mm,2.7 μm). The mobile phase consisted of acetonitrile and an aqueous solution containing 10 mmol/1 ammonium formate and 0.1% acetic acid at a flow rate of 0.3 ml/min by using gradient program. The column temperature was 35℃ and the sample injection volume was 2 μl. UV detection was set at 240 nm. A Thermo Fisher Scientific LTQ FT was connected to the LC system via an ESI interface. The compounds were detected in the positive ion mode by a full-scan mass analysis from m/z 50 to 600 with a data-dependent MS2 and MS3 analysis. HRMS data, multi-stages MS data and retention times of standards were collected. Second, MS data of standards were transformed into mass spectral trees and an illegal adulterants library was constructed. Third, health food and herbal medicine samples were pretreated and analyzed by HPLC-HRMS method, and then preliminarily screened by comparing their retention times and HRMS data with known illegal adulterants. If the samples contained a known illegal adulterant, MTSF technique was applied to confirm the target compound based on the similarity between its mass spectral tree and that of the illegal adulterants in the library. If the similarity score was greater than 950, the illegal adulterant in the sample was confirmed. The compound was then quantified against calibration curves. Last, unknown illegal adulterants could also be found by MTSF technique. After comparing the similarity between mass spectral trees of compounds in the sample and illegal adulterants in the library, a detected compound with a similarity score of more than 200 was considered as a potential illegal adulterant. Their candidate structure was obtained based on its mass spectral tree and precise molecular weight. Reference standards, which are commercially available or can be synthesized, were used to further confirm and determine them. At present,67 illegal adulterants in six categories have been input into the library. This new strategy has been tested by using 50 samples, and the illegal adulterants were rapidly and effectively detected, confirmed and quantified.The third part of this thesis chose urine as the study object. The metabolic pathways of anabolic androgenic steroids (AAS), such as stanozolol, are complex, varied and individually different, it is important to characterize their overall metabolic profiles and discover new and long-term metabolites for the aims of expanding detection windows. A new strategy for metabolic profiles characterization and long-term metabolites detection based on high performance liquid chromatography coupled with multi reaction monitoring technology was established. The analytical method for stanozolol and its metabolites in the urine was firstly developed:phase I metabolites were extracted by methyl tert butyl ether and phase II metabolites was extracted by solid phase extraction. The chromatographic separation was carried out on a Waters Symmetry C18 column (2.1 × 100mm, 3.5μm). The mobile phase was composed of an aqueous solution containing 0.1% formic acid together with acetonitrile and a gradient elution was employed. The flow rate was set at 0.3 ml/min, the column oven temperature at 35℃ and an injection volume of 5μl. A triple quadrupole mass spectrometer with an AJS ESI source was used for multiple reaction monitoring (MRM) scanning. Then utilizing the characteristic fragment ions of stanozolol and its major metabolites as the product ions, and speculating unknown precursor ions based on the possible phase Ⅰ and phase Ⅱ metabolic reactions in human body, the metabolite profiles of stanozolol could be comprehensively characterized, especially for those unknown and low concentration metabolites in human urine. Applying this strategy,27 phase Ⅰ and 21 phase Ⅱ metabolites of stanozolol were identified, in which 14 phase Ⅰ and 14 phase Ⅱ metabolites have not been reported previously.9 out of 48 metabolites could be detected over 15 days post drug administration. This strategy could be employed effectively to characterize AAS metabolic profiles and discover unknown and long-term metabolites in sports drug testing.